Formation of oxygen radicals in solutions of different 7,8- dihydropterins: Quantitative structure-activity relationships

Article abstract of DOI:10.1002/(SICI)1522-2675(20000510)83:5<954::AID-HLCA954>3.0.CO;2-9

Under certain conditions, 7,8-dihydroneopterin in aqueous solution promotes hydroxyl-radical formation. Thus, we investigated the stimulation of hydroxyl-radical formation by ten different 7,8-dihydropterins (=2-amino-7,8- dihydropteridin-4(1H)-one), i.e., 6-(1'-hydroxy) derivatives 1 and 2, methyl derivatives 3-7, and 6-(1'-oxo) derivatives 8-10. All but the 6-(1'-oxo) derivatives produced hydroxyl radicals, as measured by the amount of salicylic acid hydroxylation products. This amount was dependent on the stability of the dihydropterin used. In the presence of chelated iron ions, hydroxylation was increased in every case; even 6-(1'-oxo) derivatives showed a low hydroxylation of salicylic acid. The degree of increase, however, strongly depended on the side chain of the dihydropterin. The 7,8- dihydroneopterin (2) was investigated in more detail. Iron ions influenced both, the stability of 2 and hydroxyl-radical formation. While iron ions determined the kinetics of the reaction, the amount of 2 was responsible for the amount of hydroxyl radicals formed. Our data establish that promotion of hydroxyl-radical formation by 7,8-dihydropterins depends on the oxidizability of the dihydropterins and on their iron-chelating properties.

Full text of DOI:10.1002/(SICI)1522-2675(20000510)83:5<954::AID-HLCA954>3.0.CO;2-9

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Formation of Oxygen Radicals in Solutions of Different
7
,8-Dihydropterins: Quantitative Structure-Activity Relationships
a
b
a
by Karl Oettl )*, Wolfgang Pfleiderer ), and Gilbert Reibnegger )
a) Institute for Medical Chemistry and Pregl Laboratory, Karl-Franzens-University Graz, Harrachgasse 21/II,
A-8010 Graz
b) Faculty for Chemistry, University Konstanz, P.O. Box 5560, D-78434 Konstanz
Under certain conditions, 7,8-dihydroneopterin in aqueous solution promotes hydroxyl-radical formation.
Thus, we investigated the stimulation of hydroxyl-radical formation by ten different 7,8-dihydropterins (ˆ2-
amino-7,8-dihydropteridin-4(1H)-one), i.e., 6-(1'-hydroxy) derivatives 1 and 2, methyl derivatives 3 ± 7, and
6
-(1'-oxo) derivatives 8 ± 10. All but the 6-(1'-oxo) derivatives produced hydroxyl radicals, as measured by the
amount of salicylic acid hydroxylation products. This amount was dependent on the stability of the
dihydropterin used. In the presence of chelated iron ions, hydroxylation was increased in every case; even
6
-(1'-oxo) derivatives showed a low hydroxylation of salicylic acid. The degree of increase, however, strongly
depended on the side chain of the dihydropterin. The 7,8-dihydroneopterin (2) was investigated in more detail.
Iron ions influenced both, the stability of 2 and hydroxyl-radical formation. While iron ions determined the
kinetics of the reaction, the amount of 2 was responsible for the amount of hydroxyl radicals formed. Our data
establish that promotion of hydroxyl-radical formation by 7,8-dihydropterins depends on the oxidizability of the
dihydropterins and on their iron-chelating properties.
Introduction. ± During the oxidative burst of stimulated macrophages, reactive
O-species, as well as dihydroneopterin (ˆ2-amino-7,8-dihydro-[(1S,2R)-1,2,3-trihy-
droxypropyl]pteridin-4(1H)-one; 2) and neopterin, are secreted [1][2]. Neopterin is
used as an indicator for the activation of the cellular immune system [3]. Nevertheless,
the biological roles of neopterin and dihydroneopterin are unclear. Since about ten
years ago, the interaction of pterins with radical-mediated reactions has been under
investigation [4]. Reduced pterins were shown to act as scavengers of free radicals
generated by different chemical or biological systems: e.g., chemiluminescence of
luminol induced by macrophages or chemical systems [5 ± 8], toxicity of chloramine T
or hydrogen peroxide against bacteria [9][10], low density lipoprotein oxidation [11],
1
nitration of tyrosine by peroxynitrite [12], and electron-spin resonance (ESR) )
experiments with spin traps [13]. In all these experiments, pterins were exposed to
preformed radicals, and the action of pterins on those radicals was investigated. In
contrast, recently it was found that 7,8-dihydroneopterin (2) in high concentrations may
enhance radical-mediated reactions like luminol chemiluminescence and apoptosis
[
14 ± 16]. In addition, it was shown that, in solutions of 2, hydroxyl radicals are formed
in the absence of any radical source besides dissolved O [17][18]. Hydroxyl-radical
2
formation was dependent on the addition of chelated iron ions.
1)
Abbreviations: DHBA, 2,5-dihydroxybenzoic acid; EDTA (ˆ H
4
edta) ethylenediamine tetraacetic acid;
ESR, electron spin resonance; PBS, phosphate-buffered saline.

