Reaction between 7,8-dihydropterins and hydrogen peroxide under physiological conditions

Article abstract of DOI:10.1016/j.tet.2008.07.005

In vitiligo, a common skin disorder that produces white patches of depigmentation, 7,8-dihydropterins accumulate in the presence of high concentration of H₂O₂. In this work, we present a study of the reaction between 7,8-dihydropterins and H₂O₂. The rate of the reaction, as well as the products formed, strongly depend on the chemical structure of the substituents. Electron-donor groups as substituents are the most reactive derivatives and undergo oxidation of the pterin moiety. The corresponding bimolecular rate constants at 37 °C in neutral aqueous solutions are reported. The biological implications of the results obtained are also discussed.

Full text of DOI:10.1016/j.tet.2008.07.005

Tetrahedron 64 (2008) 8692–8699
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Reaction between 7,8-dihydropterins and hydrogen peroxide under
physiological conditions
´
M. Laura Dantola, Tobias M. Schuler, M. Paula Denofrio, Mariana Vignoni, Alberto L. Capparelli,
*
´
Carolina Lorente, Andres H. Thomas
´
´
´
Instituto de Investigaciones Fisicoquımicas Teoricas y Aplicadas (INIFTA), Departamento de Quımica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata,
CONICET, Diagonal 113 y 64, La Plata 1900, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
In vitiligo, a common skin disorder that produces white patches of depigmentation, 7,8-dihydropterins
accumulate in the presence of high concentration of H2O2. In this work, we present a study of the re-
action between 7,8-dihydropterins and H2O2. The rate of the reaction, as well as the products formed,
strongly depend on the chemical structure of the substituents. Electron-donor groups as substituents are
the most reactive derivatives and undergo oxidation of the pterin moiety. The corresponding bimolecular
rate constants at 37 ꢀC in neutral aqueous solutions are reported. The biological implications of the re-
sults obtained are also discussed.
Received 3 March 2008
Received in revised form 30 June 2008
Accepted 1 July 2008
Available online 4 July 2008
Keywords:
Reactivity
Kinetics
Ó 2008 Elsevier Ltd. All rights reserved.
Pterins
Hydrogen peroxide
1. Introduction
dihydropterins are relatively stable.7–9 The oxidation of these
compounds is of biological interest because they, under several
Pterins, heterocyclic compounds widespread in biological sys-
tems, are derived from 2-aminopteridin-4-(3H)-one or pterin. The
most common pterin derivatives are 6-substituted compounds.
Pterins can exist in different oxidation states and be divided into
two classes according to this property: (a) oxidized or aromatic
pterins containing the pyrazino[2,3-d]pyrimidine ring structure
and (b) reduced pterins (Fig. 1). Within the latter group, 7,8-dihy-
dropterins and 5,6,7,8-tetrahydropterins (denoted throughout as
dihydropterins and tetrahydropterins, respectively) are the most
important derivatives due to their biological activity, e.g., dihy-
droneopterin is secreted during the oxidative burst of stimulated
macrophages,1 dihydrobiopterin and tetrahydrobiopterin are in-
volved in the metabolism of aminoacids,2 and reduced derivatives
of folic acid participate in methionine and nucleotide biosynthesis.3
The chemistry of dihydropterins has been studied in detail.4
Depending on their oxidation state, pterins have totally different
reactivities toward oxidizing agents, e.g., whereas dihydropterins
are very efficient singlet oxygen (1O2) quenchers,5 the reactivity of
oxidized pterins toward 1O2 is relatively low.6 In air-equilibrated
aqueous solutions, tetrahydropterins are rapidly oxidized by dis-
solved molecular oxygen in its ground state (O2), whereas
pathological situations, are generated and accumulated in oxidative
environments. For instance, in the skin of patients suffering viti-
ligo,10 a common disorder that produces white patches of de-
pigmentation, several reduced and oxidized pterins accumulate in
the affected areas.11,12 Hydrogen peroxide (H2O2) participates in the
pathogenesis of this disease and is also accumulated in the depig-
mented epidermis.12,13
The oxidation of dihydrobiopterin by H2O2 to yield biopterin has
been suggested,14 but, despite the potential implications of this
reaction, it has not been studied in detail. In contrast, the reaction
between dihydroneopterin and H2O2 was investigated in more
depth and dihydroxantopterin was reported as the main oxidation
product.15 However, to the best of our knowledge, the kinetics of
these reactions and the oxidation of other dihydropterin derivatives
with H2O2 have not been investigated at all.
In the context of our investigation on reactions between dihy-
dropterins and different oxidizing agents, in this article we describe
the oxidation of a group of 6-substituted dihydropterins by H2O2 in
aqueous solutions, under physiological conditions (pH¼7.1ꢁ0.1;
T¼37 ꢀC). We investigated derivatives with substituents of different
molecular weights and with different functional groups: 6-methy-
ldihydropterin,
6,7-dimethyldihydropterin,
dihydrobiopterin,
dihydroneopterin, 6-formyldihydropterin, dihydroxanthopterin,
and dihydrofolic acid (Fig. 1). The reaction products, in most cases,
were identified. Kinetics of the reactions was analyzed and the
* Corresponding author. Tel.: þ54 221 4257291; fax: þ54 221 4254642.
