Photophysical and Photochemical Properties of 3-methylpterin as a New and More Stable Pterin-type Photosensitizer

Article abstract of DOI:10.1111/php.12931

Pterin derivatives are heterocyclic compounds which are present in different biological systems. Neutral aqueous solutions of pterins present acid–base and keto–enol equilibria. These compounds, under UV-A radiation fluoresce, undergo photooxidation, generate reactive oxygen species and photoinduce the oxidation of biological substrates. As photosensitizers, they may act through different mechanisms, mainly through an electron transfer-initiated process (type-I mechanism), but they also produce singlet molecular oxygen (¹O₂) upon irradiation (type-II mechanism). In general, upon UV-A excitation two triplet states, corresponding to the lactim and lactam tautomers, are formed, but only the last one is the responsible for the photosensitized reactions of biomolecules. We present a study of the photochemical properties of 3-methylpterin (3-Mep) which, in contrast to most pterin derivatives, exists only in the lactam form. Also an improvement in the synthesis of 3-Mep is reported. The spectroscopic properties 3-Mep in aqueous solution were similar to those of the unsubstituted pterin derivative (Ptr) in its acid form, such as absorption, fluorescent and phosphorescent emission spectra. Experiments using 2′-deoxyguanosine 5′-monophosphate (dGMP) as oxidizable target demonstrated that methylation at C-3 position of the pterin moiety does not affect significantly the efficiency of photosensitization, but results in a more photostable sensitizer.

Full text of DOI:10.1111/php.12931

DR. CAROLINA LORENTE (Orcid ID : 0000-0001-8723-3795)
Article type : Special Issue Research Article
Photophysical and Photochemical Properties of 3-
methylpterin as a New and More Stable Pterin-type
Photosensitizer†
1
1
2
Sandra Estébanez , Carolina Lorente , Teodoro S. Kaufman , Enrique L.
2
1,*
1,*
Larghi , Andrés H. Thomas , Mariana P. Serrano
1Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA),
Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata
(UNLP), CCT La Plata-CONICET, Casilla de Correo 16, Sucursal 4, (1900) La Plata,
Argentina.
2Instituto de Química Rosario (IQUIR, CONICET-UNR) and Facultad de Ciencias
Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario. Suipacha 531, Rosario
S2002LRK, Argentina
†This article is part of a Special Issue commemorating the XIII ELAFOT Conference
(23-27 October 2017 - Villa Carlos Paz, Cordoba, Argentina).
* Corresponding authors’ emails: athomas@inifta.unlp.edu.ar (Andrés Thomas) and
mariserr@inifta.unlp.edu.ar (Mariana Serrano)
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/php.12931
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ABSTRACT
Pterin derivatives are heterocyclic compounds which are present in different
biological systems. Neutral aqueous solutions of pterins presents acid-base and keto-enol
equilibria. These compounds under UV-A radiation fluoresce, undergo photooxidation,
generate reactive oxygen species, and photoinduce the oxidation of biological substrates. As
photosensitizers, they may act through different mechanisms; mainly through an electron-
transfer initiated process (type I mechanism), but they also produce singlet molecular oxygen
(1O2) upon irradiation (type II mechanism). In general, upon UV-A excitation two triplet
states, corresponding to the lactim and lactam tautomers, are formed, but only the last one is
the responsible for the photosensitized reactions of biomolecules. We present a study of the
photochemical properties of 3-methylpterin (3-Mep) which, in contrast to most pterin
derivatives, exists only in the lactam form. Also an improvement in the synthesis of 3-Mep is
reported. The spectroscopic properties 3-Mep in aqueous solution were similar to those of the
unsubstituted pterin derivative (Ptr) in its acid form, such as absorption, fluorescent and
phosphorescent emission spectra. Experiments using 2’-deoxyguanosine 5’-monophosphate
(dGMP) as oxidizable target, demonstrated that methylation at C-3 position of the pterin
moiety does not affect significantly the efficiency of photosensitization, but results in a more
photostable sensitizer.
KEYWORDS. Methylated pterin derivative; photosensitization; electron transfer;
dGMP, guanine radical.
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INTRODUCTION
Pterins and their derivatives belong to a family of heterocyclic compounds which
were uncovered over a century ago, with the isolation of its first members from the pigment
of the wings of a butterfly (Pteridae) (1). However, they are also present in different
biological systems and play various roles. Nowadays it is known that besides acting as
pigments (2), pterins participate in other relevant biological functions; this includes being the
light-harvesting antenna of DNA photolyases, enzymes involved in DNA repair processes
after UV irradiation (3–5), as well as behaving as coenzymes (6,7), enzymes inhibitors (8)
and sensitizers of different biological targets (9–14).