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The aim of this study was to compare the efficiency of different dihydropterins to
produce hydroxyl radicals, and to establish a correlation with their structures.
Furthermore, we intended to define in more detail the influence of iron ions on the
interactions of dihydropterins and free radicals.
Results and Discussion. ± In this study we investigated ten different 7,8-
dihydropterins (see 1 ± 10 in Fig. 1) for their stimulatory effect upon hydroxyl-radical
formation, as measured by the hydroxylation of salicylic acid. The compounds tested
can be divided into three groups: i) 6-(1'-hydroxy) derivatives 1 and 2, ii) methyl
Fig. 1. Structures of the studied dihydropterins: 7,8-dihydro-6-(hydroxymethyl)pterin (1), 7,8-dihydroneopterin
(
2), 7,8-dihydro-6-methylpterin (3), 7,8-dihydro-6,7-dimethylpterin (4), 7,8-dihydro-6,7,8-trimethylpterin (5), 7,8-
dihydro-6,7,7-trimethylpterin (6), 7,8-dihydro-3,6,7,7-tetramethylpterin (7), sepiapterin (8), deoxysepiapterin (9),
and 6-formyl-7,8-dihydropterin (10)

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derivatives 3 ± 7 [19][20], and iii) 6-(1'-oxo) derivatives 8 ± 10. Time-dependent
formation of salicylic acid hydroxylation products was followed up to ca. 18 h and
occurred in all pterin solutions, except in those of the 1'-oxo derivatives 8 ± 10 (Fig. 2,
right-hand) which are rather stable compounds. The source of hydroxyl radicals is
assumed to be superoxide, which could be generated from dissolved O by the
2
autooxidation of traces of metal ions or during dihydropterin oxidation and converted
to hydroxyl radicals via the Fenton reaction catalyzed by trace iron. The formation of
superoxide has been described to occur during oxidation of pterins, but only for fully
reduced tetrahydropterins [21 ± 23]. Moreover, we recently reported that, in solutions
of 7,8-dihydroneopterin, hydroxyl-radical formation can be diminished by addition of
desferrioxamine, suggesting that trace metal ions are the source of superoxide rather
than dihydropterins [17].
Parallel to the measurement of hydroxylation products, the decay of the
dihydropterins in solution was followed (Fig. 2, left-hand). The extent of hydroxylation
after completion of the assay was correlated with the decay of the corresponding
dihydropterin. A pattern of three different types of behavior according to the three
classes of dihydropterins investigated can easily be seen in the diagram shown in
Fig. 3,a. Methyl derivatives 3 ± 7 show hydroxylation yields in accordance to the extent
of the decay of the pterin, which varied considerably. The 6-(1'-oxo) derivatives 8 ± 10
show essentially no hydroxylation, and 6-(1'-hydroxy) derivatives 1 and 2 give low
yields of hydroxylation compared to their low stability. The decay of dihydropterins vs.
