Stability of 7,8-dihydropterins in air-equilibrated aqueous solutions

Article abstract of DOI:10.1002/hlca.200890046

6-Substituted 7,8-dihydropterins (=2-amino-7,8-dihydropteridin-4(1H)-ones) are heterocyclic compounds that occur in a wide range of living systems and participate in relevant biological functions. In airequilibrated aqueous solutions, these compounds react with dissolved O₂ (autooxidation). The rates of these reactions as well as the products formed strongly depend on the chemical structure of the substituents. 7,8-Dihydro-6-methylpterin and 7,8-dihydro-6,7-dimethylpterin that bear electron-donor groups as substituents are the most reactive derivatives and undergo oxidation of the pterin moiety to yield the corresponding oxidized derivatives (6-methylpterin and 6,7-dimethylpterin, resp.). The oxidations of 7,8-dihydrobiopterin, 7,8-dihydroneopterin, and 7,8-dihydrofolic acid are slower, and they yield 7,8-dihydroxanthopterin as the main product. 7,8-Dihydroxanthopterin, 6-formyl-7,8-dihydropterin, and sepiapterin are rather stable, and their consumption in air-equilibrated solutions is negligible for several days. The pseudo-first-order rate constants of the reactions between these compounds and O₂ at 25° and 40° are reported. The biological implications of the results obtained are also discussed.

Full text of DOI:10.1002/hlca.200890046

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Stability of 7,8-Dihydropterins in Air-Equilibrated Aqueous Solutions
by M. Laura Da´ntola, Mariana Vignoni, Alberto L. Capparelli, Carolina Lorente, and
Andre´s H. Thomas*
´
Instituto de Investigaciones Fisicoquímicas Teoricas y Aplicadas (INIFTA), Departamento de Química,
Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4,
CONICET, Diagonal 113 y calle 64 (1900) La Plata, Argentina
(fax: þ 54-221-4254642; e-mail: athomas@inifta.unlp.edu.ar)
6-Substituted 7,8-dihydropterins (¼2-amino-7,8-dihydropteridin-4(1H)-ones) are heterocyclic com-
pounds that occur in a wide range of living systems and participate in relevant biological functions. In air-
equilibrated aqueous solutions, these compounds react with dissolved O2 (autooxidation). The rates of
these reactions as well as the products formed strongly depend on the chemical structure of the
substituents. 7,8-Dihydro-6-methylpterin and 7,8-dihydro-6,7-dimethylpterin that bear electron-donor
groups as substituents are the most reactive derivatives and undergo oxidation of the pterin moiety to
yield the corresponding oxidized derivatives (6-methylpterin and 6,7-dimethylpterin, resp.). The
oxidations of 7,8-dihydrobiopterin, 7,8-dihydroneopterin, and 7,8-dihydrofolic acid are slower, and they
yield 7,8-dihydroxanthopterin as the main product. 7,8-Dihydroxanthopterin, 6-formyl-7,8-dihydropterin,
and sepiapterin are rather stable, and their consumption in air-equilibrated solutions is negligible for
several days. The pseudo-first-order rate constants of the reactions between these compounds and O2 at
258 and 408 are reported. The biological implications of the results obtained are also discussed.
Introduction. – Pterins are a family of heterocyclic compounds that occur in a wide
range of living systems and participate in relevant biological functions. The most
common pterin derivatives are 6-substituted compounds. The molecular weight and
functional groups of these substituents are quite different, e.g., pterins may have
substituents with one C-atom, with a short hydrocarbon chain, or larger substituents
containing a 4-aminobenzoic acid moiety (Fig. 1). Derivatives with the latter type of
substituents are frequently called conjugated pterins. Pterins can exist in different
oxidation states and be divided into two classes according to this property: a) oxidized
or aromatic pterins containing the pyrazine[2,3-d]pyrimidine ring structure and b)
reduced pterins (Fig. 1). Within the latter group, 7,8-dihydropterins (¼2-amino-7,8-
dihydropteridin-4(1H)-ones) and 5,6,7,8-tetrahydropterins are the most important
derivatives due to their biological activity, e.g., 7,8-dihydroneopterin is secreted during
the oxidative burst of stimulated macrophages [1], and 7,8-dihydrobiopterin and 5,6,7,8-
tetrahydrobiopterin participate in the metabolism of amino acids [2]. The chemistry of
7,8-dihydropterins has been studied in detail [3].
Depending on their oxidation state, pterins have totally different reactivities
towards oxidizing agents, e.g., whereas 7,8-dihydropterins are very efficient singlet-
1
oxygen (1O2) quenchers [4], the reactivity of oxidized pterins towards O2 is relatively
low [5]. In air-equilibrated aqueous solutions, tetrahydropterins are rapidly oxidized by
dissolved molecular oxygen in its ground-state (O2), whereas 7,8-dihydropterins are
ꢀ 2008 Verlag Helvetica Chimica Acta AG, Zürich