E-mail address: athomas@inifta.unlp.edu.ar (A.H. Thomas).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.07.005

´
M. Laura Dantola et al. / Tetrahedron 64 (2008) 8692–8699
8693
Dihydroderivative
Oxidized pterin
O
O
4
N
5
R
6
R
N
5
R
6
4
HN3
2
HN
3
7
1
8
CH2
1
8
7
H2N
N
N
H
2
H2N
N
N
neopterin (NPT)
biopterin (BPT)
-(CHOH)2-CH2OH
dihydroneopterin (DHNPT)
dihydrobiopterin (DHBPT)
-(CHOH)2-CH3
-CH3
6-methylpterin (MPT)
6-methyldihydropterin (MDHPT)
6,7-dimethyldihydropterin (DMDHPT)
dihydrofolic acid (DHFA)
6,7-dimethylpterin (DMPT)
-CH3
folic acid (FA)
(*)
6-formylpterin (FPT)
-CHO
6-formyldihydropterin (FDHPT)
O
O
H
OH
H
N
O
N
O
O
O
C
HN
H2N
HN
H2N
N
CH
(*)
2 H
N
H
CH2
CH
N
N
H
N
N
O
HO
dihydroxanthopterin (DHXPT)
PABA-Glu
xanthopterin (XPT)
Figure 1. Molecular structures of the dihydropterins investigated at physiological pH (neutral forms); structures of corresponding oxidized pterins are also represented. DMDHPT
and DMPT have an additional methyl group at position 7 of the pterin moiety.
corresponding bimolecular rate constants were determined. We
discuss the effects of the structural features of the substituents and
the biological implications of the results obtained.
3a, and 4a). For all compounds, the fraction of consumed reactant
converted into DHXPT was more than 90%. Therefore, cleavage of
the substituent and oxidation of C6 constitute the main pathway of
the reaction of DHNPT, DHBPT, and FDHPT with H2O2 (Scheme 1).
These results are in agreement with previous studies that reported
DHXPT as a typical oxidation product of dihydropterin derivatives
bearing a –CHOH– group linked to C6.4,16
2. Results and discussion
The chemical reactions between hydrogen peroxide (H2O2) and
dihydropterins in air-equilibrated aqueous solution (pH¼7.0–7.2)
were investigated by UV–vis spectrophotometry and HPLC analysis
at 37 ꢀC. The chemical changes after mixing the reactants were
followed as a function of time. Dihydropterins slowly react with
dissolved O2 and the corresponding half life values (t1/2) in air-
equilibrated aqueous solution have been determined.9 Taking into
account these values, the rate of the oxidation of dihydropterins by
O2 was calculated for the initial conditions of each experiment. In
almost all experiments, the rate of the consumption of reactants by
O2 was negligible in comparison with the rate of the reaction with
H2O2. In a very few experiments performed at low H2O2 concen-
tration, the oxidation of dihydropterin derivatives by O2 was sig-
nificant. In these cases, this reaction was taken into account and the
corresponding corrections were made in kinetic analysis (vide
infra).
It is interesting to compare these results to those obtained in
previous studies on oxidation of dihydropterins. In particular,
oxidation by H2O2 leads to the same product (DHXPT) as that
found in the oxidation by dissolved O2,9 although this reaction is
much slower (vide infra). In contrast, the reaction of the studied
dihydropterins with 1O2 is very fast and leads to the oxidation of
the pterin moiety, yielding the analogous oxidized pterin (bio-
pterin (BPT), neopterin (NPT), 6-formylpterin (FPT), Fig. 1), and
non-pterinic products, where the pterin moiety was oxidized
and cleaved.5
It is also worth mentioning the biological implications of these
results, especially those from the reaction of DHBPT. Both DHBPT
and H2O2 accumulate in the skin of patients affected by vitiligo.11,12
Therefore, the oxidation of DHBPT and the consequent generation
of DHXPT in vivo via the reaction described in this section should be
taken into account.
2.1. Oxidation of dihydrobiopterin, dihydroneopterin,
and 6-formyldihydropterin
2.2. Oxidation of dihydrofolic acid
Significant spectral changes were observed in aqueous solutions
of dihydrobiopterin (DHBPT), dihydroneopterin (DHNPT), and 6-
formyldihydropterin (FDHPT) after adding H2O2 aqueous solution
at 37 ꢀC (Figs. 2a, 3a, and 4a). In all cases, blue shifts were observed
for the corresponding low-energy bands. Spectral analysis sug-
gested the presence of dihydroxanthopterin (DHXPT) as the main
product of the three oxidation reactions: the normalized-experi-
mental-difference (NED) spectra were similar to the corresponding
normalized-reference-difference (NRD) spectra, obtained from
standard solutions of the reactants and DHXPT (see Section 4).
