Photochemistry of 6-formylpterin in alkaline medium

Article abstract of DOI:10.1002/1522-2675(20011219)84:12<3849::AID-HLCA3849>3.0.CO;2-F

6-formylpterin solutions at pH 11 were photolyzed at 350 nm at room temperature. The photochemical reactions were followed by UV/VIS spectrophotometry, thin layer chromatography (TLC), and high-performance liquid chromatography (HPLC). In the presence of oxygen, 6-carboxypterin is the only photoproduct detected by the analytical techniques mentioned. In the absence of oxygen, a new compound showing an absorbance maximum at 480 nm is observed. The latter compound is thermally oxidized very fast in the presence of oxygen to 6-carboxypterin. The quantum yields of substrate disappearance and of photoproduct formation are reported.

Full text of DOI:10.1002/1522-2675(20011219)84:12<3849::AID-HLCA3849>3.0.CO;2-F

Helvetica Chimica Acta Vol. 84 (2001)
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Photochemistry of 6-Formylpterin in Alkaline Medium
by Andre¬s H. Thomas, Gustavo Sua¬rez, Franco M. Cabrerizo, and Alberto L. Capparelli*1)
¬
Instituto de Investigaciones FisicoquÌmicas Teoricas y Aplicadas (INIFTA), Departamento de QuÌmica,
Facultad de Ciencias Exactas, Universidad Nacional de La Plata, CONICET, CIC. Casilla de Correo 16,
Sucursal 4, (1900) La Plata, Argentina
¬
Dedicated to Professor Dr. Andre M. Braun on the occasion of his 60th birthday
6-formylpterin solutions at pH 11 were photolyzed at 350 nm at room temperature. The photochemical
reactions were followed by UV/VIS spectrophotometry, thin layer chromatography (TLC), and high-
performance liquid chromatography (HPLC). In the presence of oxygen, 6-carboxypterin is the only
photoproduct detected by the analytical techniques mentioned. In the absence of oxygen, a new compound
showing an absorbance maximum at 480 nm is observed. The latter compound is thermally oxidized very fast in
the presence of oxygen to 6-carboxypterin. The quantum yields of substrate disappearance and of photoproduct
formation are reported
Introduction. Pterins are a very important group of natural pigments [1][2].
Although they are present in small amounts in living systems, they play an important
biological role [3]. Some aspects of their photochemistry and photophysics is not
completely described and understood [4]. Pterin is the simplest form of this family of
compounds.
Pterin and its derivatives participate in a series of processes that range from the
metabolic pathway in the biochemistry of nucleic acids to their potential capacity to act
as a natural antenna for blue light [5]. Some compounds, such as pterin itself, are also
able to induce photoinduced damage in genetic material [6][7].
Several pterins of biological interest present different substituents at C(6) of the
molecule. Among them, 6-formylpterin, 6-carboxypterin, biopterin, xanthopterin, etc.
are well-known derivatives. Most of these compounds are photosensitive to light in the
UV-A region (320 400 nm) [8][9], and are also able to chelate metal ions in aqueous
solution [10 12]. Their photoreactivity is strongly dependent on the nature of the
substituent at C(6). The excited states of these compounds are able to quench
dioxygen, leading singlet oxygen [13].
1
)
Tel/Fax: 00542214242333, e-mail: alcappa@dalton.quimica.unlp.edu.ar.

