Transformation of 6-tetrahydrobiopterin in aqueous solutions under UV-irradiation

Article abstract of DOI:10.1016/j.jphotochem.2017.07.029

Melanogenesis disturbance leads to several pathologies, including vitiligo disease. Ultraviolet (UV) narrowband phototherapy (308 or 311 nm) is used in treating vitiligo; however, the mechanism of phototherapy is not yet understood. Vitiligo is accompanied by three-fivefold increased de-novo synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (H₄Bip), its excess and its further oxidation can be considered as significant factors in the pathogenesis of vitiligo. (H₄Bip), as the phenylalanine 4-hydroxylase coenzyme, catalyzes the oxidation of phenylalanine to tyrosine (a melanin precursor). In this context, photo-transformation of H₄Bip in aqueous buffer solutions has been studied. HPLC–MS/MS has demonstrated that pterin products of H₄Bip autoxidation (7,8-dihydropterin (H₂Ptr), dihydroxanthopterin and pterin) predominate over biopterin products (7,8-dihydrobiopterin (H₂Bip) and biopterin). We have shown that UV irradiation accelerates the autoxidation while the products of oxidative degradation of H₄Bip act as photosensitizers. The distinctive feature of photooxidation of H₄Bip from autoxidation is the formation of dihydropterin (Н₂Ptr)₂ and dihydrobiopterin (Н₂Bip)₂ dimers. By means of HPLC–MS/MS it was found that formation of dihydropterin dimers is the predominant process. The signal of molecular ion of the dimer (Н₂Ptr)₂ (m/z = 331) was almost a thousand times higher than the signal of (Н₂Bip)₂ (m/z = 479). The key point of the dimerization is photoexcitation (at 310–320 nm) of the intermolecular complex (qH₂Ptr-Н₂Ptr) generated in dark. As a result of the photoreaction azacyclobutane dimers have been formed. In the case of alternation of dark and light intervals H₄Bip converted into dimers with 96 % yield. The data obtained are discussed in the context of UV-B narrowband vitiligo phototherapy.

Full text of DOI:10.1016/j.jphotochem.2017.07.029

Accepted Manuscript
Title: Transformation of 6-tetrahydrobiopterin in aqueous
solutions under UV-irradiation
Authors: Taisiya A. Telegina, Tamara A. Lyudnikova, Andrey
A. Buglak, Yulia L. Vechtomova, Mikhail V. Biryukov,
Vladimir V. Demin, Mikhail S. Kritsky
PII:
S1010-6030(17)30444-6
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2017.07.029
JPC 10755
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
7-4-2017
21-7-2017
24-7-2017
Please cite this article as: Taisiya A.Telegina, Tamara A.Lyudnikova, Andrey
A.Buglak, Yulia L.Vechtomova, Mikhail V.Biryukov, Vladimir V.Demin,
Mikhail S.Kritsky, Transformation of 6-tetrahydrobiopterin in aqueous
solutions under UV-irradiation, Journal of Photochemistry and Photobiology A:
Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.07.029
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1
Transformation of 6-tetrahydrobiopterin in aqueous solutions
under UV-irradiation
Taisiya A. Teleginaa,b*, Tamara A. Lyudnikovaa, Andrey A. Buglakс, Yulia L.
Vechtomovaa,b, Mikhail V. Biryukovd, Vladimir V. Demine and Mikhail S. Kritskya
a Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences. 33, bld. 2
Leninsky prospect, Moscow 119071, Russia
b Kazan Federal University, 18, Kremlevskaja str., Kazan 420008, Russia
c St. Petersburg State University, 7/9 Universitetskaya nab., St. Petersburg 199034, Russia
d Faculty of Biology, Lomonosov Moscow State University, Moscow 119992, Russia
e Faculty of Soil Science, Lomonosov Moscow State University, Moscow 119992, Russia
* Corresponding author. E-mail address: telegina@inbi.ras.ru (Taisiya Telegina)
Graphical Abstract
Abstract
Melanogenesis disturbance leads to several pathologies, including vitiligo disease.
Ultraviolet (UV) narrowband phototherapy (308 or 311 nm) is used in treating vitiligo; however,
the mechanism of phototherapy is not yet understood. Vitiligo is accompanied by three-fivefold
increased de-novo synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (H4Bip), its excess and
its further oxidation can be considered as significant factors in the pathogenesis of vitiligo.
(H4Bip), as the phenylalanine 4-hydroxylase coenzyme, catalyzes the oxidation of phenylalanine
to tyrosine (a melanin precursor). In this context, photo-transformation of H4Bip in aqueous buffer
solutions has been studied. HPLC-MS/MS has demonstrated that pterin products of H4Bip
autoxidation (7,8-dihydropterin (H2Ptr), dihydroxanthopterin and pterin) predominate over
biopterin products (7,8-dihydrobiopterin (H2Bip) and biopterin). We have shown that UV