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Fig. 1. Molecular structures of the 7,8-dihydropterins investigated at physiological pH (neutral forms),
together with the structures of corresponding oxidized pterins. 7,8-Dihydro-6,7-dimethylpterin and 6,7-
dimethylpterin have an additional Me group at C(7) of the pterin moiety.
relatively stable [6][7]. The oxidation of these compounds by dissolved O2 or reactive
oxygen species are of biological interest because they are generated under several
pathological situations and accumulated in oxidative environments, e.g., in the skin of
patients affected by vitiligo [8][9]. However, despite the relevance of this process from
the biological point of view, there is a lack of information on the chemical reaction
between 7,8-dihydropterins and O2 in aqueous solution at room or physiological
temperature. Moreover, in several studies reported in the literature, experiments with
these compounds are performed in the presence of O2, and their oxidation is not
controlled.
In this article, we describe the oxidation of a group of 6-substituted 7,8-
dihydropterins, with substituents of different molecular weight and with different
functional groups, by O2 in air-equilibrated aqueous solutions. We investigated the
following derivatives: 7,8-dihydro-6-methylpterin, 7,8-dihydro-6,7-dimethylpterin, 7,8-
dihydrobiopterin, 7,8-dihydroneopterin, 6-formyl-7,8-dihydropterin, sepiapterin, 7,8-
dihydroxanthopterin, and 7,8-dihydrofolic acid (Fig. 1). The pseudo-first-order rate

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constants of the reaction between these compounds and O2 at 258 and 408 were
determined, and the products of the reactions were analyzed. We discuss the effects of
the structural features of the dihydropterin derivatives and the biological implications
of the results obtained.
Results and Discussion. – Aqueous solutions (pH 7.0 – 7.3) of 7,8-dihydropterins
were prepared and kept in anaerobic conditions (Ar atmosphere) at 258 and 408 in
order to check their stability in the absence of O2. For all compounds studied, no
significant changes in the absorption spectra of solutions after more than 48 h were
observed. In the same way, HPLCmeasurements showed that the reactant concen-
trations did not decrease after 24 h of incubation in anaerobic conditions.
The chemical reactions between O2 and 7,8-dihydropterins in air-equilibrated
aqueous solution (pH 7.0 – 7.3) were investigated by UV/VIS spectrophotometry and
HPLCanalysis at 25 8 and 408. The chemical changes were followed as a function of
time during, at least, 24 h. The ꢁzero timeꢂ (t ¼ 0) corresponds to the admission of air to
the solution. In all cases, identification of products was carried out by means of HPLC
analysis: retention times and spectra, obtained using the DAD detector, of samples and
standard solutions were compared under different elution conditions.
Oxidation of 7,8-Dihydrobiopterin and 7,8-Dihydroneopterin. Significant spectral
changes were observed in aqueous solutions of 7,8-dihydroneopterin at 408 after
admission of air (Fig. 2). A blue-shift of ca. 20 nm was observed for the low-energy
band. In addition, a very slight increase in the absorbance of the solution in the VIS
region, with a maximum at ca. 423 nm, was also observed. On the other hand, slower
spectral modifications were observed at 258 (data not shown). Nevertheless, the
normalized experimental-difference (NED) spectra obtained at 258 and 408 were
similar within the experimental error, suggesting that the product(s) of the reactions
are the same at both temperatures. Very similar changes in absorption spectra, although
much slower, were observed for 7,8-dihydrobiopterin (Fig. 2). Moreover, at 258 these
changes were negligible (data not shown). This spectrophotometric analysis indicated
that both compounds undergo similar chemical transformation, but at very different
rates.
7,8-Dihydroxanthopterin was identified as the main product in the oxidation of 7,8-
dihydroneopterin and 7,8-dihydrobiopterin, by means of HPLCanalysis ( Fig. 3,a). This
observation is consistent with spectral analysis (Fig. 2): the low-energy absorption band
of 7,8-dihydroxanthopterin is blue-shifted (lmax ca. 310 nm) in comparison with the
corresponding band of the reactants (lmax ca. 330 nm).
The concentration profiles of reactants and 7,8-dihydroxanthopterin are shown in
Fig. 4. For both compounds, the fraction of consumed reactant converted into 7,8-
dihydroxanthopterin is more than 90%. Therefore, cleavage of the substituent and
oxidation at C(6) is the main pathway of the reaction of 7,8-dihydroneopterin and 7,8-
dihydrobiopterin with O2 (Scheme 1, pathway a). These results are in agreement with
previous studies that reported 7,8-dihydroxanthopterin as a typical oxidation product of
7,8-dihydropterin derivatives bearing a ꢀCHOHꢀ group at C(6) [3]. Taking into
account the general mechanism proposed for the oxidation of these compounds
[3][10], O2 most likely reacts with the covalent hydrate form of the reactants. However,
6-formyl-7,8-dihydropterin was also detected by HPLCin the air-equilibrated solutions