time (Fig. 2) can be described by an exponential curve, and the decay constants of
dihydropterins can be calculated (Table). These constants correlate with the amount of
hydroxylation and show essentially the same behavior as demonstrated in Fig. 3,a.
Table. Decay Constants of Different 7,8-Dihydropterins a)
À1
Decay constant [h
]
absence of Fe3 /EDTA
‡
presence of Fe /EDTA
3‡
1
2
8
9
7,8-Dihydro-6-(hydroxmethyl)pterin
7,8-Dihydroneopterin
Sepiapterin
0.022 Æ 0.007
0.052 Æ 0.011
0.005 Æ 0.002
0.007 Æ 0.001
0.016 Æ 0.002
0.108 Æ 0.027
0.029 Æ 0.003
0.041 Æ 0.019
0.016 Æ 0.010
0.048 Æ 0.017
0.035 Æ 0.015
0.037 Æ 0.008
0.004 Æ 0.002
0.007
0.015 Æ 0.006
0.103 Æ 0.031
0.041 Æ 0.005
0.054 Æ 0.025
0.022 Æ 0.011
0.054 Æ 0.011
Deoxysepiapterin
1
0 6-Formylpterin
3
4
5
6
7
7,8-Dihydro-6-methylpterin
7,8-Dihydro-6,7-dimethylpterin
7,8-Dihydro-6,7,8-trimethylpterin
7,8-Dihydro-6,7,7-trimethylpterin
7,8-Dihydro-3,6,7,7-tetramethylpterin
a) Dihydropterins (500 mm) were incubated in the presence of 500 mm salicylic acid and in the presence or
absence of 20/40 mm ferric ions/EDTA. After 0, 5.8, 11.6, and 17.5h, aliquots were analyzed by HPLC and the
À1
dihydropterin content measured. From the decrease of dihydropterin vs. time, the decay constants [h ] of the
dihydropterins were calculated (mean values Æ s.d. of 3 runs, except for 2 (5 runs) and 9 (1 run) in the presence
3
‡
of Fe /EDTA).
Addition of chelated ferric ions to the assay mixtures influences both the stability of
the compounds tested and hydroxylation (Fig. 2). While the effect on the stability
ranges from a moderate decrease to a 1.5 fold increase of the decay with no specific

Helvetica Chimica Acta ± Vol. 83 (2000)
957
Fig. 2. Decay (left-hand) of a) 7,8-dihydroneopterin (2), b) sepiapterin (8), and c) 7,8-dihydro-6,7,8-trimethyl-
pterin (5) and hydroxylation of salicylic acid in the presence (ꢀ) or absence (.) of ferric ions/EDTA, and
concomitant formation of 2,5-dihydroxybenzoic acid (DHBA) (right-hand). Pterins (500 mm) were incubated
together with 500 mm salicylic acid in the presence or absence of 20/40 mm ferric ions/EDTA. After the time
1
periods indicated, aliquots were analyzed by HPLC, and dihydropterins and DHBA ) were quantified (mean
values Æ s.d. of 3 runs). Note the different scale in Fig. 2,b (right-hand).

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Fig. 3. Correlation of DHBA formation with dihydropterin decay in the case of 1'-oxo derivatives 8 ± 10 (&); 1'-
hydroxy derivatives 1 and 2 (.), and methyl derivatives 3 ± 7 (ꢀ): a) absence of ferric ions/EDTA and b) presence
of ferric ions/EDTA. Dihydropterins (500 mm) were incubated in the presence of 500 mm salicylic acid without
3
‡
or with 10/20mm Fe /EDTA for 17.5 h, and then the amounts of produced DHBA and dihydropterin
determined (mean values Æ s.d. of 3 runs).