The formation of DHXPT was confirmed by means of HPLC
analysis. The concentrations of reactants (DHBPT, DHNPT and
FDHPT) and DHXPT were determined as a function of time (Figs. 2a,
The reaction between dihydrofolic acid (DHFA) and H2O2 (Fig. 5)
leads to the formation of several products. HPLC analysis revealed
that the main product is DHXPT (w60%) (Scheme 2, pathway a). In
addition, folic acid (FA) (w40%) and a very small amount of FDHPT
(<1%) were also detected (Scheme 2, pathways b and c). Oxidation
of DHFA is important from a biological point of view since folic acid
and its reduced forms are the most abundant pterin derivatives in
living systems.
In this case, there seems to be at least two parallel pathways: the
oxidation of the pyrazine ring to yield the corresponding oxidized
pterin derivative (FA) and the break of the substituent by oxidation
at the C-6 or C-9 position to yield DHXPT or FDHPT, respectively.
The former pathway was already reported for the oxidation of

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M. Laura Dantola et al. / Tetrahedron 64 (2008) 8692–8699
(a)
(a)
1.4
1.2
1.4
60
50
40
30
20
10
0
50
40
30
20
10
0
1.2
DHNPT
DHXPT
SUM
DHBPT
DHXPT
1.0
0.8
0.6
0.4
0.2
0.0
SUM
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5
6
0
1
2
3
4
t / h
5
6
7
t / h
200
250
300
350
nm
400
450
500
200
250
300
350
nm
400
450
500
0.5
0.0
(b)
0.2
0.0
(b)
[H2O2]= 2.1 mM
[H2O2]= 1.6 mM
[H2O2]= 2.7 mM
[H2O2]= 3.8 mM
[H2O2]= 5.1 mM
[H2O2]= 4.3 mM
[H2O2]= 8.1 mM
[H2O2]= 15.3 mM
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-0.5
-1.0
-1.5
-2.0
-2.5
0
2
4
t / h
6
8
0.0
0.5
1.0
1.5
2.0
t / h
2.5
3.0
3.5
4.0
Figure 2. Reaction between DHBPT and H2O2 (T¼37 ꢀC, pH¼7.1ꢁ0.1). (a) Evolution of
the absorption spectra as a function of time; arrows indicate the changes observed at
different wavelengths. Optical path length¼1 cm. Inset: evolution of the consumption
of reactant and generation of product (DHXPT). Initial concentrations: [DHBPT]0¼
Figure 3. Reaction between DHNPT and H2O2 (T¼37 ꢀC, pH¼7.1ꢁ0.1). (a) Evolution of
the absorption spectra as a function of time; arrows indicate the changes observed at
different wavelengths. Optical path length¼1 cm. Inset: evolution of the consumption
of reactant and generation of product (DHXPT). Initial concentrations: [DHNPT]0¼
60
m
M, [H2O2]0¼2.1 mM. (b) First-order plots of the oxidation of DHBPT by H2O2 at
51
m
M, [H2O2]0¼1.6 mM. (b) First-order plots of the oxidation of DHNPT by H2O2 at
different concentrations (Eq. 4, Section 4). Concentrations determined by HPLC analysis.
different concentrations (Eq. 4, Section 4). Concentrations determined by HPLC analysis.
corresponding oxidized form is very small (<5%). In addition to XPT,
three other products were detected by HPLC analysis, all of them
having short retention times. Taking into account the experimental
conditions of our chromatographic analysis (see Section 4), this fact
suggests that such additional products are very polar substances.
The corresponding absorption spectra were recorded using the
DAD detector of the HPLC equipment. Such analyses, carried out
especially for long times (t>24 hs) where conversion of the re-
actant was high, suggested that the pterin moiety was oxidized and
cleaved, yielding a group of non-pterinic products, i.e., the char-
acteristic absorption bands of oxidized pterins and dihydropterins
in the UV-A region were lost.
DHFA by 1O2,5 whereas the latter one was reported for the oxida-
tion of DHFA by dissolved O2.9
2.3. Oxidation of dihydroxanthopterin
Since DHXPT is the main product of the oxidation of the most
biologically important dihydropterins (DHNPT, DHBPT, and DHFA)
by H2O2, the reaction between this reactive oxygen species and
DHXPT in aqueous solution was also investigated. The oxidation is
very slow compared to those presented in the previous paragraphs;
e.g., after mixing DHXPT with H2O2 a slow decay of the typical
absorption band of DHXPT with a maximum at 310 nm and a very
slight increase in the absorption in the region 360–420 nm were
observed (Fig. 6a).