2
irradiation accelerates the autoxidation while the products of oxidative degradation of H4Bip act
as photosensitizers. The distinctive feature of photooxidation of H4Bip from autoxidation is the
formation of dihydropterin (Н2Ptr)2 and dihydrobiopterin (Н2Bip)2 dimers. By means of HPLC-
MS/MS it was found that formation of dihydropterin dimers is the predominant process. The signal
of molecular ion of the dimer (Н2Ptr)2 (m/z=331) was almost a thousand times higher than the
signal of (Н2Bip)2 (m/z=479). The key point of the dimerization is photoexcitation (at 310-320nm)
of the intermolecular complex (qH2Ptr-Н2Ptr) generated in dark. As a result of the photoreaction
azacyclobutane dimers have been formed. In the case of alternation of dark and light intervals
H4Bip converted into dimers with 96 % yield. The data obtained are discussed in the context of
UV-B narrowband vitiligo phototherapy.
Keywords:
Tetrahydrobiopterin,
Ultraviolet irradiation,
Autoxidation of tetrahydrobiopterin,
Photooxidation of tetrahydrobiopterin,
Azacyclobutane dimer,
Vitiligo phototherapy

3
1. Introduction
(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (H4Bip) is a coenzyme for the
series of enzymes: NO synthases, alkylglycerol monooxygenases and aromatic
amino acid hydroxylases. H4Bip is the coenzyme of phenylalanine hydroxylase (EC
1.14.16.1) catalyzing the oxidation of phenylalanine to tyrosine by molecular
oxygen in the process of melanogenesis [1]. Melanogenesis disturbance leads is a
characteristic feature of certain dermatological pathologies, including vitiligo
disease, which results in the emergence of depigmented skin patches. The patients
with vitiligo appeared to have three-fivefold increased de-novo synthesis of H4Bip;
its excess and its further oxidation can be considered as important factors in the
pathogenesis of vitiligo [2,3,4,5,6]. Pterin and its reduced forms absorb light in the
ultraviolet spectral region. Recently, tetrahydrocyanopterin has been described as a
chromophore within a photoreceptor of several cyanobacteria [7,8]. The role that
pterins perform as photoreceptors and as photosensitizers of metabolic reactions is
not well understood. It is obvious, however, that these issues are vitally important
for photobiology [9] and photomedicine. Apart from our recent work [10], the
photochemistry of H4Bip coenzyme has never been studied. We have demonstrated
that biopterin, which has been formed in H4Bip solution as a result of the dark
oxidation, acts as the photosensitizer that accelerates the oxidation of H4Bip during
its exposing to ultraviolet (UV) irradiation [10]. It is known that UV-B irradiation
(311 nm), excimer laser (308 nm) and UV-A irradiation (320-400 nm) with
photosensitizer (psoralen, etc.) is used in treating vitiligo. It should be mentioned
here, however, that the mechanism of therapeutic of 311 nm irradiation is still
unknown.
To sum up, the primary goal of the present paper is to study the photo-
transformation of tetrahydrobiopterin exposed to UV irradiation in the process of its
oxidation by oxygen in water solutions. Another focus is to discuss the obtained
results in the context of the mechanism of vitiligo UV therapy.
2. Material and methods
2.1. Chemicals.
Biopterin (Bip), 7,8-dihydrobiopterin (H2Bip), H4Bip, 5,6,7,8-
tetrahydropterin (H4Ptr), 7,8-dihydroxanthopterin (H2Xap) and pterin (Ptr) (purity >
99%) were purchased from Schirks Laboratories, Switzerland, and were used
without further purification. Other chemicals used were obtained from Sigma-
Aldrich Co.