These heterocycles can be found in living systems in different redox states. According
to this feature, they can be classified into oxidized (or aromatic) and reduced pterins. Within
the latter group, 7,8-dihydropterins and 5,6,7,8-tetrahydropterins are the most important
derivatives, due to their biological activity (15). Unconjugated oxidized pterins bear a
substituent at position 6 with one carbon atom or a short hydrocarbon chain. These
compounds are photochemically reactive; excited by UV-A radiation (320–400 nm) in
aqueous solution, they can fluoresce, undergo photooxidation to produce different
photoproducts, generate reactive oxygen species such as singlet molecular oxygen (1O2), and
photosensitize the oxidation of biomolecules (16,17).
Pterins present acid-base equilibria in aqueous solution, with pK values of ca 8. in
a
case of the aromatic pterins, the neutral form prevailing at physiological pH (16). Pterin
derivatives also exhibit tautomerism. Theoretical calculations demonstrated that the 4-keto
(lactam) tautomer is the most stable tautomer, whereas the enol (lactim) structure is ∼6
kcal/mol higher in energy, and three other neutral structures have energies within ∼4 kcal/mol
above that of the lactam (Figure 1). In addition, the studies revealed that the lactim anion is
the most stable structure in the chemistry of deprotonated pterins (18,19). Therefore, at pH >
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5, the lactam form is in equilibrium with the corresponding lactim anion.
Chahidi et al. have studied the transient absorption spectra of the excited triplet states
of pterin (Ptr), the parent unsubstituted compound of oxidized pterins, using the laser flash
photolysis technique and phosphorescence emission at 77K for the neutral molecule. Taking
into account the two absorption decays observed plus their phosphorescence data showing
two maximum bands, it was demonstrated that the triplet states of pterin have two forms (20).
It was proposed that the source of the two triplet states could be the lactim and lactam
tautomers. A similar result was observed with aqueous solutions of biopterin (Bip), which
displayed two laser-induced triplet states in the laser flash photolysis study, resulting from the
lactim and lactam tautomers (21). Furthermore, using 3-methylpterin (3-Mep) and 2-amino-4-
methoxypteridine, as substituted pterins with impeded tautomerization, Ledbetter et al.
clearly demonstrated that the two triplet states of the pterins derive from the lactim and
lactam tautomers (22).
As photosensitizers, it was reported that UV-A excitation of pterins induces DNA
damage (9,23,24). In the context of our studies on the photosensitizing properties of these
heterocycles, we have previously demonstrated that Ptr, the parent unsubstituted compound
of oxidized pterins, and the vitiligo-related pterin derivatives (Bip, formylpterin and
carboxypterin) are able to photoinduce the degradation of purine nucleotides [2’-
deoxyguanosine 5’-monophosphate (dGMP) (10,25–27) and 2’-deoxyadenosine 5’-
monophosphate (dAMP) (28,29)] in aqueous solutions. We have also demonstrated that,
under UV-A radiation, pterins photosensitize pyrimidine nucleotides (30), biomembranes
(13,31) and proteins (32).
Mechanistic analyses using dGMP as target molecule (26), established that in Ptr-
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photosensitization two competitive pathways take place: i) an electron transfer (type I
mechanism) and ii) the oxidation by 1O2 (type II mechanism). The former process is initiated
3
*
by an electron transfer from the nucleotide to the triplet excited state of Ptr ( Ptr ), yielding
•−
•+
the Ptr radical anion (Ptr ) and the nucleotide radical cation (dGMP ). On the other hand,
1
3
*
the Type II mechanism is initiated by energy transfer from Ptr to O2, yielding O2 which
reacts with the guanine moiety leading to different products (27). In the case of the
degradation of dGMP photosensitized by Ptr in neutral aqueous solution, the oxidation via the
1
•+
type I mechanism is much faster than the oxidation by O2 and the formation of dGMP
takes place exclusively by electron transfer from 3Ptr* in the lactam tautomer form; thus, this
species governs the photosensitized reaction with dGMP.