Helvetica Chimica Acta ± Vol. 83 (2000)
959
1
trend, hydroxylation is increased considerably by addition of EDTA-chelated ) ferric
ions, depending on the group of compound. Therefore, changes in the stability are
3‡
not responsible for an altered extent of hydroxylation on addition of 10 mm Fe .
In solutions of the methylated dihydropterins 3 ± 7, only a 1.4 to 3.1 fold increase
3‡
of hydroxylation is observed on addition of Fe /EDTA, while 6-(1'-hydroxy) and
-(1'-oxo) derivatives 1 and 2 and 8 ± 10, respectively, give at least a tenfold increase.
6
The relative increase is highest with the 6-(1'-oxo) derivatives (up to 65-fold), but only
because of the low basal value. In total amounts, 6-(1'-hydroxy) derivatives 1 and 2
show the highest response to the addition of chelated iron. The pattern in the
correlation of hydroxylation with the decay of the pterin changes on addition of
chelated ferric ions. There are no longer separable groups of different pterin classes, but
a single group including all tested compounds showing a good correlation of
2
hydroxylation with pterin decay with r ˆ 0.626 (Fig. 3,b).
From the consecutive oxidation of the analytes on 8 electrodes in series, the
hydrodynamic voltammogram of every 7,8-dihydropterin can be derived (Fig. 4). The
two hydroxy derivatives 1 and 2 as well as the three oxo derivatives 8 ± 10 show similar
voltammograms and nearly identical potentials for half-maximal oxidation (Fig. 4,a).
On the other hand, methyl derivatives 3 ± 7 are found to have oxidation potentials over
a broad range (Fig. 4,b), indicating a more pronounced impact on the oxidation
potential by substitution at the pterin-ring system as compared to the impact of
different side chains at position 6. Correlation of the oxidation potentials with the
amount of hydroxylation gives a pattern comparable to Fig. 3,a: in the case of methyl
derivatives 3 ± 7, hydroxylation is higher for compounds that are easily oxidized
(
Fig. 5). One exception is 3,6,7,7-tetramethyl-7,8-dihydropterin (7), which is the only
compound methylated at the pyrimidine moiety. Both other dihydropterin groups show
a clustering where, again, lower potentials are associated with higher hydroxylation. In
the experiments with added ferric ion, no more clustering or correlation could be
found.
Within groups of chemically similar dihydropterin derivatives, hydroxyl-radical
formation depends on the decay of the dihydropterin, which is in agreement with its
oxidation potential. Comparing different dihydropterin groups reveals the same trend:
those that are more easily oxidized give higher hydroxylation yields. In the presence of
chelated ferric ion, the extent of hydroxylation is well-correlated with dihydropterin
decay, including compounds of all groups. The most dramatic effect is observed for
solutions of 7,8-dihydroneopterin (2) and 6-(hydroxymethyl)-7,8-dihydropterin (1),
both with a 1'-hydroxy group. While 1 gives only ca. 10% hydroxylation compared to its
6-methyl analog 4 without added iron, in the presence of iron both 1 and 4 show about
the same hydroxylation amount. These data suggest that the effect might be due
to an enhanced binding of iron ions by dihydropterins containing a 6-(1'-hydroxy)
function.
In the case of dihydroneopterin 2, we elucidated the effects of EDTA and ferric ions
further. We found that either EDTA or ferric ions alone increase the stability of 2
considerably (Fig. 6,a). On the other hand, addition of ferric ions to a solution of 2
containing EDTA decreases the stability of 2 compared to EDTA alone. A completely
different pattern is observed for the effect on hydroxylation. While EDTA enhances
1
hydroxylation, ferric ions even decrease the amount of DHBA ) formed; the highest

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Fig. 4. Hydrodynamic voltammograms of 7,8-dihydropterins: a) 7,8-dihydro-6-(hydroxymethyl)-pterin (1; ꢀ)
7
,8-dihydroneopterin (2; .), sepiapterin, (8; 4), deoxysepiapterin (9; &) and 6-formyl-7,8-dihydropterin (10; ~)
b) 7,8-dihydro-6-methylpterin (3; .), 7,8-dihydro-6,7-dimethylpterin (4; ꢀ), 7,8-dihydro-6,7,7-trimethylpterin
(
6; 4), 7,8-dihydro-6,7,8-trimethylpterin (5; ~), and 7,8-dihydro-3,6,7,7-tetramethylpterin (7; &). After HPLC,
the dihydropterins were detected with a coulometric detector containing 8 working electrodes in series, set at
00, 210, 320, 430, 540, 650, 760, and 870 mV, respectively. From the maximum current (peak height) of the first
electrodes, the cumulative peak height was calculated for every pterin investigated.
1
7

