2134 Chem. Commun., 2012, 48, 2134–2136 This journal is c The Royal Society of Chemistry 2012

Cite this: Chem. Commun., 2012, 48, 2134–2136

An ab initio mechanism for efficient population of triplet states in

cytotoxic sulfur substituted DNA bases: the case of 6-thioguaninew

Lara Martınez-Fernandez,aLeticia Gonzalez

band Ines Corral*

a

Received 18th September 2011, Accepted 22nd December 2011

DOI: 10.1039/c2cc15775f

The deactivation mechanism of the cytotoxic 6-thioguanine, the

6-sulfur-substituted analogue of the canonical DNA base, is unveiled

by ab initio calculations. Oxygen-by-sulfur substitution leads to

efficient population of triplet states—the first step for generating

singlet oxygen—which is responsible for its cytotoxicity.

The high photostability of DNA is generally related with

ultrafast radiationless relaxation processes,1 which quickly

dissipate the absorbed electronic energy before it causes

photochemical harm. Small modifications of the molecular

DNA components can lead to very different photochemistry

causing DNA damage. 6-Thioguanine (6-TG) is a prominent

example. The sulfur analogue of the naturally occurring

guanine base (Scheme 1) has been prescribed as an effective

immunosuppressant and for treating inflammatory disorders

and cancer since the 1950s.2 Besides its medicinal significance,

6-TG has unusual optical properties. Unlike the canonical

DNA bases that absorb ultraviolet B (UVB), 6-TG is a UVA

chromophore with a maximum absorption at 340 nm. UVA

(wavelengths 320–400 nm) comprises more than 95% of the

UV incident radiation but its potential DNA damage has been

considered to be much less harmful than that of UVB

(290–320 nm). However, recent studies indicate that the

UVA energy absorbed by thiopurines such as 6-TG is able

to generate singlet oxygen (1O2) and other reactive oxygen

species (ROS) causing irreparable transcription blocking

DNA lesions and damage to proteins, lipids and nucleic acids

that are detrimental to human cells.3

Indeed, long term prescription with 6-TG has been connected

with a 65- to 250-times higher skin cancer incidence than normal.4

Considering the link between excessive oxidative DNA damage

and human cancer,5 ROS and 1O2 from 6-TG and its metabolites

have been considered as plausible contributors to skin cancer in

transplant recipients.3b,c Elucidation of the molecular mechanism

for 1O2 generation from 6-TG is, therefore, essential to under-

stand its phototoxicity and eventually design novel therapeutic

agents based on thiopurine compounds.

Very recently, time-resolved fluorescence spectroscopy

has been used to characterize quantitative 1O2 in 6-TG and

the metabolite of 6-TG, 6-thioguanosine (6-TGuo).6 Direct

observation of 1O2 luminescence indicates that these photo-

sensitizers are very efficient although the quantum yields decay

very rapidly from ca. 0.50 to zero. Additionally, femtosecond and

microsecond excited state dynamics experiments performed on

6-TGuo detected two different decay signals on the picosecond

time scale related to the bleaching and recovery of the ground

state signal.7

In this contribution, we report the first comprehensive

relaxation mechanism of 6-TG and thus the reasons for the

striking reactivity differences with its canonical counterpart. Our

calculations have been performed using multi state second order

perturbation theory on state average complete active space self-

consistent-field wavefunctions (MS-CASPT2//SA-CASSCF).

Before discussing its photoreactivity, we briefly revisit the UV

absorption spectrum of 6-TG, previously studied by Rubin

et al.8,9 and Gomzi10 with semiempirical and TD-DFT calcu-

lations, respectively. Our state-of-the-art ab initio calculations

show (Table 1) that UV irradiation populates the S2 state, which

corresponds to a pp*CS excitation (hereafter specified as pp*)within the CQS bond and the purine ring (see Fig. S1 in the

ESIw). This state is computed vertically at 4.05 eV (306 nm). The

S1 excited state is an nSp*CS excitation (hereafter specified as np*)localized in the CQS bond. Although this state is dark in the

Franck–Condon (FC) region, it can be populated via internal

conversion from the S2. Two triplet states, T1 and T2, of pp* andnp* character, respectively, are predicted below the spectroscopic

S2 state. These two states will be protagonists of the intersystem

crossing (ISC) process generating 1O2 in 6-TG.