4
2.2. Sample Preparation.
Solutions were freshly prepared for each series of experiments by dissolving
H4Bip, H2Bip, Bip, H4Ptr, H2Xap or Ptr in buffer, pH 7.2. Concentration was
determined spectrophotometrically using their molar absorption coefficients. Since
H4Bip solutions are very unstable and easily oxidized at neutral pH by atmospheric
oxygen, if necessary, aliquots of the samples stabilized by adding 0.25 M Na-citrate
buffer pH 2.7 in the ratio 1: 1. We have shown previously that H4Bip solutions were
completely resistant to oxidation by oxygen at pH ≤ 3 [11].
2.3. Experimental methods and apparatus
2.3.1. Steady-state irradiation.
The sets of steady-state experiments were performed to monitor the changes
in the electronic spectra of H4Bip aqueous solutions (pH 7.2) under UV irradiation
or without UV irradiation to study autoxidation. The freshly prepared solutions were
irradiated under air and constant stirring in 1 cm quartz cell using: (1) DRK 120
high-pressure mercury-vapor lamp (Russia) in combination with UFS-6 light filter
(transmission range 300–380 nm) the irradiance was 63 mWcm-2; (2) RF-5301
spectrofluorometer Shimadzu (Japan) with Xenon lamp was used as source of
monochromatic light at the wavelengths of 300 nm (irradiance 6.9±0.2 mWcm-2),
320 nm (irradiance 11.5±0.2 mWcm-2) and 350 nm (irradiance 26.0±0.2 mWcm-2)
with spectral bandwidth of 20 nm. Irradiance value was determined by radiometer
Argus-04 (Russia) and Hamamatsu C9386 radiometer (Japan). The first spectrum
was measured without irradiation and the following spectra were recorded
immediately after exposure.
2.3.2. UV/vis spectroscopy.
Absorption spectra were recorded using spectrophotometers SF-2000 Spektr
(Russia), Cary 300 Bio Varian (USA) and Specord 50 (Germany).
2.3.3. High-performance liquid chromatography.
A high-performance liquid chromatographic (HPLC) gradient systems from
Stayer, Aquilon (Russia) and Agilent-1200 (USA) were used. The eluate from the
HPLC column from Stayer system passed through three detectors connected in
series: (1) Stayer UVV-104 UV/Vis detector, Aquilon (Russia); (2) Fluorat-02
fluorimetric detector, Lumex (Russia) that measured fluorescence with excitation at
λ= 330 nm and emission at λ= 440 nm, and (3) Tsvet-Yauza-01 amperometric
detector, Chimavtomatika (Russia) with working electrode potential of +700 mV.
Agilent chromatograph also had spectrophotometeric and fluorimetric detectors.