Xanthopterin (XPT) (Fig. 1), in very low concentration, was
detected by HPLC analysis. The corresponding concentration pro-
files (Fig. 6a) showed that, as expected, the consumption of DHXPT
is relatively slow (vide infra) and its conversion into the
2.4. Oxidation of 6-methyldihydropterin and 6,7-
dimethyldihydropterin
Relatively fast spectral changes were observed when aqueous
solutions of 6-methyldihydropterin (MDHPT) and 6,7-

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M. Laura Dantola et al. / Tetrahedron 64 (2008) 8692–8699
8695
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(a)
(a)
1.4
DHFA
DHXPT
FDHPT
FA
40
30
20
10
0
30
20
10
0
1.2
SUM
1.0
FDHPT
DHXPT
SUM
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
0
1
2
3
4
t / h
5
6
7
t / h
200
250
300
350
400
450
500
200
250
300
350
nm
400
450
500
nm
0.5
0.0
(b)
(b)
[H2O2]= 2.3 mM
[H2O2]= 4.2 mM
[H2O2]= 7.0 mM
0
-1
-2
-3
-4
[H2O2]= 14.4 mM
-0.5
-1.0
-1.5
-2.0
-2.5
[H2O2]= 0.4 mM
[H2O2]= 2.1 mM
[H2O2]= 3.3 mM
[H2O2]= 4.2 mM
[H2O2]= 5.6 mM
0
2
4
t / h
6
8
0
1
2
3
4
5
t / h
Figure 4. Reaction between FDHPT and H2O2 (T¼37 ꢀC, pH¼7.1ꢁ0.1). (a) Evolution of
the absorption spectra as a function of time; arrows indicate the changes observed at
different wavelengths. Optical path length¼1 cm. Inset: evolution of the consumption
of reactant and generation of product (DHXPT). Initial concentrations:
Figure 5. Reaction between DHFA and H2O2 (T¼37 ꢀC, pH¼7.1ꢁ0.1). (a) Evolution of
the absorption spectra as a function of time; arrows indicate the changes observed at
different wavelengths. Optical path length¼1 cm. Inset: evolution of the consumption
of reactant and generation of products (DHXPT, FA and FDHPT). Initial concentrations:
[FDHPT]0¼42
m
M, [H2O2]0¼2.3 mM. (b) First-order plots of the oxidation of FDHPT by
[DHFA]0¼40 M, [H2O2]0¼2.1 mM. (b) First-order plots of the oxidation of DHFA by
m
H2O2 at different concentrations (Eq. 4, Section 4). DHXPT concentration determined
by HPLC, FDHPT concentration determined by HPLC and spectrophotometric analyses.
H2O2 at different concentrations (Eq. 4, Section 4). DHXPT, FA, and FDHPT concentra-
tions determined by HPLC and spectrophotometric analyses.
dimethyldihydropterin (DMDHPT) were mixed with aqueous H2O2
at 37 ꢀC (Figs. 7a and 8a). The evolution of the spectra showed an
O
H
N
O
absorbance increase at
a typical band.17 On the other hand, no absorbance increase was
observed at w310 nm. This behavior suggests that DHXPT is not
l>340 nm, where oxidized pterins show
HN
H2N
CH2
N
N
H
l
7,8-dihydroxanthopterin
the main product of the oxidation of MDHPT and DMDHPT by H2O2.
Moreover, HPLC analysis revealed that DHXPT is not present in
the reaction mixture and that 6-methylpterin (MPT) and 6,7-
dimethylpterin (DMPT) (Fig.1) are products of the reactions of H2O2
a)
O
O
N
N
N
R
N
R
HN
H2N
b)
c)
HN
H2N
+ H2O2
CH2
N
N
H
folic acid
O
N
O
N
O
7,8-dihydrofolic acid
H
N
O
CH
O
N
R
HO
H2O2
HN
H2N
HN
H2N
O
HN
H2N
O
C
CH2
CH2
N
N
H
N
H
R:
CH2
N
H
CH2
N
H
N
N
H
O
7,8-dihydroxanthopterin
HO
6-formyl-7,8-dihydropterin
Scheme 1. Oxidations of DHNPT, DHBPT, and FDHPT by H2O2 in neutral aqueous so-
lution at 37 ꢀC.
Scheme 2. Reaction between DHFA and H2O2 in neutral aqueous solution at 37 ꢀC.

´
8696
M. Laura Dantola et al. / Tetrahedron 64 (2008) 8692–8699
(a)
(a)
50
40
30
20
10
0
DHXPT
MDHPT
MPT
XPT
40
30
20
10
0
SUM
2.0
1.2
1.0
0.8
0.6
1.5
1.0
0.5
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2
t / h
0
1
2
3
4
t / h
5
6
7
8
0.4
0.2
0.0
200
250
300
350
nm
400
450
500
200
250
300
350
nm
400
450
500
0.2
0.0
(b)
0.5
0.0
(b)
[H2O2]= 62 mM
[H2O2]= 154 mM
[H2O2]= 250 mM
[H2O2]= 0.54 mM
[H2O2]= 0.75 mM
[H2O2]= 1.0 mM
[H2O2]= 1.7 mM
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-0.5
-1.0
-1.5
-2.0
-2.5
-3.0
-3.5
0
1
2
3
4
5
6
7
0.0
0.4
0.8
1.2
t / h
1.6
2.0
2.4
t / h
Figure 6. Reaction between DHXPT and H2O2 (T¼37 ꢀC, pH¼7.1ꢁ0.1). (a) Evolution of
the absorption spectra as a function of time; arrows indicate the changes observed at
different wavelengths. Optical path length¼1 cm. Inset: evolution of the consumption
Figure 7. Reaction between MDHPT and H2O2 (T¼37 ꢀC, pH¼7.1ꢁ0.1). (a) Evolution of
the absorption spectra as a function of time; arrows indicate the changes observed at
different wavelengths. Optical path length¼1 cm. Inset: evolution of the consumption
of reactant and generation of product (MPT). Initial concentrations:
of reactant and generation of product (XPT). Initial concentrations: [DHXPT]0¼50
mM,
[H2O2]0¼62 mM. (b) First-order plots of the oxidation of DHXPT by H2O2 at different
concentrations (Eq. 4, Section 4). DHXPT and XPT concentrations determined by HPLC
analyses.