Scheme 1 2-Amino-9H-purine-6-thiol (6-thioguanine, 6-TG).

aDepartamento de Quımica, Facultad de Ciencias,Universidad Autonoma de Madrid, Campus de ExcelenciaUAM-CSIC, Modulo 13, Cantoblanco, 28049 Madrid, Spain.E-mail: ines.corral@uam.es; Fax: +34 914975238

b Institute of Theoretical Chemistry, University of Vienna,Wahringerstrasse 17, 1090 Vienna, Austria

w Electronic supplementary information (ESI) available: Furthercomputational details and full set of computational data. See DOI:10.1039/c2cc15775f

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This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 2134–2136 2135

The deactivation mechanism of 6-TG starting from the

spectroscopic S2 is shown in Fig. 1. The energies are relative

to the equilibrium geometry in the electronic S0 state. Deactivation

from the initially populated S2 state leads directly to a surface

crossing (conical intersection) with the S1,1(pp*)/1(np*), located at

3.86 eV, white arrows in Fig. 1. This conical intersection requires

the stretching of the CS bond, the twisting of the NH2 group, a

slight pyramidalization of C2, and a small reorganization of the

purine skeleton (Fig. S2, ESIw). The passage through this conical

intersection brings the population to the S1 state, where it can

relax into two different local minima: the 1(pp*)min and the1(np*)min, at 3.80 and 3.14 eV, respectively. The 1(pp*)min is

characterized by further pyramidalization and twisting of the C2

and amino substituent (Fig. S2, ESIw). Relaxing into the 1(np*)minimum requires on the other hand the synchronized stretching

and out-of-plane movement of the CQS bond and the recovery of

the NH2 group in its FC initial orientation (Fig. S2, ESIw).Notoriously, the probability of ISC at the 1(pp*) and 1(np*)minima is already very significant: both states are separated from

the triplet state T2 by less than 0.2 eV and the calculated spin–orbit

coupling exceeds 200 cm�1 at both points of the potential energy

surface (PES). Indeed, the corresponding minimum energy singlet–

triplet crossing points, 1(pp*)/3(np*) and 1(np*)/3(pp*), are locatedvery close to the 1(pp*)/1(np*) conical intersection and the 1(np*)minimum, respectively, see Fig. 1. Therefore, ISC to the triplet

states can proceed in two different ways: the first one consists

of reaching the triplet manifold through the 1(pp*)/3(np*)

crossing (red arrows in Fig. 1). This path allows a non-

adiabatic distribution of the population into 3(pp*)min1,3(np*)min and 3(pp*)min2 minima. The second possibility to

populate the triplet manifold is through the 1(np*)/3(pp*) ISCpoint (yellow arrows in Fig. 1). The molecules reaching the1(np*)min would populate the 3(pp*)min2 in the T2 that, in turn,

would immediately internally convert to the 3(np*)min in the T1

(second yellow arrow). In general, the high density of states

accumulated in the region of the (np*) minima allows for an

efficient population transfer from the singlet to the triplet PES.

The minima 3(np*)min and 3(pp*)min1 lie vertically 2.18 and

1.96 eV above the S0 potential at these particular geometries.

This implies that enough energy is available to excite ground

state triplet oxygen molecules (0.98 eV)11 and produce 1O2 and

other ROS.

The population of the triplet states competes with internal

conversion to the S0.7 Two S1/S0 conical intersections have

been optimized starting from the S11pp* and 1np* potentials.

Given the calculated energies (3.82 vs. 4.97 eV for the 1(pp*)/S0and 1(np*)/S0 conical intersections, respectively), the internal

conversion process will more likely proceed via the 1(pp*)/S0surface crossing (white arrows, Fig. 1). Deactivation through this

funnel requires the folding of the CQS bond perpendicularly to

the purine skeleton.

In view of the above results and assuming similar excited

state dynamics for 6-TG and 6-TGuo, a clear-cut assignment

of the delay lifetimes measured by Reichardt et al.7 can be

made. Fig. 2 shows a kinetic model. We ascribe the maximum

of the ground state bleaching, experimentally recorded at a

time delay of 0.3 ps (t1), to the consecutive decays of S2 to S1,

of S1 to T2 and of T2 to T1 via the1(pp*)/1(np*), 1(np*)/3(pp*),

and 3(pp*)/3(np*) crossing cascade. Given the shape of the

minimum energy profile from the 1(pp*)/1(np*) intersection

point, see Fig. S5 of the ESIw, and the relative energies of the1(pp*) and 1(np*) minima, the former pathway is expected to

compete, although to a lesser extent, with the population of the

triplet manifold from the 1(pp*) minimum via the 1(pp*)/3(np*)ISC point. Along this path, the conical intersection 3(pp*)/3(np*)will distribute the population into the three triplet minima