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Fig. 2. Oxidation of 7,8-dihydropterin derivatives in air-equilibrated aqueous solutions. Evolution of the
absorption spectra as a function of time. Spectra were recorded at 0, 2, 4.5, 9, and 24 h after admission of
air for 7,8-dihydrobiopterin, 7,8-dihydrofolic acid, 7,8-dihydro-6-methylpterin, 7,8-dihydro-6,7-dime-
thylpterin; at 0, 2.5, 6, 21, and 25 h for 7,8-dihydroneopterin; and at 0, 1.33, 4, 7, 22, and 26 h for 7,8-
dihydroxanthopterin. Arrows indicate the changes observed at different wavelengths. Optical path
length 1 cm. T¼ 408. Initial concentrations: [7,8-dihydroneopterin]0 ¼ 107 mm, [7,8-dihydrobiopterin]0 ¼
177 mm, [7,8-dihydrofolic acid]0 ¼ 66 mm, [7,8-dihydro-6-methylpterin]0 ¼ 120 mm, [7,8-dihydro-6,7-dime-
thylpterin]0 ¼ 104 mm, [7,8-dihydroxanthopterin]0 ¼ 96 mm.
of 7,8-dihydroneopterin and 7,8-dihydrobiopterin (Scheme 1, pathway b; Fig. 3,a). The
concentration of this product, although very low, increased as a function of time
(Fig. 4). This result is also consistent with spectral analysis (Fig. 2), since the spectrum
of 6-formyl-7,8-dihydropterin presents an absorption band in the VIS region (lmax ca.
423 nm) [4]. Finally, no H2O2 was detected as a product of the studied reactions.
It is worth mentioning the biological implications of these results because both
compounds can be produced in an oxidant environment [8][9]. Therefore, the

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Fig. 3. Chromatograms obtained in HPLC analysis: a) 7,8-dihydroneopterin (0 and 21.5 h after admission
of air), b) 7,8-dihydro-6,7-dimethylpterin (0 and 5.5 h after admission of air)

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Scheme 1. Oxidation of 7,8-Dihydroneopterin, 7,8-Dihydrobiopterin, and 7,8-Dihydrofolic Acid by O2 in
Neutral Aqueous Solution
Fig. 4. Oxidation of 7,8-dihydroneopterin and 7,8-dihydrobiopterin in air-equilibrated aqueous solution
(pH 7.2) at 258 and 408. Evolution of the consumption of reactants and generation of products (7,8-
dihydroxanthopterin and 6-formyl-7,8-dihydropterin). Concentrations determined by HPLC analysis.
generation of 7,8-dihydroxanthopterin in vivo via the reactions described in this Section
should be taken into account. It is also interesting to compare the process studied with