5
Fractionation was performed using Partisil SCX and Synergi 4u Polar-RP 80A
(250x4.6 mm) columns, Phenomenex (USA). The mobile phases were Ar-saturated.
2.3.4. Liquid chromatography / electrospray ionization mass spectrometry.
HPLC/mass spectrometry (HPLC/MS) was performed by HPLC-ESI-qTof
method, using HPLC system (Agilent-1260 Infinity (USA)) consisting of binary
pump, thermostat, photometric detector VWD, and the mass spectrometer Agilent
6520 Accurate-Mass quadrupole time-of-flight (Q-TOF) (USA).
2.4. Quantum-chemical calculations.
Quantum-chemical calculations were performed using Orca 3.0.2 program
package [12]. Geometry optimization of H2Ptr dimers and hessian calculation were
performed using the density functional theory method (DFT) and the hybrid
functional B3LYP [13,14] with a 6-31G(d,p) basis set. The effect of the solvent was
taken into account using COSMO continuum solvation model [15,16] with a
dielectric constant for water (?? = 80).
3. Results and discussion
3.1. Autoxidation of tetrahydropterins.
Both in vivo and in aqueous solution in vitro, H4Bip is subjected to
autoxidation, i.e. oxidation by molecular oxygen [17]. The process of autoxidation
is initiated by spontaneous reaction: electron transfers from H4Bip to oxygen
●-
molecule, which results in the formation of superoxide anion radical O2 and
tetrahydrobiopterin cation radical H4Bip•+ [18]:
+
-
•
H4Bip + O2  H4Bip• + O2
(1
or in the formation of trihydrobiopterin radical H3Bip• and hydroperoxyl radical
HOO• [19]:
H4Bip + O2  HOO• + H3Bip•
(2)
Free radicals and reactive oxygen species that participate in further reactions
activating molecular oxygen and oxidizing H4Bip. In general, the entire process of
autoxidation has a radical chain character [19, 20]. The first molecular product of
H4Bip autoxidation is quinonoid 6,7-dihydrobiopterin (qH2Bip) with the halftime
(t ) about 1.5 minutes [17]. Then qH2Bip isomerizes to more stable H2Bip or is
½
transformed into 7,8-dihydropterin (H2Ptr) by side-chain cleavage. H2Ptr is
subsequently oxidized by O2 to H2Xap [17] and to pterin. Then H2Bip may be
oxidized to Bip [17] (Scheme 1).

6
O
N
OH
OH
H
N
HN
H2N
OH
N
H
H4Bip (298 nm)
O2
H2O2
O
N
N
N
OH
qH2Bip (301 nm)
H2N
N
H
O
N
OH
O
N
N
HN
HN
H2N
+ CH3CHOHCHO
OH
H Bip (330 nm)
H2N
N
H
N
N
H2Ptr (325 nm)
2
H
O2
O2
O2
H2O2
H2O2
H2O2
O
O
N
O
OH
H
N
N
O
N
HN
HN
HN
OH
H2N
H2N
H2N
Pterin (340nm)
N
N
N
H
N
N
H2Xap (310 nm)
Bip (346 nm)
Scheme 1. Scheme of tetrahydrobiopterin autoxidation pathway. Within the brackets: the long-
wave maximuma of absorption spectra
Temperature, nature and pH of buffer influence the rearrangement qH2Bip
up to a certain dihydroform. Armarego et al. [21] have stated that while Н4Bip is
being autoxidized, Н2Ptr tends to prevail in tris-buffer (pH 7.6). In addition,
according to Davis et al. [17], in tris-buffer (pH 6.8) Н2Ptr as well tends to prevail
considerably; while in phosphate buffer (pH 6.8) it is H2Bip which prevails. The rise
of temperature from 0oС to 50oС promotes eliminating of the side radical from
qH2Bip and accumulating of H2Ptr in 0.1 M phosphate buffer both with pH 6.8 and
with pH 7.6. We have studied the kinetics of the initial stage of autoxidation Н4Bip
and Н4Ptr with the concentration 1.5 × 10-4М in tris-buffer and in phosphate buffer
(pH 7.2). The products of oxidation in tris-buffer and in the phosphate buffer were
analysed by means of HPLC method with photometric and fluorimetric detectors on