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We have recently studied the photochemistry, in aqueous solution, of 6-
carboxypterin and folic acid [14 16]. The latter compound can be considered a
derivative of pterin, where the substituent is the para-aminobenzoylglutamic acid.
When solutions of folic acid are photolyzed in acid media, 6-formylpterin is obtained
with a quantum yield of F ˆ 2.5  10À2. 6-Formylpterin is further oxidized to 6-
carboxypterin and pterin. The quantum yields of these processes have been reported,
and the effect of oxygen and singlet oxygen on folic acid has also been discussed in the
literature [16].
The photochemistry of this family of compounds is strongly dependent on pH. In
the present papter, we report and discuss the photochemistry of 6-formylpterin in
alkaline medium in order to obtain more detailed information about this kind of
compounds and about the effect of the pH of the fate of the photoexcited species.
Experimental. 6-Formylpterin, 6-carboxypterin as well as other pterins, were provided by Schircks
Laboratories (Switzerland) and used without further purification. Other chemicals from Sigma-Aldrich were
used as received. The pH of solns. was adjusted to 11 by adding drops of conc. solns. of NaOH. The ionic strength
was ca. 10À3 m in all the experiments.
UV/VIS Spectra were performed on a CARY 3 (VARIAN) spectrophotometer, with a program for
smoothing and averaging signals. Measurements were made using quartz cells of 1-cm optical length and double
cells, one of them with an optical path of 1 cm for irradiation purposes, whereas the second one was of 0.2 cm for
absorbance measurements.
The continuous photolysis of 6-formylpterin solns. were carried out in the presence and absence of air.
Deaerated solns. were obtained by bubbling with O2-free N2 for 20 min. Rayonet RPR 3500 A lamps (Southern
N.E. Ultraviolet Co.) were employed.
Products were identified by analyzing the absorbance change during irradiation, as well as by TLC and
HPLC.
Absorption spectra of the solns. were recorded at regular intervals during the irradiation time. As explained
in [14][16], experimental-difference (ED) spectra were obtained by subtracting the spectrum at time t ˆ 0 from
the subsequent spectra recorded at different times. Each ED spectrum was normalized relative to the maximum
absolute value of the absorbance difference, yielding the normalized experimental difference (NED) spectrum.
Reference-difference (RD) and normalized reference-difference (NRD) spectra were obtained from aq.
solns. of commercial standards. The RD spectra were calculated as follows: the absorption spectrum of the
reactant in a given experiment was subtracted from the spectrum of standard solns. of the commercial
compounds assumed to be possible products of the reaction. These spectra were recorded using solns. of equal
concentrations of both compounds, at concentrations similar to those used in the photolysis experiments. NRD
Spectra were obtained by normalizing RD spectra as described above.
The procedures described previously were carried out for all experimental conditions reported in this
paper. The comparison between NED and NRD spectra allows us to characterize the major photolysis products.
TLC Experiments were performed using DEAE-cellulose plates (100 mm) and aq. NH4Cl 0.3% (p/v) soln.
as eluent. Irradiated 6-formylpterin (1.5 Â 10À4 m) and aq. solns. of pteridin-derivative standards were developed
by exposing them to 350 nm light. Under the present conditions, pterin derivatives show blue fluorescent spots.
Rf values have already been reported in [16].
HPLC (Konic Instruments 500 B) was employed to monitor the reaction and identify photoproducts: with a
C18 Spherisorb S5 ODS2 (250 Â 46 mm) column, eluent: 10% MeCN/H2O 60 :40, 90% aq. soln. of 20 mm
K3PO4 (pH 5.5), and 2.5 mm EDTA (soln. 2). A Lambda 1000 Bischoff HPLC spectrophotometer was used as
detection system. For irradiated 6-formylpterin solns., HPLC runs were monitored at 280 and 340 nm. The same
eluent was used to identify 6-carboxypterin.
The quantum yields of 6-formylpterin disappearance and 6-carboxypterin formation were determined in
experiments performed under different conditions. A chemical actinometer, ([CoIII(NH3)5)Br]2‡) was
employed for the measurements of photon rates [17]. When possible, the initial concentrations of reactants
were adjusted so that more than 99.99% of the incident radiation at 350 nm was absorbed. The evolution of the
concentrations of reactants and photoproducts during the irradiation time was followed by HPLC. Aq. solns. of
commercial standards were employed to obtain the corresponding calibration curves.