[MDHPT]0¼54 M, [H2O2]0¼1.0 mM. (b) First-order plots of the oxidation of MDHPT
m
by H2O2 at different concentrations (Eq. 4, Section 4). Concentrations determined by
HPLC analysis.
Therefore, at least two reaction pathways have to be considered
for the reaction of H2O2 with MDHPT and DMDHPT: oxidation of
the dihydropyrazine ring yielding the aromatic pyrazine moiety
(Scheme 3, pathway a) and oxidation and cleavage of the dihy-
dropterin to yield non-pterinic substances (Scheme 3, pathway b).
This pattern was already observed for oxidation by O2,9 although
the rate of this latter reaction is much slower.
with MDHPT and DMDHPT, respectively (Figs. 7a and 8a; Scheme 3,
pathway a). However, the concentration profiles show that only
a fraction of the dihydropterin consumed is transformed into the
corresponding oxidized derivative (Figs. 7a and 8a). For MDHPT,
about 10% of the reactant is oxidized to MPT, whereas for DMDHPT
only about 5% of the reactant is oxidized to DMPT.
Besides oxidized pterins, several additional products were
detected by HPLC analysis, 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 be-
cause of incorporation of oxygen into their structures. Spectral
analysis of these additional products formed after long reaction
times (t>5 h) suggested that the pterin moiety was oxidized and
cleaved, yielding a group of non-pterinic products. These non-
pterinic substances cannot originate from the further oxidation of
6-methylpterin and 6,7-dimethylpterin because these compounds
are stable.
2.5. Rate constants of the chemical reaction between H2O2
and dihydropterin derivatives
The apparent pseudo-first-order rate constants of the chemical
reaction between H2O2 and several dihydropterin derivatives (kapp
)
in H2O at pH 7.0ꢁ0.1 were determined. Each solution containing
a given dihydropterin derivative and H2O2 was incubated at 37 ꢀC
and monitored as function of time by means of HPLC and spectral
analyses. Under the experimental conditions used, disappearance

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M. Laura Dantola et al. / Tetrahedron 64 (2008) 8692–8699
8697
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(a)
12
DDHPT
DHXPT
40
30
20
10
0
0.8
0.6
0.4
0.2
0.0
DPT
SUM
10
8
MDHPT
DDHPT
DHBPT
DHNPT
FDHPT
DHFA
0
50 100 150 200 250
[H2O2] / mM
6
0.0
0.4
0.8
1.2
1.6
t / h
4
2
0
200
250
300
350
nm
400
450
500
0
2
4
6
8
[H2O2] / mM
10
12
14
16
(b)
Figure 9. Chemical reaction between dihydropterins and H2O2 in neutral aqueous
solution at 37 ꢀC: plot of the apparent pseudo-first-order rate constants (kapp) as
a function of H2O2 concentration.
[H2O2]= 1.1 mM
[H2O2]= 1.9 mM
[H2O2]= 2.9 mM
0
-1
-2
-3
-4
revealed that the reactivity of dihydropterins toward H2O2 is
strongly dependent on the chemical structure of their 6-
substituent.
Comparison of k values indicates that the most reactive com-
pounds within the studied series are MDHPT and DMDHPT. This
result suggests that the higher electronic density on the pyrazine
ring induced by the methyl group(s) favors the attack of H2O2. It is
noteworthy that k is higher for MDHPT ((0.66ꢁ0.03) Mꢂ1 sꢂ1) than
for DMDHPT ((0.32ꢁ0.02) Mꢂ1 sꢂ1), probably due to steric effects of
the methyl group in C7. However, the same behavior was observed
for the reaction between O2 and dihydropterins.9
It is interesting to assess the reactivity of DHNPT, DHBPT, and
DHFA, the most relevant derivatives from a biological point of view,
toward H2O2. The k values reported in the present work show that
the reaction of the mentioned derivatives with H2O2 will be sig-
nificant if the H2O2 concentration is high. This is the case of vitiligo
(vide supra), where millimolar concentrations of H2O2 has been
reported.12 Therefore, the oxidation of DHBPT, that accumulates in
the skin of patients suffering vitiligo, should be taken into
account.11
0.0
0.5
1.0
t / h
1.5
2.0
Figure 8. Reaction between DMDHPT and H2O2 (T¼37 ꢀC, pH¼7.1ꢁ0.1). (a) Evolution
of the absorption spectra as a function of time; arrows indicate the changes observed
at different wavelengths. Optical path length¼1 cm. Inset: evolution of the con-
sumption of reactant and generation of product (DMPT). Initial concentrations:
[DMDHPT]0¼40 M, [H2O2]0¼1.9 mM. (b) First-order plots of the oxidation of DMDHPT
m
by H2O2 at different concentrations (Eq. 4, Section 4). Concentrations determined by
HPLC analysis.