Table 1 Vertical absorption and emission energies (DE) and oscillatorstrengths (f) from the low-lying singlet and triplet states of 6-thioguaninecalculated at theMS-CASPT2//CASSCF(14,12)/ANO-L level of theory.Energies in eV (nm)

State DE vertical absorption f DE vertical emission

S11(nsp*CS) 3.36 (369) 0.000 2.07 (599)

S21(pp*CS) 4.05 (306) 0.535 2.35 (528)

S31(pp*) 4.90 (253) 0.144 —

T13(pp*CS) 3.10 (400) — min1: 1.96 (633)

min2: 2.18 (569)T2

3(nsp*) 3.31 (375) — 2.18 (569)T3

3(pp*) 4.24 (292) — —

Fig. 1 Singlet (white arrows) and triplet (red and yellow arrows)

decay paths for 6-thioguanine, predicted by CASPT2 calculations.

Relative energies (in eV) refer to the equilibrium ground state

minimum.

Fig. 2 Kinetic model for 6-thioguanine. Excitation of 6-thioguanine

to the S2 state results in an ultrafast internal conversion (IC) from the

S2 to the S1. From this potential, deactivation to the ground state in

tens of picoseconds7 (t2) is explained via IC to the singlet manifold and in

hundreds of nanoseconds7 (t3) through intersystem crossing (ISC) from

the triplet manifold. The triplet manifold is populated in 0.3 ps7 (t1)preferably from the 1(np*)min via the 1(np*)/3(pp*) crossing.

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2136 Chem. Commun., 2012, 48, 2134–2136 This journal is c The Royal Society of Chemistry 2012

3(pp*)min1,3(pp*)min2 and

3(np*)min. In turn, the partial recovery

of the ground state signal, competing with ISC to the triplet

states, and experimentally observed7 to take place in the timescale

of tens of picoseconds (t2) must be assigned to internal conversion

mediated by the 1(pp*)/S0 conical intersection. The alternative1(np*)/S0 internal conversion funnel to the S0 lies 0.9 eV above

the S2 spectroscopic FC energy and it is energetically inaccessible.

Finally, the experimental lifetime of hundreds of nanoseconds (t3)is ascribed to the decay to the S0 from the triplet minima.12

The extreme photoreactivity of 6-TG can be now compared

with the mechanism responsible for the high photostability of the

canonical DNA base. The photostability of the biologically active

guanine tautomer relies on an extremely favourable internal

conversion from the S11(pp*CC) spectroscopic state to the S0.

The pathway to the 1(pp*CC)/S0 conical intersection is barrierless

and does not involve any other stationary point.14

In the natural DNA base, deactivation to the triplet manifold

plays a minor role15 since the spin–orbit coupling terms at the

FC geometry are negligible and the large energy gap between

the 1(pp*CC) and3(nOp*) states makes ISC highly improbable

after vertical excitation. Moreover, the spin–orbit couplings

along the spectroscopic state’s minimum energy path with the

two lower-lying 3(pp*CC) states and far away from this path

with the 3(nOp*) state are insignificant. This situation is

drastically different in 6-TG where the calculated spin–orbit

couplings at the FC region amount to 100 cm�1, even if the

energy gap between the S2 and T2 states of 6-TG is still 0.7 eV.

In 6-TG the singlet and triplet nSp*CS and pp*CS states are

lower in energy than the guanine’s pp*CC spectroscopic state.

This stabilization forces the spectroscopic states of guanine

and 6-TG to be different, both in character and in energy.

Moreover, the destabilization of the 3(nSp*CS) state in 6-TG

along the 1(pp*CS) minimum energy path, added to the high

density of singlet and triplet states in the region of 1(np*CS)minimum, gives rise to energetically and mechanistically acces-

sible singlet–triplet crossings with spin–orbit couplings twice as

large as those at the FC point, therefore, providing a funnel for

very efficient ISC.

In summary, we have shown that the dramatic change from

the photostability of guanine to the phototoxicity of 6-TG can

ultimately be ascribed to both the heavy atom effect and the

strong stabilization experienced by the np* and pp* singlet

and triplet excitations involving the thiocarbonyl group

(B2 eV) as compared to that of other pp*CC states upon

sulfur substitution. The mechanistic path to 1O2 generation

from 6-TG is expected to shed new light on the biological

implications of thionucleobases in mutagenic and cancer

processes.