The main goal of this work was to change the excited states of pterin moiety by
modifying its chemical structure, resulting in better photosensitizing properties. The
observation that one of the two triplet states, the lactam form, is the active form during the
photosensitized reactions of biomolecules (26), prompted us to synthetized and study a
photosensitizer which exists only in the lactam form. The acylation at the C-3 position of the
pterin moiety (Scheme 1) prevents the formation of the lactim tautomer, which would result
in a more efficient formation of the lactam triplet excited state upon irradiation. If this
hypothesis is true, methylation at the C-3 position might improve the photosensitizing
properties of Ptr. Therefore, in this work we have improved the synthesis of 3-methylpterin
(3-Mep) and we have studied its photophysical properties, photostability and photosensitizing
properties using a nucleotide as target.
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MATERIALS AND METHODS
Pterin (Ptr) was purchased from Schircks Laboratories (Jona, Switzerland) and used
without further purification. The nucleotide dGMP, formic acid, 1,4-dioxane, DMF-DMA,
MeOH, NH OH and other chemicals were provided by Sigma-Aldrich.
4
Anhydrous dioxane was prepared by refluxing the commercial product over sodium to
which a small amount of benzophenone was added and distilling the solvent from the
resulting sodium-benzophenone ketyl.
The preparative TLC chromatographies were run on silica gel 60 GF
254
plates
(Merck, Germany), eluting with CHCl3:MeOH (9:1). The compounds were recovered
according to Sherma and Fried (33). The new compound gave single spots on analytical TLC
plates (silica gel 60 GF ) run in CHCl :MeOH (9:1). The chromatographic spots were
254
3
detected by exposure to 254 nm UV light, followed by spraying with Dragendorff reagent
(Munier and Macheboeuf modification), or with ethanolic p-anisaldehyde/sulfuric acid
reagent and final careful heating of the plates for improving selectivity.
1
The H NMR spectra were acquired at 300.13 MHz with the samples dissolved in
CDCl3 or DMSO-d6 on a Bruker Avance 300 (Bruker, Germany) NMR spectrometer. The
solvent residual signals (CHCl3 in CDCl3, δ 7.26; DMSO in DMSO-d6, δ 2.49) were used as
the internal standard. The chemical shifts are reported in parts per million on the δ scale.
Signals are informed as singlet (s), broad singlet (bs), doublet (d). The scalar couplings (J-
values) are given in Hertz (Hz).
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The photophysical and photochemical experiments were performed in aqueous
solution, at pH around 6.0. The pH values of the solutions were registered at a pH-meter
PHM220 (Radiometer Copenhagen), fitted with a pH electrode pHC2011-8 (Radiometer
Analytical). The pH of the solutions was adjusted by adding very small aliquots (few μL) of
concentrated (0.1 – 2 M) HCl or NaOH solutions with a micropipette.
Synthesis of 3-methylpterin. A solution of compound 2 (Scheme 1), 22.5 mg, 0.097
mmol in MeOH (2 mL) was treated with concentrated NH OH (2 mL). The reaction was
4
stirred under an argon atmosphere, at room temperature during 17h. Then, it was gently
concentrated under reduced pressure, leaving a solid residue, which was purified by
preparative TLC (CHCl :MeOH, 9:1) to give 3-methylpterin (4, Scheme 1, 13.1 mg, 77%),
3
1
as a yellowish solid, m.p.> 300°C; Lit.: 325-326°C. H NMR (DMSO-d ): 3.37 (s, 3H, Me-
6
3), 7.56 (bs, 2H, NH ), 8.35 (d, J= 2.1, 1H, H-7) and 8.65 (d, J= 2.1, 1H, H-6). The spectral
2
data for 4 were in agreement with the literature (34).
Emission measurements. Steady-state and time-resolved fluorescence measurements
were performed using a single-photon-counting equipment FL3TCSPC-SP (Horiba Jobin
Yvon), described elsewhere (35). All the experiments were carried out using quartz cells.
Fluorescence. To obtain the fluorescence spectra the sample solution was irradiated
with a Xenon source at room temperature and the luminescence was registered at 90° with
respect to the incident beam using a R928P detector.
The fluorescence quantum yields (ΦF) were determined from the corrected
fluorescence spectra using Equation 1:
ΦF = ΦF I AR/IRA
(1)
R
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where I is the integrated intensity, A is the absorbance at the excitation wavelength (λ ) and
exc
the superscript R refers to the reference fluorophore. In our experiments Ptr in acidic media
(Φ = 0.33) (16) was used as a reference. To avoid inner filter effects, the absorbance of the
F
solutions, at the excitation wavelength, was kept below 0.10.
