Reactivity of nucleosides with a hydroxyl radical in non-aqueous medium

Article abstract of DOI:10.1002/chem.201201090

An experiment was conducted to demonstrate the reactivity of nucleosides with a hydroxyl radical (HO) in non-aqueous medium. Five nucleosides and three purine-derived lesions were taken as targets for HO attack. The reactivity with HO radical was determined by laser flash photolysis (LFP) of N-hydroxypyridine-2-thione (NPT) at 355 nm. Following an established kinetic model, the rate constants for the reaction between HO and the modified nucleobases were determined by using naphthalene as a standard. The values obtained for the silylated 2 -deoxyribonucleosides in acetonitrile are of the same order of magnitude and remarkably lower than diffusion control in this solvent, pointing to a similar reactivity pattern. The result confirmed that the reaction of a hydroxyl radical with 2'-deoxyribonucleosides is in general somewhat slower in acetonitrile than in water.

Full text of DOI:10.1002/chem.201201090

DOI: 10.1002/chem.201201090
Reactivity of Nucleosides with a Hydroxyl Radical in Non-aqueous Medium
Gemma M. Rodrꢀguez-MuÇiz, M. Luisa Marin, Virginie Lhiaubet-Vallet,* and
[
a]
Miguel A. Miranda*
The hydroxyl radical (HOC) is an important reactive
ronments, such as liposomes (lipoplexes) and polymers (pol-
yplexes) as vectors for gene delivery, in the non-viral ap-
oxygen species (ROS), which is usually present at very low
levels in biological systems, mainly arising from oxygen me-
tabolism. Its concentration is markedly enhanced upon ex-
posure of cells to exogenous chemical and physical agents,
such as ionizing radiation. It is well established that HOC
[7]
proaches to gene therapy. To enhance the transfection effi-
[8]
ciency, photochemical internalization has been developed.
It is based on improved endolysosomal release by photoacti-
vation of a sensitizer. However, a limitation of this tech-
nique relies on the potential loss of gene integrity by oxida-
mediated damage to biomolecules is involved in deleterious
phenomena such as aging, chronic inflammation, ischemia,
[9]
tively generated damage.
[1]
autoimmune disease, cancer.
Indeed, DNA oxidation
A quantitative estimation of the reactivity of HOC with
mediated by HOC may lead to sugar and base modifications
nucleic acids or their building blocks can be obtained by de-
that threaten genomic integrity due to their mutagenic po-
tential.
termining the reaction rate constants (kHO
medium, the kHO values have been determined by means of
pulse radiolysis; for nucleosides they range from 4.0 to 6.0ꢀ
C
). In aqueous
[2,3]
C
From a chemical point of view, HOC is an electrophilic
radical that reacts with most targets at high rates. It may un-
dergo addition to nucleobases and, in the case of thymine or
guanine, H-abstraction from the C-5 methyl or the C-2
9
À1 À1
[4b,10,11]
10 m s , close to the diffusion control limit.
Related kinetic information in non-aqueous medium is es-
sentially lacking. Hence, it seems relevant to check for the
stability of nucleic acid components against oxidative degra-
dation within lipophilic gene delivery vectors. Reactivity of
DNA bases through another type of mechanism, that is, one
electron oxidation, has been recently reported in organic
solvents. In the present work, acetonitrile has been select-
ed as a simple non-aqueous system. An important advantage
of this solvent is that it exhibits a low reactivity towards
[3,4]
amino group, respectively.
In general, the reactivity of
HOC with bases, nucleosides or nucleotides has been investi-
gated in aqueous medium by means of ionizing radiation (g-
radiolysis) or by the Fenton reaction with iron(II) and hy-
drogen peroxide. Interestingly, radiation experiments lead
mainly to nucleobase oxidation, whereas Fenton chemistry
favors hydrogen abstraction from the sugar. Thus, the reac-
tion conditions appear to play an important role in oxida-
tively generated DNA damage mediated by HOC. Indeed,
hydrogen abstraction by HOC from ethanol or isopropanol is
[12]
6
À1 À1 [6]
HOC (kHO
ca. 10 m s ). In addition, the diffusion rate
C
constant in acetonitrile is higher than in water; this would
provide a broader dynamic range for a possible differentia-
tion between the intrinsic reactivity of the nucleobases.
Here, five nucleosides and three purine-derived lesions
have been taken as targets for HOC attack. As their solubili-
ty in acetonitrile is insufficient to reach the concentrations
required for kinetic experiments, they have been employed
as their silylated 2’-deoxyribonucleosides (Figure 1, see
preparation in the Supporting Information).
[5]
2
5 times slower in acetonitrile than in water, whereas addi-
tion of HOC to an aromatic system such as naphthalene is
[6]
only 5 times slower in the organic solvent. Therefore, it
seems meaningful to investigate whether the reactivity of
HOC with nucleosides in non-aqueous systems is actually do-
minated by addition to the base.
In this context, considerable effort has been devoted in
recent years to include nucleic acids within lipophilic envi-
The reactivity with HOC radical was determined by laser
flash photolysis (LFP) of N-hydroxypyridine-2-thione (NPT)
[13]
at 355 nm (Scheme 1). Homolytic cleavage of this thione
generated HOC together with the unreactive pyrithiyl radical
[
a] Dr. G. M. Rodrꢁguez-MuÇiz, Dr. M. L. Marin, Dr. V. Lhiaubet-Vallet,
Prof. Dr. M. A. Miranda
(
lmax =490 nm), which provided a quantitation of the pro-
cess. Since HOC is undetectable, trans-stilbene (TS) was used
as a trap; the adduct [TS-OH]C exhibited a maximum at
Instituto de Tecnologꢁa Quꢁmica UPV-CSIC
Universidad Politꢂcnica de Valencia
Consejo Superior de Investigaciones Cientꢁficas
Avenida de los Naranjos, s/n, 46022 Valencia (Spain)
Fax : (+34)963877807
[5,6,13,14]
390 nm.
The kinetic traces at this wavelength were
monitored in the absence and in the presence of increasing
amounts of the modified nucleosides (0–34 mm). Following
an established kinetic model, the rate constants for the reac-
tion between HOC and the modified nucleobases were deter-
E-mail: lvirgini@itq.upv.es
mmiranda@qim.upv.es
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201090.
mined by using naphthalene as a standard.
8024
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 8024 – 8027