The authors thank the MICINN (Spain) for FPU grant

(LMF), a Juan de la Cierva postdoctoral contract (IC), the

Deutsche Forschungsgemeinschaft and the COST Action

CM0702 for financial support. Generous allocation of time

from the Centro de Computacion Cientıfica-UAM is gratefully

acknowledged.

Notes and references

1 (a) T. Schultz, E. Samoylova, W. Radloff, I. V. Hertel,A. L. Sobolewski and W. Domcke, Science, 2004, 306, 1765;(b) H. Satzger, D. Townsend, M. Z. Zgierski, S. Patchkovskii,S. Ullrich and A. Stolow, Proc. Natl. Acad. Sci. U. S. A., 2006,103, 10196; (c) N. K. Schwalb and F. Temps, Science, 2008,322, 243; (d) M. Barbatti, A. J. A. Aquino, J. J. Szymczak,D. Nachtigallova, P. Hobza and H. Lischka, Proc. Natl. Acad.Sci. U. S. A., 2010, 107, 21453.

2 G. B. Elion, Science, 1989, 244, 41.3 (a) P. O’Donovan, C. M. Perrett, X. Zhang, B. Montaner,Y.-Z. Xu, C. A. Harwood, J. M. McGregor, S. L. Walker,F. Hanaoka and P. Karran, Science, 2005, 309, 1871;(b) B. Montaner, P. O’Donovan, O. Reelfs, C. M. Perrett,X. Zhang, Y.-Z. Xu, X. Ren, P. Macpherson, D. Frith andP. Karran, EMBO Rep., 2007, 8, 1074; (c) X. Zhang, G. Jeffs,X. Ren, P. O’Donovan, B. Montaner, C. M. Perrett, P. Karran andY.-Z. Xu, DNA Repair, 2007, 6, 344; (d) P. Karran and N. Attard,Nat. Rev. Cancer, 2008, 8, 24; (e) R. Brem, F. Li and P. Karran,Nucleic Acids Res., 2009, 37, 1951.

4 S. Euvrard, J. Kanitakis and A. Claudy, N. Engl. J. Med., 2003,348, 1681.

5 N. Al-Tassan, N. H. Chmiel, J. Maynard, N. Fleming,A. L. Livingston, G. T. Williams, A. K. Hodges, D. R. Davies,S. S. David, J. R. Sampson and J. P. Cheadle, Nat. Genet., 2002,30, 227.

6 Y. Zhang, X. Zhu, J. Smith, M. T. Haygood and R. Gao, J. Phys.Chem. B, 2011, 115, 1889.

7 C. Reichardt, C. Guo and C. E. Crespo-Hernandez, J. Phys. Chem.B, 2011, 115, 3263.

8 Y. V. Rubin, Y. P. Blagoi and V. A. Bokovoy, J. Fluoresc., 1995,5, 263.

9 M. J. Stewart, J. Leszczynski, Y. V. Rubin and Y. P. Blagoi,J. Phys. Chem. A, 1997, 101, 4753.

10 V. J. Gomzi, J. Theor. Comput. Chem., 2009, 8, 71.11 G. Herzberg,Molecular Spectra and Molecular Structure I: Spectra

of Diatomic Molecules, 2nd edn, Van Nostrand, New York, 1950.12 This assignment substantially differs from that tentatively

proposed by Reichardt et al.7 using density functional theory atthe FC region and qualitative El-Sayed rules.13 These authorsascribe the t1 lifetime to both the population of the triplet manifoldvia a singlet–triplet crossing between the 1(pp*) spectroscopic stateand a T3 state, incorrectly assigned as 3(np*) and the simultaneousinternal conversion S2 - S1. In agreement with the correctcharacter of the T3 state (pp*) and the El-Sayed rules, we findthese two states negligibly coupled (spin–orbit couplingB20 cm�1)both at the FC and at the S2 minimum structures, where thecalculated energy gaps amount to 0.2–0.5 eV. The second lifetime,t2, was assigned to the relaxation pathway 1np* - S0. However,our accurate quantum chemical calculations demonstrate that thisfunnel is thermodynamically inaccessible. Instead, the systemshould proceed via the 1(pp*)/S0 conical intersection.

13 M. A. El-Sayed, J. Chem. Phys., 1963, 38, 2834.14 L. Serrano-Andres, M. Merchan and A. C. Borin, J. Am. Chem.

Soc., 2008, 130, 2473.15 R. Gonzalez-Luque, T. Climent, I. Gonzalez-Ramırez,

M. Merchan and L. Serrano-Andres, J. Chem. Theory Comput.,2010, 6, 2103.

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