In time-resolved experiments, a NanoLED source (maximum at 341 nm) was used for
excitation and the emitted photons were detected by a TBX-04 detector and counted by a
FluoroHub-B module.
Phosphorescence. The absolute ethanol solution was placed in a quartz capillary tube
(5 mm diameter), which was then immersed in the liquid-nitrogen-filled Dewar flask and
cooled to 77 K. The sample, with an absorbance of about 1.0 at the excitation wavelength,
was excited at 360 nm with a pulsed Xenon source (flash duration pulse 3 µs). The emission
spectra were obtained on using a single-photon-counting equipment FL3TCSPC-SP (Horiba
Jobin Yvon) spectrofluorimeter equipped with a and a TBX-04 detector. The apparatus was
operated in time-resolved mode, with a delay time of 10 ms. Excitation and emission
monochromator band-passes of 10 nm were used.
1
Singlet oxygen measurements. The experiments for O2 detection were carried out at
1
room temperature using deuterated water (D2O) as solvent, since the lifetime of O2 (τΔ)
1
under this condition is long enough to detect it (36). The O2 emission in the near-infrared
(NIR) region was registered using a NIR PMT Module H10330-45 (Hamamatsu) coupled to
the FL3 TCSPC-SP equipment mentioned above, as described elsewhere. (37). Briefly, the
sample solution (3 mL) in a quartz cell (1 cm x 1 cm) was irradiated with a CW 450W Xenon
source through an excitation monochromator (330 nm blaze grating). The luminescence in
the NIR region, after passing through an emission monochromator (1000 nm blaze grating),
was detected at 90o with respect to the incident beam. Corrected emission spectra obtained by
1
excitation at 350 nm were recorded between 950 and 1400 nm, and the total integrated O2
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phosphorescence intensities (IP) were calculated by integration of the emission band centered
at ca. 1270 nm.
1
For determining quantum yields of O2 production (Φ ), the IP of each compound
∆
x
R
(IP ) and that of phenalenone (IP ) used as a reference sensitizer in the same solvent were
measured, using matched absorbances at the wavelength(s) of excitation. Knowing the Φ of
∆
1
R
the reference (Φ ) and assuming that the quenching of O2 by 3-Mep and by the reference
∆
sensitizer is negligible compared with 1O2 deactivation by the solvent, the Φ
value of a
app
∆
given compound x is given by Equation 2
Φ∆x = Φ∆ IPx/IPR
(2)
R
Steady-state irradiation. The continuous photolysis of 3-Mep was carried out by
irradiating air-equilibrated aqueous solutions of the heterocycle at pH 6.0, in quartz cells (1
cm optical path length). A Rayonet RPR 3500 lamp (Southern N.E. Ultraviolet Co.) with
emission centered at 350 nm (band width (FWHM) 20 nm) was employed as the radiation
source.
Aberchrome 540 (Aberchromics Ltd.) was used as an actinometer for the
measurement of the incident photon flux density (q0,Vλ,p) at the excitation wavelength. The
method for the determination of q0,V has been described in detail elsewhere (38,39). The
λ,p
obtained value was 4.6 ( 0.3) × 10-6 Einstein L−1 s−1. Values of the photon flux absorbed the
reactant (qVλ,p) were calculated from q0,Vλ,p according to the Lambert–Beer law:
qVλ,p = q0,Vλ,p x (1-10-A)
(3)
where A is the absorbance of the reactant at the excitation wavelength.
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Analysis of irradiated solutions by UV⁄Vis spectrophotometry. The electronic
absorption spectra were recorded on a Shimadzu UV-1800 spectrophotometer, using quartz
cells of 1 cm optical path-length. The absorption spectra of the solutions were recorded at
regular intervals of irradiation time.
High-performance liquid chromatography. A high-performance liquid chromatograph
Prominence from Shimadzu (solvent delivery module LC-20AT, on-line degasser DGU-
20A5, communications bus module CBM-20, auto sampler SIL-20A HT, column oven CTO-
10AS VP and photodiode array detector SPD-M20A) was employed for monitoring the
reaction. A Synergi Polar-RP column (ether-linked phenyl phase with polar end-capping, 150
x 4.6 mm, 4 μm, Phenomenex) was used for product separation. Solutions containing 3% of
methanol and 97% of 25 mM formic acid (pH = 3.2) were used as mobile phase.