These results are in agreement with previous works reporting
that these compounds, especially DHNPT, due to their reactivities
toward reactive oxygen species, can be considered as antioxi-
dants18,19 and, consequently, might have a protective roll against
oxidative stress. However, there are other studies that claim that
when 6-formylpterin (FPT) is reduced by, for example, NADH the
resulting 6-formyldihydropterin reacts with O2 to yield H2O2.20 In
of the dihydropterins followed in all cases first-order kinetics (Figs.
2b, 3b, 4b, 5b, 6b, 7b, and 8b).
For each compound, series of experiments were performed at
different H2O2 concentrations. In all cases, linear relationships be-
tween kapp and the H2O2 concentration were found (Fig. 9), thus
revealing a first kinetic order for H2O2. As a result, the bimolecular
rate constants (k) were determined from the slopes of the corre-
sponding plots kapp versus [H2O2]. The results obtained (Table 1)
Table 1
Rate constants of the chemical reaction between dihydropterins and H2O2 (k) in air-
equilibrated aqueous solutions; pH¼7.0ꢁ0.1; T¼37 ꢀ
C
O
N
O
N
Compound
k/Mꢂ1 sꢂ1
N
CH3
+ H2O2
N
N
CH3
a)
b)
HN
H2N
HN
H2N
Dihydrobiopterin
Dihydroneopterin
6-Methyldihydropterin
6,7-Dimethyldihydropterin
Dihydroxanthopterin
Dihydrofolic acid
(2.7ꢁ0.2)ꢃ10ꢂ2
(3.7ꢁ0.4)ꢃ10ꢂ2
0.66ꢁ0.03
CH
R
N
H
R
0.32ꢁ0.02
(2.5ꢁ0.2)ꢃ10ꢂ4
(6.1ꢁ0.7)ꢃ10ꢂ2
(1.5ꢁ0.1)ꢃ10ꢂ2
Non-pterinic products
Scheme 3. Reaction between MDHPT (R: H) and DMDHPT (R: –CH3) and H2O2 in
6-Formyldihydropterin
neutral aqueous solution at 37 ꢀC.

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M. Laura Dantola et al. / Tetrahedron 64 (2008) 8692–8699
8698
this case the dihydropterin derivative, that was not identified yet,
would be acting as H2O2 generator and not as antioxidant. Such
reduced pterin certainly is not 6-formyl-7,8-dihydropterin, which
is rather stable in air-equilibrated solutions.9 Alternatively, it
could be a 5,8-dihydropterin derivative. It has been recently
shown that these compounds, that can also be formed photo-
chemically from oxidized pterins, react with O2 very fast to yield
H2O2.21,22
Although DHXPT also undergoes oxidation, its k value is much
smaller than those determined for the other studied compounds.
This fact is logical taking into account that in DHXPT the elec-
tronic density on the pyrazine ring is low due to the oxygen in
position 6. Therefore, the electrophilic attack should be much less
efficient than in the case of the rest of the series. It is also worth
mentioning that since DHXPT is the product of the oxidation of
those dihydropterins present in biological systems (DHBPT,
DHNPT, and DHFA) and its oxidation is relatively slow, this
compound might be produced and accumulated in vivo under
oxidative conditions.
and cleavage of the dihydropterin molecule to yield non-pterinic
substances (Scheme 3).
4. Experimental
4.1. General
Dihydropterins and the corresponding oxidized pterins were
purchased from Schircks Laboratories, and used without further
purification. Deionized water was further purified in a Milli Q Re-
agent Water System apparatus. The specific resistance measured
was w18 MU cm. H2O2 (30%) was from Merck. The pH of the
aqueous solutions 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 2 M. The pH measurements
were performed using a pH meter PHM220 (Radiometer Copen-
hagen) with a combined pH electrode pHC2011-8 (Radiometer
Analytical).
4.2. UV–Vis analysis
3. Conclusions
Electronic spectra were recorded on a Cary-3 (Varian) spec-
trophotometer or on a diode array spectrophotometer 8452A
(Hewlett Packard). Measurements were made in quartz cells of
1 cm optical path length. The absorption spectra of the solutions
were recorded at regular intervals of time after mixing the re-
actants. Experimental-difference (ED) spectra were obtained by
subtracting the spectrum at time t¼0 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. Reference-difference (RD) spectra and normal-
ized-reference-difference (NRD) spectra were obtained from
aqueous solutions of commercial standards. The comparison be-
tween NED and NRD spectra allows the characterization of the
major products. The analysis based on these difference spectra is
described elsewhere.23,24
The chemical reaction between H2O2 and a group of 7,8-dihy-
dropterins, one of the biologically active forms of pterins, has been
investigated in aqueous solution at 37 ꢀC, and physiological pH (w7).