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The O2 consumption during the photolysis was measured with an O2-selective electrode (Orion, model 37-
08-99). The exper. set-up for these measurements was described in [18].
Photolysis experiments were carried out in the presence of furfuryl alcohol (10 mm; Riedel-de Hae»n). The
high reactivity of the latter compound towards singlet oxygen [19] greatly reduces its concentration during the
photolysis. In this group of experiments, 6-formyl- and 6-carboxypterin concentrations as a function of
irradiation time were determined by HPLC measurements. Results are compared with those obtained in the
absence of the selective singlet-oxygen scavenger.
Results and Discussion. Alkaline solutions of 6-formylpterin show very strong
changes when they are irradiated at 350 nm. The effect of oxygen on the photochemical
transformation is quite remarkable.
Photolysis in the Presence of Oxygen. Spectral changes on photolyzed solutions can be
observed as shown in Fig. 1,a. ED Spectra were obtained as described under Experimental.
Solutions were irradiated under steady conditions for almost 50 min. An isosbestic
point at 274 nm can be observed during the whole irradiation time. For simplicity, only
the behavior during the first 20 min of continuous photolysis is shown in Fig. 1,b.
No further changes are detected in irradiated solutions when stored in the dark.
After 50 min of irradiation, 6-formylpterin is photolyzed to yield a product that
accumulates in the solution. Upon further photolysis, this product itself suffers
photochemical transformation.
In a previous study of the photochemistry of folic acid in acidic medium [16], 6-
formylpterin is observed as one of the photoproducts. Under these pH conditions
(pH ˆ 6), this compound is photolyzed to yield 6-carboxypterin. This compound is also
the main photoproduct at pH 11, as seen in Fig. 2, though the rate of photo-
transformation of 6-formylpterin is slower than in acidic conditions. As already
described in [14][16], a NED spectrum can be directly compared with a NRD spectrum
obtained from standard solutions of 6-formylpterin and 6-carboxypterin. A direct
comparison allows us to propose 6-carboxypterin as the main photoproduct under these
experimental conditions.
Moreover, TLC analysis performed on the irradiated solutions shows only the
presence of two fluorescent substances. The Rf values correspond to the standards of 6-
forylpterin and 6-carboxypterin (Fig. 3).
In a similar way, HPLC experiments show the decrease of 6-formylpterin and the
appearance of a photoproduct that shows a similar tR value as that observed for
solutions of 6-carboxypterin.
Finally, in Fig. 4, the spectrum observed for solutions after 50 min of irradiation is
compared with that of 6-carboxypterin. These facts support strongly the conversion of
6-formyl- to 6-carboxypterin.
Linear behavior for the time evolution of the concentrations of 6-formylpterin and
6-carboxypterin as measured by HPLC is observed and depicted in Fig. 5,a and b.
The rate of degradation of 6-formylpterin is 3.7 Æ 0.3 mm/min, whereas the rate of
formation of 6-carboxypterin is 3.7 Æ 0.2 mm/min. The agreement between the two
values suggests that the photoproduct arises from the 6-formylpterin.
Actinometry measurements were performed as described under Experimental. The
quantum yield obtained under the present conditions is 9.0 Â 10À3, corrected by the
absorbance of the solution. This value is smaller than the value reported at pH 6 (ca.
3.8 Â 10À2).

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Fig. 1. a) Time evolution of the absorption spectrum of a 150 mm aqueous solution of 6-formylpterin at pH 11 and
in the presence of oxygen. Each spectrum was taken every 4 min, and the arrows show the direction of the
recorded changes. Optical cell length 1 cm; b) Experimental difference (ED) spectrum.
Effect of Oxygen on Photolysis. The O2 evolution during irradiation was monitored
in a closed cell. Experiments were performed with an oxygen electrode as described
under Experimental. As can be seen in Fig. 6, [O2] decreases steadily with the
irradiation time.
To investigate whether singlet oxygen is involved in the reaction, some experiments
were performed in the presence of furfuryl alcohol [20]. However, no conclusive
evidence can be reached from these experiments because a strong photoinduced
interaction was observed between the substrate and the singlet-oxygen scavenger. In
fact, mixtures of 6-formylpterin and furfuryl alcohol are stable in the dark, but, when