Helvetica Chimica Acta ± Vol. 83 (2000)
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Fig. 5. Correlation of DHBA formation with dihydropterin oxidation potential: 1'-oxo derivatives 8 ± 10 (&), 1'-
hydroxy derivatives 1 and 2 (.), and methyl derivatives 3 ± 7 (ꢀ). The amount of DHBA formed after 17.5 h
incubation of 7,8-dihydropterins with salicylic acid without added ferric iron (same values as in Fig. 3,a) was
correlated with the potential necessary for half-maximal oxidation from the hydrodynamic voltammogram.
hydroxylation yields are achieved in the assay containing both EDTA and ferric ions
(
Fig. 6,b). The enhancement of hydroxylation by EDTA is attributable to an effective
conversion of superoxide to hydroxyl radicals by iron/EDTA. Despite the enhanced
hydroxylation, 2 is much more stable in EDTA-containing solution, indicating that 2
does not serve as a scavenger of hydroxyl radicals.
In solutions containing 7,8-dihydroneopterin (2) and EDTA with increasing
amounts of ferric ions, both the decay rate of 2 and the formation rate of DHBA are
increased with increasing iron concentrations. As a consequence, DHBA formation is
2
correlated with the rate constant of dihydroneopterin decay linearily with r ˆ 0.97 and
a slope of 1.1 (Fig. 7). The maximum hydroxylation rate, which can also be calculated
from an exponential fit of the amount of DHBA formed vs. time, is only slightly
influenced by the increase in iron concentration. We have previously shown that the
amount of hydroxylation products formed depends on the amount of 7,8-dihydro-
neopterin (2) added [17]. When the dihydropterin is consumed, hydroxylation ceases
and starts again after addition of new dihydropterin (Fig. 8).
Different dihydropterins of similar structure lead to hydroxylation of salicylic acid
in an amount according to the decay of the dihydropterin. The 7,8-dihydroneopterin
(2), under conditions of differing stability, also induces hydroxylation dependent on its
decay. The faster decay of dihydropterins is not attributable to the formation of more
hydroxyl radicals, as shown for 2, which is stabilized by EDTA despite a much higher
hydroxylation rate. Hydroxylation is dependent not only on the oxidizability of the
dihydropterin used, which is determined primarily by the dihydropterin ring system,
but also by different side chains at position 6, especially in the presence of iron ions.
This may be due to different iron-chelating properties influenced by different
substituents at C(1').

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Fig. 6. Effect of iron and EDTA on the stability of 7,8-dihydroneopterin (2) and hydroxylation: a) decay of 2 and
b) formation of DHBA. Up to 55 h, 2 (50 mm) was incubated with 500 mm salicylic acid in the absence (A) or
presence of 200 mm EDTA (B), 50 mm ferric ions (C) or both (D). After 7 periods of time, the amount of
DHBA and 2 in the mixture was measured. From an exponential fit of the decay of 2 and the formation of
DHBA, the decay rate constants (a)) and formation constants (b)) were calculated (mean values Æ s.d. of 2
runs).

Helvetica Chimica Acta ± Vol. 83 (2000)
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Fig. 7. Correlation of the DHBA-formation rate constant (., left-hand axis) and maximum hydroxylation (&,
right-hand axis) in the case of 7,8-dihydroneopterin (2) decay. Up to 55 h 2 (50 mm) was incubated with 500 mm
salicylic acid in the presence of 200 mm EDTA and 10, 20, 50, and 100 mm ferric ions. From an exponential fit of
the decay of 2 and the formation of DHBA, the decay constants, formation constants (.) and maximum
hydroxylation (&) were calculated.
Fig. 8. The 7,8-dihydroneopterin (2) as electron donor: amounts of 2 and DHBA vs. time. Dihydroneopterin 2
(100 mm) was incubated with 200 mm ferric ions and 400 mm EDTA. After the time periods indicated, aliquots
were analyzed and 2 (.) and 2,5-DHBA (ꢀ) quantified. After 18 h, the amount of 2 was brought to the initial
concentration and the incubation prolonged for a further 16 h.