The series includes: dihydrobiopterin (DHBPT), dihydroneopterin
(DHNPT), 6-formyldihydropterin (FDHPT), dihydrofolic acid (DHFA),
dihydroxanthopterin (DHXPT), 6-methyldihydropterin (MDHPT),
and 6,7-dimethyldihydropterin (DMDHPT) (Fig. 1). The rates of the
reaction with H2O2, as well as the products formed, strongly depend
on the chemical structure of the substituents.
The cleavage of the substituent and oxidation of C6 to yield
DHXPT (>90%) is the main pathway of the reaction of DHNPT,
DHBPT, and FDHPT with H2O2 (Scheme 1). These results show that
biopterin (BPT), an oxidized pterin detected in the skin of patients
suffering vitiligo, is not the product of the reaction between DHBPT
and H2O2, as suggested in previous works.14 The corresponding
bimolecular rate constants (k) (Table 1) vary in the narrow range
(1.5–3.7ꢃ10ꢂ2 Mꢂ1 sꢂ1) and reveal that the consumption of DHNPT,
DHBPT, and FDHPT is significant in the presence of H2O2, at con-
centration of the same order of magnitude as that measured in
tissues affected by vitiligo.12
The reaction between DHFA and H2O2 also leads to the forma-
tion of DHXPT as the main product (w60%) (Scheme 2, pathway a).
However, FA (w40%) and a very small amount of FDHPT (<1%) were
also detected (Scheme 2, pathways b and c, respectively). Oxidation
of DHFA is important from a biological point of view since folic acid
and its reduced forms are the most abundant pterin derivatives in
living systems. The corresponding k value is of the same order of
magnitude than those of the compounds described in the previous
paragraph.
DHXPT also reacts with H2O2, the reaction is much slower,
the corresponding k value being about 2 orders of magnitude
smaller than those reported for the derivatives analyzed in the
previous paragraphs (Table 1). Since DHXPT is the product of the
oxidation of those dihydropterins present in biological systems
(DHBPT, DHNPT, and DHFA) and its reaction with H2O2 is rela-
tively slow, this compound might be produced and accumulated
in vivo.
4.3. High-performance liquid chromatography (HPLC)
A high-performance liquid chromatograph Prominence from
Shimadzu (solvent delivery module LC-20AT, on-line degasser
DGU-20A5 and UV/VIS photodiode array detector SPD-M20A)
was employed for monitoring the reactions. A Pinnacle-II C18
column (250ꢃ4.6 mm,
5 mm; Restek) was used for product
separation. Solutions containing 3–5% of acetronitrile and 95–97
of potassium phosphate aqueous solution (20 mM, pH 5.5) were
used as eluent.
It should be noted that in the case of DHBPT, DHFA, and DHXPT
the peak of the reactant could not be well separated from that of the
corresponding product (DHXPT, FA, and XPT, respectively).
Therefore, calibration curves were established and integrations of
the peaks 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:
Areal ¼ flR1CR þ flP1CP
(1)
(2)
1
MDHPT and DMDHPT present the higher k values of the series
(Table 1), which suggest that the higher electronic density on the
pyrazine ring induced by the methyl group(s) favors the attack of
H2O2. At least two reaction pathways have to be considered for the
oxidation of these compounds: (a) oxidation of the dihydropyr-
azine ring yielding the aromatic pyrazine moiety, and (b) oxidation
Areal ¼ flR2CR þ flP2CP
2
where areal1 and areal2 are the values resulting from integration of
the chromatograms at analysis wavelengths
l1 and
l
2, CR and CP are
P
the concentrations of the reactant and the product, flR1, fl , fl2R, and
1

´
M. Laura Dantola et al. / Tetrahedron 64 (2008) 8692–8699
8699
P
fl are the factors obtained from the calibration curves for the
Acknowledgements
The present work was partially supported by Consejo Nacional
2
reactant and the product at l1 and l2. Although only two equations
are required for calculating CR and CP, more equations were used in
´
de Investigaciones Cientificas y Tecnicas (CONICET-Grant PIP 6301/
order to check the results obtained.
´
´
´
05), Agencia de Promocion Cientıfica y Tecnologica (ANPCyT Grants
PICT 06-12610 and PICT 33919), and Universidad Nacional de La
Plata (UNLP). M.L.D. and M.V. thank CONICET, and M.P.D. thanks
ANPCyT for graduate research fellowships. C.L., A.L.C. and A.H.T. are
research members of CONICET.
4.4. Determination of the rate constant of the chemical
reaction between H2O2 and dihydropterins
In all experiments the concentration of H2O2 was much higher
than that of the studied dihydropterin ([H2O2]>10[DHPT]). There-
fore, pseudo-first-order conditions for the consumption of
dihydropterins can be assumed and the rate of disappearance of
reactant should be given by the following pseudo-first-order rate
equation:
References and notes
1. Huber, C.; Fuchs, D.; Hausen, A.; Margreiter, R.; Reibnegger, G.; Spielberger, M.;
Wachter, H. J. Immunol. 1983, 130, 1047.