Helvetica Chimica Acta Vol. 84 (2001)
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Fig. 2. Difference experimental spectra (NDE) normalized at 280 nm. In spectrum (1), a NDE obtained after
10 min of irradiation is shown. Spectrum (2) is a typical NRD obtained from 100 mm solutions, pH ˆ 11, of 6-
formylpterin and 6-carboxypterin.
irradiated, deep changes in the absorbance spectrum can be detected with a very small
production of 6-carboxypterin. These changes are shown in Fig. 7.
Photoexcited 6-formylpterin is quenched by O2 to yield singlet oxygen [13][21].
Therefore, the results shown in Fig. 7 suggest that furfuryl alcohol, after reacting with
singlet oxygen, produces intermediates that react with 6-formylpterin, leading to its
degradation by an alternative pathway. This behavior is quite different from that
reported for the photolysis of folic acid in acidic medium in the presence of this singlet-
oxygen scavenger.
Moreover, the depletion rate of 6-formylpterin is faster in the presence of furfuryl
alcohol, i.e., after 5 min of irradiation, the degradation rate of the substrate reaches
25.4 mm/min, that is almost 7 times higher. The amount of 6-carboxypterin determined
by HPLC is very small, thus indicating that it is not the main photoproduct (see Fig. 8).
Therefore, in the presence of furfuryl alcohol, the formation of 6-carboxypterin is
partially inhibited.
Photolysis in the Absence of Oxygen. To evaluate the role of oxygen in the
photochemistry of 6-formylpterin, solutions previously bubbled with ultrapure
dinitrogen were irradiated. Under these conditions, visible changes in the color of
the solutions can be observed. The originally yellowish solutions developed an orange-
red color after irradiating during more than 50 min. This compound will be referred to
as −red intermediate or compound×. These changes are seen in Fig. 9, where the
spectrum shows a new band around 480 nm (Fig. 9,b).

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Fig. 3. TLC Runs. These lanes were obtained after dropping
solutions of standards and irradiated solutions: 1: 6-carbox-
ypterin (150 mm); 2: formylpterin (200 mm); 3 and 4: irradiated
150 mm of 6-formylpterin solutions at pH 11.0 after 10 and
20 min of continuous irradiation, respectively.
Fig. 4. 1) Normalized spectrum of a solution of 6-formylpterin after 50 min of continuous irradiation. 2)
Normalized spectrum of a solution of 6-carboxypterin (100 mm, pH 11.0).

Helvetica Chimica Acta Vol. 84 (2001)
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Fig. 5. Time vs. concentration profiles of a) 6-formylpterin (initial concentration 150 mm) photolyzed at pH 11
and b) 6-carboxypterin. Calibration curves were previously obtained in HPLC experiments as described under
Experimental.
Fig. 6. Time evolution of the O2 concentration in irradiated solution of 6-formylpterin (150 mm and pH 11.0)

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Fig. 7. a) Spectral changes observed in solutions with initial concentrations of 125 mm in 6-formylpterin and
10 mm in furfuryl alcohol at pH 11, before irradiation, and after 2 and 5 min of continuous irradiation at 350 nm.
Experiments were performed in standard cells with an optical length of 1 cm. b) Difference spectra obtained
after subtracting the spectrum at t ˆ 0 from those recorded of irradiated solutions.
The photoproduct obtained in oxygen-free solutions is stable in closed oxygen-free
cells. However, it readily suffers a thermal reaction as soon as air or O2 is admitted into
the cell. The band around 480 nm disappears. Difference experimental spectra match
the normalized theoretical spectra of the reference solution of 6-formylpterin and 6-
carboxypterin. These observations show that 6-carboxypterin is the oxidation product
of the −red compound× (Fig. 10).
TLC Runs performed on the aerated solutions show a new fluorescent spot with an
Rf value identical to that of 6-carboxypterin (not shown). The intensity of the spot of 6-
formylpterin decreases with the irradiation time. No other spots or fluorescent products
can be detected by this procedure.
HPLC Experiments confirm the same tendency as observed in TLC runs. The rate
of degradation of 6-formylpterin is 4.7 Æ mm/min (quantum yield of 1.1 Â 10À2),
whereas the rate of formation of 6-carboxypterin is 3.3 Æ 0.1 mm/min (quantum yield of