7
cation-exchange column Partiсil SCX (pH 2.7) in the manner that has already been
described [10]. Chromatographic analysis with fluorimetric detection has showed
that Н2Ptr and Н2Bip are formed simultaneously since the very beginning of H4Bip
autoxidation. In order to find out the way buffer characteristics affect the elimination
of dihydroxypropyl radical from qH2Bip, the kinetics of accumulating Н2Bip and
Н2Ptr have been studied. Fluorimetric detection of Н2Bip and Н2Ptr made it possible
to compare the constants of the initial rates at which these dihydropteridins were
formed. Thus, the initial rate of H2Ptr formation (according to the results of
fluorescence) in tris-buffer tends to threefold exceed the rate of H2Bip formation (the
constants of rate are equal to 8.5 × 10-4 sec-1 and 2.9 × 10-4 sec-1, respectively). Rate
constants were evaluated as the rate constants of the first-order reactions. In
phosphate buffer the initial rate of H2Ptr formation also exceeded the rate of H2Bip
formation. The constants of the rates of H2Bip formation in phosphate buffer and
tris-buffer are equal to 1.6 × 10-4 sec-1 and 2.9 × 10-4 sec-1, respectively.
We suppose that fluorescence detection results do not show the actual
relation of pterin and biopterin products, because Н2Xap (as an example) does not
fluoresce and, thus, does not contribute to the total sum of the oxidized pterin
derivatives. That is why we use HPLC/MS to identify the products of the H4Bip
autoxidation. The oxidation of H4Bip (1.5 × 10-4М) was performed in three 0.1M
buffers (ammonium-acetate buffer, phosphate buffer, and tris-buffer) with pH 7.2.
The products of the oxidation were divided on Synergy Polar column (0.05M
ammonium formate, pH 6.2, 0-60% acetonitrile gradient) and further by mass-
spectrometry (MS/MS) were identified. Table 1 presents results of mass-
spectrometry analysis.
Table 1.
The effect of the type of buffer on the product distribution from the autooxidation of H4Bip at
25oC, pH 7.2, 60 min.
Component in the sum of products, (%)
(The average of three experiments)
H4Bip derivatives
H4Ptr derivatives
H2Xap
H2Bip
Bip
H2Ptr
67.8
44.1
35.3
Ptr
2.1
Buffer
Tris
27.6
34.6
15.7
0.8
1.7
11.3
7.6
Phosphate
3.7
6.0
Ammonium-
acetate
24.2
17.2
It is clear from the table that in each of three buffers (pH 7.2) the prevailing
type of H4Bip oxidation was the one accompanied by the elimination of the side

8
radical. Hence, pH can be considered as the one of the main factors that determines
the process of H4Bip autoxidation. In acid medium (pH 3) H4Bip is completely
resistant to the oxidation by oxygen; as pH is shifted to either neutral or alkaline
medium, its resistance to oxygen is decreasing. The rate of the quinonoid
rearrangement depends on the structure of the buffer anion that interacts with the
quinonoid and shifts its electron density. Thus, in tris-buffer ratio of the sum (in %)
of the pterin oxidized products (Н2Ptr + Н2Xap + Ptr) to the sum of the biopterin
oxidized products (Н2Bip + Bip) is equal to 71.6/28.4; in phosphate buffer –
61.7/38.3; in ammonium-acetate buffer – 60.1/39.9.
To conclude, the autoxidation of H4Bip in neutral water solutions leads to
the formation of biopterin derivatives (Н2Bip and Bip) and, simultaneously, oxidized
pterin derivatives (Н2Ptr, Ptr and Н2Xap) are formed.
3.2. Acceleration of tetrahydrobiopterin autoxidation by UV radiation.
Light action directly on H4Bip (max = 298 nm) accelerates the reaction with
O2 and, consequently, accelerates H4Bip autoxidation. We have shown that UV
irradiation of H4Bip affected its oxidation rate (quantum yield Φ300 = (2.7±0.4) × 10-
3). The effect of UV irradiation at  = 350 nm on H4Bip oxidation was found to be
stronger (Φ350 = (7.7 ± 1.2) × 10-3), especially in the presence of Bip (Φ350 = (9.7±1.5)
× 10-3) [10]. The rate of H4Bip oxidation linearly depends on Bip concentration.
Experiments with KI, a selective quencher of triplet pterins at micromolar
concentrations, have demonstrated that the oxidation is sensitized by the triplet state
3
biopterin Bip. Apparently, electron transfer sensitization (Type-I mechanism) is
dominant. Energy transfer (Type-II mechanism) and singlet oxygen generation play
only a secondary role (Scheme 2). Bip is formed as a result of both autoxidation and
photooxidation of H4Bip. The mechanisms of these photoprocesses are described in
detail in our article [10]. The process of photosensitized H4Bip oxidation is induced
by Bip (obviously, other oxidized photosensitizing pterins besides Bip, such as
6-carboxypterin, 6-formylpterin and pterin, can photoinduce H4Bip oxidation)
[22,23,24].