Quantum yield determinations. The quantum yields of reactant consumption (Φ- )
R
were determined in experiments performed under different conditions using Equation 4,
Φ- = (d[R]/dt)0/ qV
(4)
λ,p
R
where (d[R]/dt)0 (mol L-1 s-1) is the initial rate of reactant consumption. The initial rates were
obtained from the slope of the corresponding plots of concentration vs. irradiation time.
Transient absorption experiments. Laser flash photolysis (LFP) experiments were
performed using a LP980 equipment. Briefly, the excitation of the pterins was performed
with the third harmonic at 355 nm of a Continuum Nd:YAG Surelite II-10 laser (6 ns FWHM,
10 mJ per pulse). The transient absorption spectra of aqueous solutions of 3-Mep and Ptr (90
µM), previously saturated by bubbling Ar, were recorded with the LP980 laser-flash
photolysis apparatus (Edinburgh Instruments) linked to a 300 MHz Tektronik TDS 312C
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digital oscilloscope for signal acquisition. The signal analysis was done with the OriginPro
8.5 software from OriginLab Corporation.
RESULTS AND DISCUSSION
Synthesis and Characterization of 3-Methylpterin
3-Methylpterin (3-Mep, 4) was initially prepared in 17% yield, by condensation of the
hydrochloride of N -methyltriamino-2,4,5-oxo-1,6-pyrimidine (5) with glyoxal (40) under
1
different conditions (41) (Scheme 1). However, it has been later reported that the starting
hydrochloride is easily oxidizable, a drawback that was overcame employing the
corresponding sulfate (42), in which case the yield increased to 25%. 3-Mep has also been
prepared more conveniently in two steps (34% overall yield) by the selective methylation of
pterin (1) with N,N-dimethylformamide dimethyl acetal (DMF-DMA) (34).
Accordingly, as shown in Scheme 1, the synthesis of 3-Mep was carried out by the
selective methylation of N-3 with DMF-DMA (43) in anhydrous 1,4-dioxane. Under the
reaction conditions, compound 1 undergoes N-methylation (44) and its amino moiety suffers
a concomitant nucleophilic substitution by DMF-DMA, followed by a two-fold elimination
of methanol, to afford the N,N-dimethylformimidamido derivative 2. In turn, this was
submitted to amino group deprotection by ammonolysis with concentrated ammonia in
methanol.
Presumably, the deprotection takes place through attack of the ammonia to the N,N-
dimethylformimidamide moiety of 2, which easily forms the more labile N -
2
(aminomethylene) congener 3; this is less soluble and soon precipitates, slowly re-dissolving
to afford the expected 3-Mep (4).The use of carefully dried reactants for the first stage and
gentle removal of the volatiles under reduced pressure were key for obtaining an improved
overall yield of 68%.
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Spectroscopic properties
The absorption spectra of 3-Mep were recorded in aqueous solutions, at different pH
values between 4.0 and 11.0. As shown in Figure 2, all the recorded spectra were equal within
the experimental error, and presented two broad bands, one centered at ca. 350 nm and the
other more intense at ca. 270 nm. The spectra have features, very similar to those of the
neutral form of other aromatic pterins derivatives (45). The typical acid base equilibrium of
aromatic pterins was not observed, indicating that the methyl group attached at position 3
effectively avoids the formation of the anionic form.
The fluorescence spectra of 3-Mep by excitation at 350 nm were obtained in aqueous
solution at pH 6.0 and 11.0. Both emission spectra were equal within the experimental error
(Figure 3) and similar to the corresponding emission spectra of the neutral form of Ptr. The
fluorescence quantum yield (Ô ) of 3-Mep was determined using Ptr as reference, and a
F
value of 0.41 ( 0.02) was obtained. This value is higher than that previously reported for Ptr
in acidic media (45). In the time resolved fluorescence experiments, a first-order rate law was
observed in emission decays registered at different wavelengths between 350 nm to 650 nm,
after excitation at 341 nm (Figure 3). The fluorescence lifetime was ôF = 11.1 ( 0.2) ns and
pH-independent. This value is slightly longer than that reported for Ptr and other
unconjugated oxidized pterins (45,46) and explains why ÔF of 3-Mep is higher than that
reported for Ptr.