Helvetica Chimica Acta Vol. 84 (2001)
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Fig. 8. a) Time evolution of a photolyzed solution of 6-formylpterin (140 mm at pH 11.0) in the presence of
furfuryl alcohol (10 mm). b) Time evolution of 6-carboxypterin in the same system. Concentrations were
determined by HPLC.
0.8 Â 10À2) (see Fig. 11). The quantum yield is of the same order of magnitude as that
measured in the presence of O2 (i.e., 0.9 Â 10À3).
The −red intermediate× detected in the absence of O2 seems to be a relevant
intermediate in the oxidation of 6-formyl- to 6-carboxypterin. The similar quantum
yields measured in the presence and the absence of O2 support this hypothesis. In air
atmosphere, the −red intermediate× oxidizes immediately to 6-carboxypterin, explaining
why the −red intermediate× can be detected only under anaerobic conditions. If this is
the reaction pathway, singlet oxygen is not essential in the later stage of photooxidation
of 6-formyl- to 6-carboxypterin, because this process takes place in the absence of light.
As reported in the literature, 6-carboxypterin suffers a slow photodegradation in
acidic medium to yield pterin as a major photoproduct. However, at pH 11.0, pterin or
other pteridinic compounds are not formed [14], the presence of oxygen being essential
to observe photochemical changes. Moreover, 6-carboxypterin does not suffer any
chemical modification as a consequence of the absorption of UV-A light in the absence
of O2.
Conclusions. Photo-oxidation of 6-formylpterin in alkaline medium show similar
patterns to those observed in acidic medium. After absorption of a photon and without
participation of dioxygen, a −red intermediate× is generated, which shows spectral
characteristics different than those observed for the substrate and 6-carboxypterin.
−Pterins× derivatives that show similar spectral characteristics than those observed in
this paper have been reported in the photochemical studies on biopterin [22][23].
In a second stage, but corresponding to a themal oxidation processes, 6-
carboxypterin is produced by reaction of this −red intermediate× as seen in the Scheme.
As it was proposed in literature [22][23], the long-wavelength absorption at 480
500 nm of low intensity seems to be characteristic for 6-acyl-5,8-dihydropterins. These
compounds suffer spontaneous dehydrogenation to corresponding 6-acylpterin upon

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Fig. 9. a) Time evolution of the absorption spectra of irradiated solutions of 6-formylpterin (150 mm, pH 11.0) in
the absence of oxygen. Spectra were recorded after irradiating these solutions 4, 9, 17, and 33 min in 1-cm-
optical-length cells. Arrows shows the change direction recorded. The insert (b) show the recorded spectrum in
the wavelength region 400 700 nm. c) Difference spectra corresponding to those shown in a.
exposure to O2. Due to its spectral features and reactivity, the −red intermediate×
detected in our experiments could be 6-carboxy-5,8-dihydropterin. Further investiga-
tions are in progress to obtain additional evidence on the nature of the intermediate
detected in this study.

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Fig. 10. Photolysis of a oxygen-free solution of 6-formylpterin (150 mm, pH 11.0). 1: Spectrum of the started
solution, 2: Spectrum of the solution after 33 min of continuous irradiation. See the band between 400 to
600 nm. 3: Same solution as that shown previously after aeration.
Fig. 11. Time vs. concentration profiles of a) 6-formylpterin photolyzed at pH 11 in deaerated solutions (initial
concentration 140 mm) and b) 5-carboxypterin

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Scheme
¬
The present work was supported in part by Consejo Nacional de Investigaciones Cientificas y Tecnologicas
¬
¬
(CONICET-PIP 4354), Agencia de Promocion CientÌfica y Tecnologica (ANPCyT Grant No. PICT 06-03531),
¬
Comision de Investigaciones CientÌfificas de la Provincia de Buenos Aires (CIC), SETCIT-BMFB, and
Universidad Nacional de La Plata. A. H. T. wishes to thank CONICET for a research graduate fellowship. The
authors thank the authorities of CINDECA for the HPLC facilities, as well as to Lic. R. Martino for his technical
assistance. We are deeply thankful Prof. Dr. A. M. Braun and Dr. Esther Oliveros for the facilities given to
A. H. T. during his stay at the Engler Bunte-Institut, University of Karlsruhe, Germany. A. L. C. is a research
member of the CONICET. A. H. T. and F. M. Cabrerizo thank CONICET and CIC for their fellowships,
respectively.
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Received May 31, 2001