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1
the oxidation by O2. The reaction of 7,8-dihydrobiopterin and 7,8-dihydroneopterin
1
with O2 is very fast and leads to the oxidation of the pterin moiety, yielding the
analogous oxidized pterin (biopterin and neopterin, resp.; Fig. 1), non-pterinic
products, where the pterin moiety was oxidized and cleaved, and H2O2 [4].
Oxidation of 7,8-Dihydro-6-methylpterin and 7,8-Dihydro-6,7-dimethylpterin. Much
faster spectral changes were observed when solutions of 7,8-dihydro-6-methylpterin
and 7,8-dihydro-6,7-dimethylpterin were equilibrated with air at 258 and 408 (Fig. 2). In
this case, the NED spectra obtained at 258 and 408 were also similar, suggesting that the
reactions are the same at both temperatures (data not shown). The evolution of the
spectra showed an absorbance increase at l > 340 nm, where oxidized pterins show a
typical band [11]. On the other hand, no absorbance increase was observed at l ca.
310 nm (Fig. 2). This behavior indicates that the products of the reaction of 7,8-
dihydro-6-methylpterin and 7,8-dihydro-6,7-dimethylpterin by O2 are oxidized pterins,
instead of 7,8-dihydroxanthopterin.
This hypothesis was confirmed by HPLCanalysis: oxidation of 7,8-dihydro-6-
methylpterin and 7,8-dihydro-6,7-dimethylpterin leads to 6-methylpterin and 6,7-
dimethylpterin, respectively (Fig. 3,b; Scheme 2, pathway a). The reaction is faster for
7,8-dihydro-6-methylpterin (Fig. 5; vide infra). The Me group(s) seems to protect the
oxidation at C(6) and, at the same time, makes the pterin moiety easier to oxidize. The
concentration profiles show that only a fraction of the dihydropterin consumed is
transformed into the corresponding oxidized derivative (Fig. 5). For 7,8-dihydro-6-
methylpterin, ca. 50% of the reactant is oxidized to 6-methylpterin at both temper-
atures, whereas, for 7,8-dihydro-6,7-dimethylpterin, only ca. 20% of the reactant is
oxidized to 6,7-dimethylpterin. Therefore, this reaction is not the only pathway
responsible for the oxidation of 7,8-dihydro-6-methylpterin and 7,8-dihydro-6,7-
dimethylpterin.
Scheme 2. Oxidation of 7,8-Dihydro-6-methylpterin (R ¼ H) and 7,8-Dihydro-6,7-dimethylpterin (R ¼
Me) by O2 in Neutral Aqueous Solution
Besides oxidized pterins, several additional products were detected by HPLC
analysis (Fig. 3,b), all of them having retention times lower than those corresponding
to both reduced and oxidized pterins. They must, therefore, be very polar substances,
most probably because of incorporation of oxygen into their structures. Spectra of these
additional products were recorded by using the DAD detector of the HPLCequipment.
Such analyses, carried out especially for long times (t > 20 h), where conversion of the
reactants was high, indicated that the pterin moiety was oxidized and cleaved, yielding
a group of non-pterinic products, i.e., the characteristic absorption bands of pterins and

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Fig. 5. Oxidation of 7,8-dihydro-6-methylpterin and 7,8-dihydro-6,7-dimethylpterin in air-equilibrated
aqueous solution (pH 7.2) at 258 and 408. Evolution of the consumption of reactants and production of
the corresponding oxidized pterins (6-methylpterin and 6,7-dimethylpterin). Concentrations determined
by HPLCanalysis.
7,8-dihydropterins in the UV-A disappeared. These non-pterinic substances cannot
originate from the further oxidation of 6-methylpterin and 6,7-dimethylpterin because
these compounds are stable. Therefore, at least two reaction pathways have to be
considered for the reaction of O2 with 7,8-dihydro-6-methylpterin and 7,8-dihydro-6,7-
dimethylpterin: oxidation of the dihydropyrazine ring yielding the aromatic pyrazine
moiety (Scheme 2, pathway a), and oxidation and cleavage of the dihydropterin to yield
non-pterinic substances (Scheme 2, pathway b). In this case, no H2O2 was detected.
Oxidation of 7,8-Dihydrofolic Acid. The spectra of solutions of 7,8-dihydrofolic
acid change very slowly after admission of air (Fig. 2). Small changes in the absorption
bands of 7,8-dihydrofolic acid and a very slight increase in the absorbance in the VIS
region, with a maximum at ca. 423 nm, were observed. HPLCAnalysis revealed that
the main product of the oxidation of 7,8-dihydrofolic acid is 7,8-dihydroxanthopterin
(Scheme 1, pathway a). Therefore, this compound seems to be the major oxidation
product of the autooxidation of many 7,8-dihydropterin derivatives. The analysis of the
corresponding concentration profiles (Fig. 6) showed that the fraction of consumed
reactant converted into 7,8-dihydroxanthopterin is more than 80%. 6-Formyl-7,8-
dihydropterin was also detected in the air-equilibrated solutions of 7,8-dihydrofolic
acid (Scheme 1, pathway b). The concentration of this product, although very low,
increased as a function of time (Fig. 6). This result is also consistent with spectral
analysis (Fig. 2). Finally, no H2O2 was detected as a product of the studied reactions.
Oxidation of 7,8-dihydrofolic acid is important from a biological point of view, since
folic acid and its reduced forms are the most abundant pterin derivatives in living
systems.