The triplet excited state of 3-Mep
In laser flash photolysis (LFP) experiments, transient absorption spectra of an Ar-
saturated 3-Mep aqueous solution at pH 6.0 (A355 = 0.45) were registered between 0 and 8 µs
after the 355 nm laser pulse (Figure 4a). The spectra show a major absorption maximum at
430 nm and a second one, with lower intensity, at 600 nm. These features do not change
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during the time window analyzed, suggesting a single transient specie. Hence, the decay of
the maximum transient absorption band at 430 nm (Figure 4b), and over the spectral range
(380-650 nm) could be adjusted using a mono-exponential fitting function, with recovered
lifetime τT = 7 ( 1) μs in average, confirming the existence of only one transient specie. On
the contrary, LFP experiments performed with a Ptr solution at pH 6.0, which exists as the
lactam tautomer in the ground state, show a biexponential dependence with time, indicating
that both, lactam and lactim tautomers are present after the laser excitation. Considering that
the methyl group in position 3 has replaced the hydrogen required for the tautomeric
3
*
equilibrium, the transient could be assigned to triplet excited state of 3-Mep ( 3-Mep ),
corresponding to the lactam form. Moreover, the τT value of 3-Mep is comparable to the
component with longer lifetime observed for other pterin derivatives (20,25,29).
As shown in Figure 5, the corresponding decay rate increases in the presence of O2.
3
This behavior serves to substantiate the assignment of the 3-Mep*. The interaction between
33-Mep* with O2 at pH 6.0 was analyzed by the calculation of the corresponding quenching
rate constant of the transient (kqO2), using the Stern-Volmer equation (Equation 5),
τ0 / τT = 1+ τ0 kqO2 [Q]
(5)
T
T
where τ0
and τT are the lifetimes of the triplet state in the absence and presence of the
T
3
quencher Q (Q= O2) (Figure 5 inset). Ground state molecular O2 quenches efficiently 3-
Mep* with kqO2 =1.7 ( 0.4) x 109 M-1s-1.
Figure 6 shows the phosphorescence excitation spectrum and decay at 77 K recorded
for 3-Mep, which has similar spectroscopic features to those previously reported for Ptr.
However, the phosphorescence lifetime for 3-Mep, τP = 860 ( 50) ms was shorter than that
for Ptr, τP =1100 ( 50) ms. The determined triplet excited state energy of 3-Mep was E =
T
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269 kJ mol-1, determined from the maximum wavelength and the half-width of the
phosphorescence band observed (47), using the Equation 6,
ET = hc (νp + Δνp)
(5)
where νp is the frequency at the maximum intensity of phosphorescence and Δνp is the
frequency at half-width. Table 1 summarizes the determined parameters for 3-Mep in
comparison with Ptr.
1
3-Mep was found to generate O2 in air-equilibrated D2O solutions by monitoring its
1
near-infrared luminescence at 1270 nm. Figure 7 shows the O2 emission that was detected
for 3-Mep, with Ptr as reference. In air equilibrated and O2-saturated aqueous solutions of 3-
1
Mep, the quantum yields of O2 production (Φ∆) were 0.14 ( 0.02) and 0.15 ( 0.02),
respectively. The values were calculated using the Equation 2
Photostability
In addition to a longer τT, photostability of the photosensitizer would be desired for its
use as a photosensitizer. Therefore, the photostability of 3-Mep was investigated, with
solutions of the heterocycle, which were exposed to UV-A irradiation (350 nm) and the
corresponding absorption spectra were recorded at different times. For comparative purposes,
Ptr solutions were exposed to UV-A irradiation under the same experimental conditions
(Figure S1a). It was observed that almost no spectral changes were registered in the case of 3-
Mep, over 150 minutes of irradiation time (Figure S1b). HPLC experiments confirmed that 3-
Mep was not consumed during the UV-A irradiation period; on the contrary, they
demonstrated that Ptr was degraded within the same time (Figure 8). These results revealed
that 3-Mep is considerably more photostable than Ptr under the same experimental
conditions.
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Photosensitization of dGMP by 3-Mep
To evaluate the photosensitizing properties of 3-Mep, dGMP was used as an
oxidizable target molecule. Air-equilibrated aqueous solutions (pH 5.5) containing dGMP
(500 µM) and 3-Mep (A350 = 0.6) were steadily irradiated at 350 nm during different periods
of time (up to 240 min). Under these experimental conditions dGMP did not absorb radiation.
The consumption of both compounds was followed by HPLC (Figure 9). The results were
compared to those obtained in experiments carried out under the same conditions (pH,
temperature, irradiation intensity and absorbance), using Ptr as a photosensitizer. In
particular, the mechanism of the photosensitization process of dGMP mediated by Ptr and
other pterin derivatives has been studied in detail and the reaction photoproducts have been
identified (27). Figure 9 shows that the consumption rate of dGMP was the same, within the
experimental error, when 3-Mep was used in place of Ptr as photosensitizer. The calculation
of the quantum yield consumption of the substrate was equal between both photosensitizers
(ΦdGMPPtr ≈ ΦdGMP3-Mep = 0.12 0.02) confirmed that 3-Mep is an efficient photosensitizer as
Ptr and other pterin derivatives (48).