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This work was financially supported by the Austrian National Science Foundation (project P12366CHE).
Experimental Part
General. 7,8-Dihydro-6-methylpterin (3), 7,8-dihydro-6,7,7-trimethylpterin (6), 7,8-dihydro-6,7,8-trimeth-
ylpterin (5), and 7,8-dihydro-3,6,7,7-tetramethylpterin (7) were synthesized as described elsewhere [19][20]. All
other pterins were obtained from Dr. Schircks Laboratory (Jona, Switzerland). The 2,3-dihydroxybenzoic acid
and 2,5-dihydroxybenzoic acid (DHBA) were purchased from Sigma (Vienna, Austria). Phosphate buffered
saline (PBS), consisting of 1.15 g/l of Na
.2 g/l of KCl, 8 g/l of NaCl, pH 7.4, was obtained from PAA (Linz, Austria). All other reagents were obtained
from Merck (Darmstadt, Germany).
2 4 2 4 2 2 2
HPO , 0.2 g/l of KH PO , 0.1 g/l of CaCl , 0.1 g/l of MgCl ´ 6 H O,
0
Analyses of Dihydropterins and Hydroxylation Products of Salicylic Acid by HPLC. Hydroxylation assays
were carried out in brown 2-ml Chromacol autosampler vials with incubation times up to 24 h. After the
indicated time intervals, 20-ml aliquots of the mixture were analyzed by HPLC. The system consisted of an ERC
vacuum degasser (Kawaguchi City, Japan), a Flux-Rheos-4000 quaternary gradient pump (Basel, Switzerland),
two pulse dampeners in series, a Spark-Triathlon autosampler (Emmen, Netherlands) with an inert sample
needle and a 20-ml sample loop, a Spark Marathon column oven and an ESA-Coularray multielectrode
coulometric detector (Chelmsford, MA, USA), equipped with eight graphite working electrodes. The potentials
of the electrodes were set at 100, 210, 320, 430, 540, 650, 760, and 870 mV against a Pd reference electrode.
Separation was performed on a Bischoff column (150 Â 3 mm) filled with Waters Spherisorb ODS2 (3 mm
particles). Mobile phase was delivered at a flow rate of 0.5 ml/min by a gradient elution of 100% solvent A for
5
5
min, then raising solvent B within 10 min to 30%. After 5 min, solvent A was raised again to 100% within
min. Before the next run, the column was equilibrated for 10 min with 100% solvent A. Solvent A: 30 mm
NaOAc, 30 mm sodium citrate (pH 4.6); solvent B: MeOH. Column and electrodes were kept at 358.
With this HPLC system, the different dihydropterins were separated from their degradation products as
well as 2,3- and 2,5-dihydroxybenzoic acid (DHBA) and salicylic acid. DHBA was used as a quantitative probe
for hydroxyl-radical formation in all experiments. For the quantitation of each compound, the chromatogram
obtained by the dominant channel was used.
The incubation mixtures consisted of 500 mm dihydroneopterin and 500 mm salicylic acid in PBS (pH 7.4),
unless otherwise stated. After addition of 500 mm salicylic acid, the pH of the mixture decreased to 7.2.
Incubations were carried out at r.t. Stock solns. of 7,8-dihydroneopterin (2) and iron salts were prepared
immediately before starting the assay.
Solutions of ferric ions were prepared by dissolving FeSO
4
in PBS, which autooxidized immediately [21].
For the preparation of the solns., H O deionized by a Millipore MilliQ 185 (Vienna, Austria) was used.
2
Computations. The kinetics of the formation of hydroxylated products and the decay of dihydropterins were
Àkt
Àkt
evaluated by fitting simple exponential functions of the form c(t) ˆ cmax ´ (1 À e ) and c(t) ˆ c
0
´ e , respectively,
to the temporal profiles (c(t) ˆ time-dependent concentration of hydroxylation products or dihydropterins,
c
max ˆ finally reached concentration of hydroxylation products, c ˆ initial dihydropterin concentration, k ˆ rate
0
constant). Fitting of the parameters cmax and k was performed with the program SCIENTIST (MicroMath, Inc.,
Salt Lake City, Utah, USA). Notably, the standard estimation errors of the parameter estimates are provided by
this program.
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Received February 4, 2000