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Fig. 6. Oxidation of 7,8-dihydrofolic acid in air-equilibrated aqueous solution (pH 7.2) at 258 and 408.
Evolution of the consumption of reactant and generation of products (7,8-dihydroxanthopterin and 6-
formyl-7,8-dihydropterin). Concentrations determined by HPLC analysis.
7,8-Dihydroxanthopterin. Since 7,8-dihydroxanthopterin is the main product of the
oxidation of the most important dihydropterins in biological systems (7,8-dihydro-
neopterin, 7,8-dihydrobiopterin, and 7,8-dihydrofolic acid), the stability of this
compound in air-equilibrated aqueous solutions was also investigated. The spectral
changes observed in the presence of air were very small at 408 (Fig. 2) and negligible at
258 (data not shown). However, the appearance of a band with maximum at ca. 390 nm
was registered (Fig. 2). This observation together with the analysis of NED spectra
suggests the formation of xanthopterin, the analogously oxidized form of the reactant
(Fig. 1).
This hypothesis was confirmed by HPLCanalysis: oxidation of 7,8-dihydroxan-
thopterin leads to xanthopterin (Scheme 1, pathway c). This reaction is similar to those
observed in the cases of 7,8-dihydro-6-methylpterin and 7,8-dihydro-6,7-dimethylpter-
in; however, as revealed by concentration profiles obtained by means of HPLCanalysis
(Fig. 7), the oxidation is much slower (vide infra). In addition, the conversion of 7,8-
dihydroxanthopterin into the corresponding oxidized form is complete; i.e., the amount
of xanthopterin produced is equal, within the experimental error, to the consumption of
the reactant. In agreement with this result, no additional products were detected by
HPLCanalysis.
6-Formyl-7,8-dihydropterin and Sepiapterin. No modification of the spectra and no
consumption measured by HPLCwere registered for 6-formyl-7,8-dihydropterin and
sepiapterin after 48 h of incubation at 258 and 408 in the presence of O2. The C¼O
group in the substituent seems to make the chemical structure less reactive.
Rate of the Chemical Reaction between O2 and 7,8-Dihydropterin Derivatives. The
apparent pseudo-first-order rate constants of the chemical reaction between O2 and
several 7,8-dihydropterin derivatives (kapp) in H2O at pH 7.0 ꢁ 0.1 were determined,

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Fig. 7. Oxidation of 7,8-dihydroxanthopterin in air-equilibrated aqueous solution (pH 7.2) at 408.
Evolution of the consumption of reactant and generation of product (xanthopterin). Concentrations
determined by HPLCanalysis.
from HPLCanalyses of the disappearance of the reactants during the incubation of the
air-equilibrated aqueous solutions at 258 and 408. Under the experimental conditions
used, the disappearance of the 7,8-dihydropterins followed in all cases first-order
kinetics (Fig. 8). The results obtained (Table) revealed that the reactivity of 7,8-
dihydropterins towards O2 is strongly dependent on the chemical structure of the
substituent at C(6).
Taking into account that the chemical structure of 7,8-dihydrobiopterin and 7,8-
dihydroneopterin differs only in a OH group (Fig. 1), and the oxidation process is
exactly the same (Scheme 1), it seems to be surprising that the kapp values are quite
different, 7,8-dihydroneopterin being much more easily oxidizable by O2 than 7,8-
dihydrobiopterin. Nevertheless, 7,8-dihydroneopterin has been reported to be espe-
cially reactive and very effective in scavenging radicals [12]. Moreover, it has been
observed that 7,8-dihydroneopterin reacts with reactive species, such as 1O2 [4] and the
stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) [13], faster than 7,8-dihydrobiop-
terin. Their different biological functions is maybe related to the very different
reactivity of both compounds, i.e., whereas 7,8-dihydrobiopterin acts as a cofactor in
several metabolic reactions [2][14], 7,8-dihydroneopterin does not participate in
enzymatic reactions and is synthesized mainly in activated macrophages during cell-
mediated immune responses [1][15].
For 7,8-dihydro-6-methylpterin and 7,8-dihydro-6,7-dimethylpterin, the kapp values
are relatively high, which suggests that the higher electronic density on the pyrazine
ring induced by the Me group(s) favors the electrophilic attack of O2. The same
behavior was observed for the reaction between 1O2 and oxidized pterins: 6,7-
dimethylpterin and 6-methylpterin have the highest rate constant within a series of 6-
substituted unconjugated pterin derivatives [5][16]. However, it is noteworthy that the