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3. Lucock, M. Mol. Genet. Metab. 2000, 71, 121.
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Wachter, H., Blair, J. A., Eds.; Walter de Gruyter: Berlin, New York, NY, 1987; Vol.
5, pp 3–21.
5. Da´ntola, M. L.; Thomas, A. H.; Braun, A. M.; Oliveros, E.; Lorente, C. J. Phys. Chem.
A 2007, 111, 4280.
6. Cabrerizo, F. M.; Da´ntola, M. L.; Petroselli, G.; Capparelli, A. L.; Thomas, A. H.;
Braun, A. M.; Lorente, C.; Oliveros, E. Photochem. Photobiol. 2007, 83, 526.
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Chim. Acta 2008, 91, 411.
ꢂðd½DHPTꢄ=dtÞ ¼ kapp½DHPTꢄ
(3)
Where [DHPT] is the concentration of a given dihydropterin de-
rivative and kapp is the pseudo-first-order rate constant of the
chemical reaction between H2O2 and DHPT. Therefore, integration
of Eq. 3 leads to:
À
Á
ln ½DHPTꢄ=½DHPTꢄ0 ¼ kapp
t
(4)
and first-order kinetics should be observed for the disappearance of
DHPT.
10. Kovacs, S. O. J. Am. Acad. Dermatol. 1998, 38, 647.
11. Schallreuter, K. U.; Wood, J. M.; Pittelkow, M. R.; Gutlich, M.; Lemke, K. R.; Rodl,
W.; Swanson, N. N.; Hitzemann, K.; Ziegler, I. Science 1994, 263, 1444.
12. Schallreuter, K. U.; Moore, J.; Wood, J. M.; Beazley, W. D.; Peters, E. M. J.; Marles,
L. K.; Behrens-Williams, S. C.; Dummer, R.; Blau, N.; Tho¨ny, B. J. J. Invest. Der-
matol. 2001, 116, 167.
13. Schallreuter, K. U.; Moore, J.; Wood, J. M.; Beazley, W. D.; Gazeg, D. C.; Tobin,
D. J.; Marshall, H. S.; Panske, A.; Panzig, E.; Hibberts, N. A. J. Invest. Dermatol.
Symp. Proc. 1999, 4, 91.
For determining kapp
, air-equilibrated aqueous solutions
(pH¼7.1ꢁ0.1) of the dihydropterin derivatives were incubated at
37 ꢀC, using a low temperature bath/circulator R1 (Grant In-
struments) or a thermostat bath D8/17V (MGW Lauda). Consump-
tion of the dihydropterin as a function of time was analyzed by
HPLC (vide supra). kapp was calculated from the slope of the plot
ln([DHPT]/[DHPT]0) versus t.
14. Moore, J.; Wood, J. M.; Schallreuter, K. U. J. Raman Spectrosc. 2002, 33, 610.
15. Murr, C.; Baier-Bitterlich, G.; Fuchs, D.; Werner, E. R.; Esterbauer, H.; Pfleiderer,
W.; Wachter, H. Free Radical Biol. Med. 1996, 21, 449.
If the reaction is of first order for H2O2, the rate of disappearance
of a given dihydropterin derivative is given by Eq. 5,
16. Armarego, W. L. F.; Randles, D.; Taguchi, H. Eur. J. Biochem. 1983, 135, 393.
17. Cabrerizo, F. M.; Petroselli, G.; Lorente, C.; Capparelli, A. L.; Thomas, A. H.;
Braun, A. M.; Oliveros, E. Photochem. Photobiol. 2005, 81, 1234.
18. Gieseg, S. P.; Maghzal, G.; Glubb, D. Free Radical Res. 2001, 34, 123.
19. Gieseg, S. P.; Whybrow, J.; Glubb, D.; Rait, C. Free Radical Res. 2001, 35, 311.
20. Arai, T.; Endo, N.; Yamashita, K.; Sasada, M.; Mori, H.; Ishii, H.; Hirota, K.;
Makino, K.; Fukuda, K. Free Radical Biol. Med. 2001, 30, 248.
21. Petroselli, G.; Bartsch, J. M.; Thomas, A. H. Pteridines 2006, 17, 82.
22. Vignoni, M.; Cabrerizo, F.M.; Lorente, C.; Thomas, A. H., in preparation.
ꢂðd½DHPTꢄ=dtÞ ¼ k½DHPTꢄ½H2O2ꢄ
where k is the bimolecular rate constant of the chemical reaction
between H2O2 and DHPT. Therefore, k can be determined from the
slope of the plot kapp versus [H2O2] (Eq. 6).
(5)
´
23. Thomas, A. H.; Suarez, G.; Cabrerizo, F. M.; Martino, R.; Capparelli, A. L.
J. Photochem. Photobiol., A: Chem. 2000, 135, 147.
24. Sua´rez, G.; Cabrerizo, F. M.; Lorente, C.; Thomas, A. H.; Capparelli, A. L.
J. Photochem. Photobiol., A: Chem. 2000, 132, 53.
kapp ¼ k½H2O2ꢄ
(6)