COMMUNICATION
Figure 1. Structure of the modified 2’-deoxyribonucleosides dNu(Si) syn-
thesized for the kinetic studies in acetonitrile.
Scheme 1. Experimental approach to determine the rate constants for the
reaction of HOC with the 2’-deoxyribonucleosides dNu(Si).
Figure 2. A) Kinetic traces recorded at 390 nm after laser flash photolysis
(
l
exc =355 nm) of NPT (0.29 mm), in the presence of TS (7.5 mm) and in-
creasing concentrations of Thd(Si) in deaerated acetonitrile solutions.
B) Stern–Volmer plots obtained as the ratio DA /DA of the traces mea-
sured at 390 nm versus concentration of each modified nucleoside,
Thd(Si) (
), dCyd(Si) (~), dUrd(Si) (*) and naphthalene (^), used as
a reference. Inset: Stern–Volmer plots of dAdo(Si) (~), 8-oxodAdo(Si)
*), dI(Si) (&) and naphthalene (^).
o
The traces due to [TS-OH]C in the presence of increasing
concentrations of Thd(Si) are shown in Figure 2A. The com-
petitive Stern–Volmer analysis for the three modified nu-
cleosides (together with naphthalene) is given in Figure 2B.
It was performed by plotting the ratio between the transient
&
(
absorbance at 390 nm in the absence (DA ) and in the pres-
ence (DA) of each substrate versus concentration.
o
Table 1. Rate constants for the reaction of HOC with 2’-deoxyribonucleo-
sides.
Determination of the absolute rate constants for the reac-
tion between the modified nucleosides and HOC (Table 1)
À1 À1
[a]
À1 À1
[a]
Nu(Si)
k
HOC [m
s
] CH
3
CN
k
HOC [m
s
2
] H O
[
b]
Thd(Si)
4.0
3.3
3.0
3.6
n.d.
4.7
n.d.
2.9
4.7
6.0
5.2
4.6
was based on the comparison between the obtained slopes
and the known absolute rate constant for naphthalene
[b]
[b]
[b]
[b]
dCyd(Si)
dUrd(Si)
dAdo(Si)
dGuo(Si)
9
À1 À1 [14a]
(
kHO
The values obtained for the silylated 2’-deoxyribonucleo-
C
=1.8ꢀ10 m s ).
[
[
c]
c]
[d]
4.1 /5.7
[
e]
8
8
-oxodAdo(Si)
-oxodGuo(Si)
4.3
4.8
–
sides in acetonitrile are of the same order of magnitude and
remarkably lower than diffusion control in this solvent,
pointing to a similar reactivity pattern. Furthermore, they
are in general slightly lower than those reported in the liter-
ature for the natural nucleosides in water. It has been re-
ported that HOC addition to aromatics is much faster in
[e]
dI(Si)
9
[
a] Rate constants are stated values ꢀ10 . [b] These values correspond to
the natural 2’-deoxyribonucleosides from ref. [10]. [c] Not determined:
compounds not soluble in CH CN. [d] This value corresponds to the nat-
3
ural 2’-deoxyguanosine from ref. [4b]. [e] These values correspond to the
-oxo-7,8-dihydro-2’-deoxyguanosine and 8-oxo-7,8-dihydro-2’-deoxyade-
8
water than in acetonitrile, due to a differential stabilization
nosine from ref. [11].
[6]
of the relatively polar transition state. In our case, this
effect should be less pronounced, since the substrates are al-
ready markedly polar.
the base, since selected ion monitoring (SIM) at m/z
127.05Æ0.05 (corresponding to unaltered thymine) gave
a trace identical to unreacted starting material (Figure 3A).
Conversely, no significant reaction at the sugar moiety was
detected, as demonstrated by the facts that 1) no free thy-
mine was observed (see the Supporting Information), and
2) SIM at m/z 359.21Æ0.05 (corresponding to unaltered sily-
lated 2-deoxyribose) gave a complex chromatogram with
two main peaks (1 and 2), in addition to starting material
To determine whether the base or the sugar moiety are in-
volved in the reaction with the HOC radical, steady-state ir-
radiations of the 2’-deoxyribonucleosides dNu(Si) were car-
ried out in the presence of equimolar amounts of NPT as
a source of HOC in air-equilibrated acetonitrile. The result-
ing reaction mixtures were analyzed by UPLC-MS; typical
chromatographic patterns are shown in Figure 3 for the case
of Thd(Si). It became clear that the reaction takes place at
Chem. Eur. J. 2012, 18, 8024 – 8027
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8025