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Fig. 8. First-order plots of the oxidation of the 7,8-dihydropterin derivatives (DHPT) in air-equilibrated
aqueous solutions (Eqn. 2, Exper. Part). Concentrations of dihydropterins determined by HPLC analysis,
pH 7.0 ꢁ 0.1. a) T¼ 408. b) T¼ 258.
Table. Apparent Pseudo-First-Order Rate Constants of the Chemical Reaction between 7,8-Dihydropter-
ins and O2 (kapp) and Half Life (t1/2) of 7,8-Dihydropterins in Air-Equilibrated Aqueous Solutions.
pH 7.0 ꢁ 0.1.
Compound
T¼ 258
T¼ 408
kapp [10ꢀ2 hꢀ1
]
t1/2 [h]
kapp [10ꢀ2 hꢀ1
]
t1/2 [h]
7,8-Dihydrobiopterin
7,8-Dihydroneopterin
7,8-Dihydro-6-methylpterin
7,8-Dihydro-6,7-dimethylpterin
7,8-Dihydroxanthopterin
7,8-Dihydrofolic acid
0.04 ꢁ 0.02
1.2 ꢁ 0.3
11 ꢁ 1
ca. 1700
ca. 60
0.13 ꢁ 0.02
2.3 ꢁ 0.3
27 ꢁ 2
ca. 530
30
2.6
6.5
2.5 ꢁ 0.2
0.05 ꢁ 0.02
0.8 ꢁ 0.1
< 0.02
28
ca. 1400
ca. 90
> 3000
> 3000
5.4 ꢁ 0.4
0.11 ꢁ 0.04
1.5 ꢁ 0.6
< 0.02
13
ca. 630
46
> 3000
> 3000
6-Formyl-7,8-dihydropterin
Sepiapterin
< 0.02
< 0.02
kapp value is higher for 7,8-dihydro-6-methylpterin than for 7,8-dihydro-6,7-dimethyl-
pterin. Taking into account that the reaction occurs on the C(7)ꢀC(8) bond, steric
effects due to the Me group at C(7) of 7,8-dihydro-6,7-dimethylpterin might hinder the
attack of O2 onto the pyrazine ring.
Although 7,8-dihydroxanthopterin also undergoes the oxidation of the pyrazine
ring to yield the corresponding oxidized derivative xanthopterin, its kapp value is much
smaller than those determined for 7,8-dihydro-6-methylpterin and 7,8-dihydro-6,7-
dimethylpterin. However, this observation is expected taking into account the
hypothesis proposed in the previous paragraph; i.e., in xanthopterin, the electronic
density on the pyrazine ring is low due to the O-atom at C(6). Therefore, the
electrophilic attack of O2 should be much less efficient than in the case of 7,8-dihydro-