Quenching experiments were carried out using 3-Mep as photosensitizer, to provide
direct evidence of the interaction between dGMP and 33-Mep*. The Stern-Volmer analysis of
3
the results obtained by LFP experiments for the quenching of the 3-Mep* by dGMP are
shown in Figure 10. The value of the corresponding bimolecular quenching rate constant,
determined from the slope of the Stern–Volmer plot (Equation 3, Q = dGMP), was
(kqdGMP=1.9 ( 0.6) x 109 M-1 s-1, characteristic of a diffusion-controlled limit. It is worth
are quite similar, thus suggesting that in vivo 3-Mep* can
O2
dGMP
3
mentioning that kq and kq
react with both O2 and dGMP to yield 1O2 and dGMP radical.
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The radical cation of guanosine nucleosides or nucleotides, produced by one-electron-
7
oxidation, rapidly deprotonates to form the neutral G radical with a rate constant of 1.8×10
-1
s
at pH 7.0. (49). The resulting neutral radical has been well characterized, and the
•
formation of 2´-deoxyguanosine radicals [dGMP(-H) ] in solutions containing dGMP and Ptr
upon UV-A excitation have already been reported (26).
Under our experimental conditions, the differential transient absorption spectra
showed the characteristic narrow absorption band centered at 320 nm of 2´-deoxyguanosine
radicals. The formation of the neutral dGMP radical (dGMP(-H)•) can be monitored by the
time-evolution of the ÄA at 320 nm after the laser pulse. The traces recorded for different
dGMP concentrations followed in all cases first-order kinetics and showed an increase in the
absorbance at infinite time with the dGMP concentration (Figure 11, inset). The lifetime
value obtained for the formation of the dGMP radical recorded at 320 nm, decreased with the
nucleobase concentration and was equal, within the experimental error, to that obtained for
3
the decay of the 3-Mep* at 430 nm at the corresponding dGMP concentration (Figure 11).
Taking into account that the fluorescence lifetime of 3-Mep is 11 ns, the participation of
singlet excited states can be discarded and, consequently, the results indicate that the
formation of the dGMP radical is exclusively by electron transfer from the triplet excited
state of 3-Mep.
CONCLUSION
As in the case of the recently synthesized decyl chain-pterins (50), in this work we
have demonstrated that by changing the chemical structure, the photochemical properties of
pterin sensitizers can be modulated. One of the main problems of the sensitizers used for
photodegradation studies is the photostability. We have synthesized a new compound that is
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an efficient photosensitizer and, at the same time, it is more resistant to its own
photodegradation than most pterin derivatives.
In the present work we report the synthesis and photochemical characterization of a
new photosensitizer, 3-methylpterin (3-Mep). The study of the photophysical features of 3-
Mep in aqueous solution revealed that its spectroscopic properties were similar to those of the
unsubstituted pterin derivative in its acid form, such as absorption, fluorescent and
phosphorescent emission spectra. On the other hand, in contrast to Ptr, 3-Mep has only one
triplet excited state, corresponding to the lactam tautomer and it is more photostable. The
photosensitization properties of 3-Mep were also studied and it was demonstrated that upon
UV-A irradiation this compound generates singlet oxygen (1O2), with a quantum yield of 1O2
production [Ö∆=0.14 ( 0.02)], which is lower than that corresponding to Ptr [Ö∆=0.18
( 0.02)]. The efficiency of the photoinduced degradation of dGMP used as oxidizable target,
was similar to Ptr. This study confirms that when the lactim-lactam equilibrium is impaired
(as is in this case due to the methyl substitution) only one triplet state, corresponding to the
lactam tautomer, is formed. In turn, this is responsible for the energy transfer to O2 to produce
1O2.
Supporting Information
Additional Supporting Information is available in the online version of this article:
Figure S1. Time evolution of the absorption spectra of irradiated (λexc = 350 nm) solutions of
(a) Ptr and (b) 3-Mep. The absorption spectra were recorded at 0, 30, 60, 90 and 150 min. The
cell optical path length was 1 cm.