M. A. Miranda, V. Lhiaubet-Vallet et al.
toin derived from the cleavage of the 5,6-bond were not de-
tected (see the Supporting Information).
In summary, the reaction of a hydroxyl radical with 2’-de-
oxyribonucleosides is in general somewhat slower in aceto-
nitrile than in water. Therefore, loss of gene integrity by oxi-
datively generated damage in lipoplexes or polyplexes is ex-
pected to be similar or even slightly lower than in aqueous
medium. In addition, the main reaction pathway involves
addition to the nucleobase, rather than hydrogen abstraction
from the sugar. This is interesting in connection with the re-
activity of nucleic acids within lipophilic gene delivery vec-
tors in non-viral approaches to gene therapy.
Acknowledgements
The Spanish Government (CTQ2009-13699 and Ramon y Cajal contract
(
RyC-2007-00476) to V.L.V) is gratefully acknowledged.
Keywords: DNA damage
·
laser flash photolysis
·
nucleobases · photochemistry · time-resolved spectroscopy
[
[
1] a) T. Finkel, N. J. Holbrook, Nature 2000, 408, 239–247; b) H. Sies
in Oxidative Stress: Oxidants and Antioxidants, Academic, New
York, 1991; c) B. N. Ames, M. K. Shigenaga, T. M. Hagen, Proc.
Natl. Acad. Sci. USA 1993, 90, 7915–7922.
2] a) A. Romieu, S. Bellon, D. Gasparutto, D. J. Cadet, Org. Lett. 2000,
2, 1085–1088; b) Q. Zhang, Y. Wang, Nucleic Acids Res. 2005, 33,
1593–1603; c) H. Ding, M. M. Greenberg, Chem. Res. Toxicol. 2007,
20, 1623–1628; d) K. Randerath, E. Randerath, C. V. Smith, J.
Chiang, Chem. Res. Toxicol. 1996, 9, 247–254; e) D. R. Lloyd, D. H.
Phillips, P. L. Carmichael, Chem. Res. Toxicol. 1997, 10, 393–400.
3] a) C. J. Burrows, J. G. Muller, Chem. Rev. 1998, 98, 1109–1151; b) J.
Cadet, M. Berger, T. Douki, J. L. Ravanat, Rev. Physiol. Biochem.
Pharmacol. 1997, 131, 1–87; c) W. K. Pogozelski, T. D. Tullius,
Chem. Rev. 1998, 98, 1089–1107; d) J. R. Wagner, C. Decarroz, M.
Berger, J. Cadet, J. Am. Chem. Soc. 1999, 121, 4101–4110; e) J.
Cadet, T. Douki, J. L. Ravanat, Acc. Chem. Res. 2008, 41, 1075–
[
[
1
083; f) G. Pratviel, B. Meunier, Chem. Eur. J. 2006, 12, 6018–6030;
g) R. J. Wagner, J. Cadet, Acc. Chem. Res. 2010, 43, 564–571.
4] a) J. Cadet, T. Delatour, T. Douki, D. Gasparutto, J. P. Pouget, J. L.
Ravanat, S. Sauvaigo, Mutat. Res. 1999, 424, 9–21; b) C. Chatgilialo-
glu, M. DꢄAngelantonio, M. Guerra, P. Kaloudis, Q. G. Mulazzani,
Angew. Chem. 2009, 121, 2248–2251; Angew. Chem. Int. Ed. 2009,
Figure 3. UPLC-MS analysis of the reaction mixture obtained after
steady-state UVA-irradiation (60 min) of NPT (50 mm) in the presence of
equivalent amounts of Thd(Si) in air-equilibrated acetonitrile. A) Select-
ed ion monitoring (SIM) at m/z 127.05Æ0.05, corresponding to the unal-
tered base; B) SIM at m/z 359.21Æ0.05, corresponding to the unaltered
sugar; C) SIM at m/z 541.23Æ0.05, corresponding to the base peak of
4
8, 2214–2217.
5] S. Mitroka, S. Zimmeck, D. Troya, J. M. Tanko, J. Am. Chem. Soc.
010, 132, 2907–2913.
[
[
[
1
and 2. Exact mass, together with the molecular formulae, are given.
2
6] M. P. De Matteo, J. S. Poole, X. Shi, R. Sachdeva, P. G. Hatcher,
C. M. Hadad, M. S. Platz, J. Am. Chem. Soc. 2005, 127, 7094–7109.
7] a) M. A. Kay, Nature Rev. 2011, 12, 316–328; b) M. Cavazzana-
Calvo, A. Thrasher, F. Mavilio, Nature 2004, 427, 779–781; c) S.
Bhattacharya, A. Bajaj, Chem. Commun. 2009, 4632–4656; d) M. A.
Mintzer, E. E. Simanek, Chem. Rev. 2009, 109, 259–302.
(
Figure 3B). Exact mass analysis of peaks 1 and 2 revealed
that they are isobaric, with molecular formula
C H N O Si (Figure 3C). This is compatible with the for-
a
22
42
2
8
2
[
8] a) T. Nagasaki, A. Taniguchi, S. Tamagaki, Bioconjugate Chem.
mation of thymidine glycols, which are well-known products
of HOC addition to natural thymidine in aerated solution.
Moreover, 5-formyl-2’-deoxyuridine and 5-(hydroxymethyl)-
2
003, 14, 513–516; b) P. K. Selbo, A. Weyergang, A. Hogset, O.-J.
Norum, M. B. Berstad, M. Vikdal, K. Berg, J. Controlled Release
2010, 148, 2–12; c) J. Guo, L. Bourre, D. M. Soden, G. C. OꢄSulli-
van, C. OꢄDriscoll, Biotechnol. Adv. 2011, 29, 402–417; d) K. Berg,
M. Berstad, L. Prasmickaite, A. Weyergang, P. K. Selbo, I. Hedfors,
A. Hoegset, Top. Curr. Chem. 2010, 296, 251–281.
2
’-deoxyuridine (marked with an asterisk in Figure 3B) re-
sulting from H-abstraction from the C5 methyl group were
also observed as minor products (see the Supporting Infor-
mation), whereas formamide or 5-hydroxy-5-methylhydan-
[
9] S. Rudiuk, S. Franceschi-Messant, N. Chouini-Lalanne, E. Perez, I.
Rico-Lattes, Photochem. Photobiol. 2011, 87, 103–108.
8026
www.chemeurj.org
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 8024 – 8027