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6-methylpterin and 7,8-dihydro-6,7-dimethylpterin. It is also worth mentioning that,
since 7,8-dihydroxanthopterin is the product of the oxidation of those dihydropterins
present in biological systems (7,8-dihydrobiopterin, 7,8-dihydroneopterin, and 7,8-
dihydrofolic acid) and its oxidation is relatively slow, this compound and xanthopterin
might be produced in vivo under oxidative conditions.
Finally, taking into account the kapp values, the half life (t1/2), the time required for
the concentration of a given 7,8-dihydropterin to decay to half of its initial value, was
calculated at 258 and 408 for each studied compound (Eqn. 3 in Exper. Part and Table).
These t1/2 values provide a direct evaluation of the stability of air-equilibrated aqueous
solutions of 7,8-dihydropterins and should be taken into account when experiments
with these compounds are carried out in air-atmosphere.
Conclusions. – The chemical reaction between dissolved O2 and a group of 7,8-
dihydropterins, one of the biologically active forms of pterins, have been investigated in
aqueous solution at 258 and 408, and physiological pH (ca. 7). The series includes 7,8-
dihydro-6-methylpterin, 7,8-dihydro-6,7-dimethylpterin, 7,8-dihydrobiopterin, 7,8-dihy-
droneopterin, 6-formyl-7,8-dihydropterin, sepiapterin, 7,8-dihydroxanthopterin, and
7,8-dihydrofolic acid. Whereas 7,8-dihydropterins are stable under anaerobic condi-
tions, the rates of the reaction with O2 as well as the oxidation products strongly depend
on the chemical structure of the substituents.
The oxidations of 7,8-dihydrobiopterin, 7,8-dihydroneopterin, and 7,8-dihydrofolic
acid yield 7,8-dihydroxanthopterin as the main oxidation product (> 80%) and 6-
formyl-7,8-dihydropterin as a minor product (Scheme 1). The values of the apparent
pseudo-first-order rate constant (kapp) revealed significant differences in the chemical
reactivity toward O2. Considering that the chemical structure of 7,8-dihydrobiopterin
and 7,8-dihydroneopterin differs only in a OH group (Fig. 1), and the oxidation process
is exactly the same, it is noteworthy that kapp(7,8-dihydroneopterin)ꢂkapp(7,8-
dihydrobiopterin). The half-life values (t1/2 ¼ ln 2/kapp; Table) show that, in experiments
performed with 7,8-dihydroneopterin and 7,8-dihydrofolic acid, two important pterin
derivatives in biological systems, the autooxidation of these compound should be
controlled.
7,8-Dihydro-6-methylpterin and 7,8-Dihydro-6,7-dimethylpterin present the higher
kapp values of the series (Table), which suggests that the higher electronic density on the
pyrazine ring induced by the Me group(s) favors the electrophilic attack of O2. At least,
two reaction pathways have to be considered for the oxidation of these compounds: a)
oxidation of the dihydropyrazine ring yielding the aromatic pyrazine moiety, and b)
oxidation and cleavage of the dihydropterin molecule to yield non-pterinic substances
(Scheme 2).
Finally, 7,8-dihydroxanthopterin, 6-formyl-7,8-dihydropterin, and sepiapterin are
the most stable compounds of this series, and their consumption in air-equilibrated
solutions is negligible for several days. Since 7,8-dihydroxanthopterin is the product of
the oxidation of those dihydropterins present in biological systems (7,8-dihydrobiop-
terin, 7,8-dihydroneopterin, and 7,8-dihydrofolic acid), and its oxidation to xanthopter-
in (Scheme 1) is relatively slow, this compound and xanthopterin might be produced in
vivo under oxidative conditions.