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ACKNOWLEDGEMENTS
The present work was partially supported by Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET-Grant PIP 112-200901-00425; PUE IQUIR 2016),
Agencia de Promoción Científica y Tecnológica (ANPCyT-Grant PICT-2012–0508; PICT-
2015-2173), Universidad Nacional de La Plata (UNLP-Grant X712). S.E thanks ANPCyT
and CONICET for her doctoral research fellowship. C.L., T.S.K., E.L.L., M.P.S. and A.H.T.
are research members of CONICET.
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SCHEME and FIGURE CAPTIONS
Scheme 1. Reagents and conditions: a) DMF-DMA, 1,4-dioxane, reflux, 5 h (88%); b)
NH OH
, MeOH, r.t., 17 h (77%); c) Glyoxal; see ref (40).
4
(conc.)
Figure 1. Lactam and lactim tautomers of the neutral form and anionic form of pterins in
aqueous solutions.
Figure 2. Normalized absorption spectra of 3-Mep at different pH values.
Figure 3. Corrected fluorescence spectra of 3-Mep obtained by excitation at 350 nm of an
aqueous solution (A350 = 0.1), at pH 6.0 and 11.0. Inset: Fluorescence decay (λexc = 341 nm,
λan = 440 nm).
Figure 4. Laser flash photolysis experiments carried out with Ar-saturated aqueous solutions
of 3-Mep (A355=0.45). (a) Differential transient absorption spectra recorded at different times
after the 355-nm laser pulse. Inset: (b) Time dependence of the absorbance at 430 nm;
adjusted decay using a mono-exponential fitting function and the residual distribution.
Figure 5. Laser flash photolysis experiments: time dependence of the absorbance at 430 nm,
adjusted using a mono-exponential fitting function. Inset: Stern–Volmer plots of the
quenching of the 3-Mep triplet states by dissolved O2. τT values were calculated analyzing the
transient absorbance ÄA vs. t; ëexc = 355 nm, A355= 0.45.
Figure 6. Phosphorescence emission spectra at 77 K of 3-Mep. Inset: time dependence of the
phosphorescence intensity at 505 nm of 3-Mep in absolute ethanol solution, adjusted using a
mono-exponential fitting function. ë = 360 nm, A = 1.0.
exc
360
Figure 7. Singlet oxygen emission spectra of pterin and 3-Mep air-equilibrated D2O solutions
(λexc= 350 nm; A350= 1).
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Figure 8. Evolution of the concentrations of 3-Mep and Ptr in air-equilibrated aqueous
solutions exposed to UV-A radiation (λexc = 350 nm, A350= 0.8) as a function of the elapsed
irradiation time. The concentrations were determined by HPLC analysis, pH = 6.0.
Figure 9. Evolution of the concentration of dGMP in air-equilibrated aqueous solutions
exposed to UV-A radiation (λexc = 350 nm; A350=0.6) as a function of the elapsed irradiation
time using 3-Mep (■) or Ptr (□) as photosensitizer. The concentrations were determined by
HPLC analysis, pH = 5.5.
3
Figure 10. Quenching of 3-Mep* by dGMP. Time dependence of the absorbance, adjusted
using a mono-exponential fitting function, recorded in LFP experiments at different dGMP
concentrations. Inset: Stern–Volmer plot. λ
= 355 nm; λ = 430 nm, pH = 6.0, A355
=
exc
an
0.45.
Figure 11. Lifetimes for the decay of the triplet state of 3-Mep and for the formation of the
neutral radical dGMP(-H)• as a function of the dGMP concentration. The experiments were
performed in Ar-saturated aqueous solutions (λexc= 355 nm). Inset: time dependence of the
absorbance at 320 nm and 430 nm at 200 μM of dGMP concentration.
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TABLES
Table 1. Spectroscopic properties of 3-Mep and their comparison with Ptr.. Fluorescence (τF)
and phosphorescence lifetimes (τP), triplet excited state energy (ET), fluorescence quantum
yields (ΦF) and singlet oxygen quantum yields (ΦΔ).
ET / KJ mol-1
τF / nsb
( 0.2)
τP / msc
( 50)
ΦF
ΦΔ
a
d
Compound
( 15)
( 0.02)
( 0.02)
11.1
7.6
860
269
270
0.41
0.14
3-Mep
Ptre
1100
0.33
0.18
aλexc= 350 nm
bλexc= 341 nm, λem= 450 nm
cλexc= 360 nm, λem= 505 nm, in ethanol
dλexc= 350 nm
e Acidic form, data from reference (16).
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SCHEME
Scheme 1.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

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