Reactivity of Nucleosides with a Hydroxyl Radical
COMMUNICATION
[
[
[
[
10] C. von Sonntag, Free Radical Induced DNA Damage and Its Repair,
Springer, Berlin, 2006, Chapter 10, pp. 212–334.
11] T. A. Singh, B. S. Madhava Rao, P. OꢄNeill, J. Phys. Chem. B 2010,
[14] a) J. S. Poole, X. Shi, C. M. Hadad, M. S. Platz, J. Phys. Chem. A
2005, 109, 2547–2551; b) M. L. Marin, V. Lhiaubet-Vallet, L. Santos-
Juanes, J. Soler, J. Gomis, A. Arques, A. M. Amat, M. A. Miranda,
Appl. Catal. B Environ. 2011, 103, 48–53.
1
14, 16611–16617.
12] A. Capobianco, M. Carotenuto, T. Caruso, A. Peluso, Angew. Chem.
009, 121, 9690–9692; Angew. Chem. Int. Ed. 2009, 48, 9526–9528.
13] B. M. Aveline, I. E. Kochevar, R. W. Redmond, J. Am. Chem. Soc.
996, 118, 10113–10123.
2
Received: March 30, 2012
Published online: May 30, 2012
1
Chem. Eur. J. 2012, 18, 8024 – 8027
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www.chemeurj.org
8027