Helvetica Chimica Acta – Vol. 91 (2008)
423
The present work was partially supported by Consejo Nacional de Investigaciones Cientificas y
´
´
´
Tecnicas (CONICET-PIP 02470/00 and 6301/05), Agencia de Promocion Científica y Tecnologica
(ANPCyT Grant PICT 06-12610), and Universidad Nacional de La Plata (UNLP). M. L. D. and M. V.
thanks CONICET for graduate research fellowships. We deeply acknowledge Prof. A. M. Braun and
Prof. E. Oliveros for offering their facilities to A. H. T., C. L., and M. L. D. during their stays at the
Engler-Bunte Institute, University of Karlsruhe, Germany, and for their crucial contributions to this
work. A. L. C., A. H. T., and C. L. are research members of CONICET.
Experimental Part
General. 7,8-Dihydropterins and the corresponding oxidized pterins were purchased from Schircks
Laboratories, and used without further purification. The pH of the aq. solns. was adjusted by adding drops
of HCl or NaOH from a micropipette. The concentrations of the acid and base used for this purpose
ranged from 0.1 to 2m. The pH measurements were performed with a pH-meter PHM220 (Radiometer
Copenhagen) and a combined pH electrode pHC2011-8 (Radiometer Analytical).
To check the oxidation of 7,8-dihydropterins during preparation and manipulation of solns., control
solns. were prepared under anaerobic conditions (Ar atmosphere), using a glovebox. The O2
concentration in the H2O employed for preparing the 7,8-dihydropterin solns. within the air-free
compartment was monitored using an O2-selective electrode (Celiox 325) and kept below 0.02 ppm. The
same experimental setup was used to control the stability of the studied compounds in the absence of O2.
Determination of the Apparent First-Order Rate Constant of the Chemical Oxidation of 7,8-
Dihydropterins by O2. In all experiments, the concentration of O2 was higher than that of the studied
dihydropterin. In addition, the reactions took place in open cells with stirring. Taking into account these
experimental conditions, and that the consumption of O2 was in all cases relatively slow, the O2
concentration can be considered constant during the experiments. Therefore, pseudo-first-order
conditions for the consumption of dihydropterins can be assumed, and the rate of consumption of
reactant should be given by the following pseudo-first-order rate equation:
ꢀ (d[DHPT]/dt) ¼ kapp [DHPT]
(1)
where [DHPT] is the concentration of a given 7,8-dihydropterin derivative, and kapp is the pseudo-first-
order rate constant of the chemical reaction between O2 and DHPT. Therefore, integration of Eqn. 1
leads to:
ln([DHPT]/[DHPT]0) ¼ kapp
t
(2)
and first-order kinetics should be observed for the disappearance of DHPT. For determining kapp, air-
equilibrated aq. solns. (pH 7.1 ꢁ 0.1) of the 7,8-dihydropterin derivatives were incubated at 258 and 408,
using a low-temp. bath/circulator R1 (Grant Instruments) or a thermostat bath D8/17V (MGW Lauda).
Consumption of the dihydropterin as a function of time was analyzed by HPLC (vide infra). kapp was
calculated from the slope of the plot ln([DHPT]/[DHPT]0) vs. t.
The half life (t1/2), the time taken for the concentration of a reactant to fall to half of its initial value,
was calculated using Eqn. 3.
t1/2 ¼ ln 2/kapp
(3)
High-Performance Liquid Chromatography. High-performance liquid chromatography (HPLC;
System Gold chromatographic system from Beckman Instruments) was employed for monitoring the
reaction. A Pinnacle-II C18 column (250 ꢃ 4.6 mm, 5 mm; Restek) was used for product separation. Solns.
containing 3 – 5% of MeCN and 95 – 97 of potassium phosphate aq. soln. (20 mm, pH 5.5) was used as
eluent. HPLCRuns were monitored with a UV/VIS photodiode array detector ( SPD-M20A, Shimadzu).
It should be noted that, in the case of 7,8-dihydrobiopterin and 7,8-dihydroxanthopterin, the peak of
the reactant could not be well separated from that of the corresponding product (7,8-dihydroxanthopter-
in and xanthopterin, resp.). Therefore, calibration curves were established, and integrations of the peaks

424
Helvetica Chimica Acta – Vol. 91 (2008)
at different wavelengths were performed. Assuming that the peak considered is only due to the reactant
and one known product, the concentration of both compounds can be calculated by resolving sets of
equations as follows:
Areal1 ¼ flR1 CR þ flP1 CP
Areal2 ¼ flR2 CR þ flP2 CP
(4)
(5)
where Areal1 and Areal2 are the values resulting from integration of the chromatograms at analysis
wavelengths l1 and l2, and CR and CP are the concentrations of the reactant and the product,
respectively, f Rl1, f lP1, f lR2, f lP2 are the factors obtained from the calibration curves for the reactant and the
product at l1 and l2. Although only two equations are required for calculating CR and CP, more equations
were used in order to check the results obtained.
UV/VIS Analysis. Electronic spectra were recorded on a Varian Cary-3 spectrophotometer.
Measurements were conducted in quartz cells of 1-cm optical-path length. The absorption spectra of the
solns. were recorded at regular intervals of time after admission of air, and the signals were averaged and
smoothed with the Varian software. Experimental-difference (ED) spectra were obtained by subtracting
the spectrum at time t ¼ 0 from the subsequent spectra recorded at different times t. Each ED spectrum
was normalized relative to the maximum absorbance value of the absorbance difference, yielding the
normalized experimental-difference (NED) spectrum.
Determination of the Concentration of H2O2. For the determination of H2O2, a Cholesterol Kit
(Wiener Laboratorios S. A. I. C.) was used. H2O2 was quantified after reaction with 4-aminophenazone
and phenol [17][18]. The main features of the method have already been described in [19].
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Received August 14, 2007