9
Bip·-
H4Bip
3О2
H4Bip·+
H4Bip
Type I
3О2
Type II
1О2
hν
H3Bip·
3О2
1О2
Bip (T1)
ISC
qH2Bip
Bip (S1)
hν
3О2
H2Bip
Bip (S0)
hν
Scheme 2. Proposed tetrahydrobiopterin oxidation pathways under UV-irradiation.
Reactions that occur under UV irradiation are shown by dotted arrows; reactions that
occur without UV are shown by solid arrows.
We believe that our data may help to shed light on the pathological processes
occurring during vitiligo, because the processes described in this article may occur
in vivo and lead to the progression of vitiligo pathology.
3.3. The photodimerization of dihydropterins
The effect of photoexcitation on the oxidation of H4Bip was investigated by
means of irradiation of its solutions (pH 7.2) with UV light (300 - 380 nm) using (1)
and (2) light sources. It was found that the products of H4Bip (Fig. 1b)
photooxidation differ from the products of autoxidation (Fig. 1a). Spectral analysis
of the photooxidation process has shown that in this case absorption band that is
typical of H2Bip does not appear, while this band arises at autoxidation (Fig. 1a).
During the photooxidation process, the increase of absorption at 240-250 nm was
observed.
HPLC analysis of the photooxidation products (Fig. 2) has revealed that
among the products a considerable amount of a new compound occurs, while it is
not present among the products of dark oxidation. It should be noted that the process
of accumulating the new compound (Fig. 2b) and the increase in absorption at 240-
250 nm (Fig. 1b) unfold simultaneously. A difference spectrum (Fig. 1b, inset)
reflects the accumulation of product with the maximum absorption at 245 nm and
an arm at 275 nm. The spectral characteristics of the compound under discussion are
close to dimers of H2Bip (the maximum absorption on 246 nm and an arm on 280
nm), a small amount of which is formed by photoexcitation of Н2Bip performed
without oxygen [25,26]. It should be specially mentioned here that 280 nm
corresponds with the maximum point of the penultimate band in the absorption

10
spectrum of H2Bip, while for H2Ptr the maximum point is at 275 nm. A question
arises, what are physicochemical characteristics of new compound? This compound
does not fluoresce; in absorption spectra it has an unclearly expressed band at 275
nm. During the chromatography on cation-exchange column Particil with pH 2.7
(Fig. 2b), its retention time is considerably longer than that of the other
photooxidation products which have the similar structure (Н2Bip, Н2Ptr, Bip, Ptr).
Consequently, there is a large positive charge on the compound. This may be
conditioned by protonation in acidic medium of two pterin structures at once within
one compound. Taking into account the identified physicochemical characteristics,
we supposed that the dimers of dihydropterin were formed as a result of
photooxidation of H4Bip. We studied action spectrum of dimerization. It was
ascertained that the maximum of the action spectrum is at 310-320 nm.
There is an experimental evidence to support our assumption that it is
predominantly photoexcited molecules of dihydropterin, which tend to dimerize. To
prove the point Н4Bip and Н4Ptr were irradiated at equal concentrations with
monochromatic light at 320 nm. The total exposure time was 20 minutes for Н4Bip
and 16 minutes for Н4Ptr (Fig. 3). After every two minutes of exposure the
absorption spectra were recorded. At that, dark intervals lasted for 2 minutes as well.
As a result, the absorption at 298 nm, which is typical for tetrahydroforms of pterins,
decreased for both Н4Bip and Н4Ptr, at the same time the absorption at 240-250 and
275 nm increased. The rate of dimer formation during Н4Bip oxidation (2.0 × 10-7
M sec-1) was lower than that at Н4Ptr oxidation (3.5 × 10-7 M sec-1). The difference
in the rates of dimer formation probably derives from an amount of time taken to
eliminate the side chain from C6 atom of qН2Bip.
Differences in the reaction rate of dimerization can also be explained by steric
hindrance in the case of Н4Bip. During the cycloaddition reaction, side substituent
can prevent overlapping of the frontier orbitals of the two reactants.
Chromatography of the products of Н4Bip and Н4Ptr photooxidation on the
Synergy Polar column (0.05 M ammonium acetate, pH 7.0, 0-40% acetonitrile
gradient) showed that the main formed dimer was the dimer of dihydropterin
(Н2Ptr)2 with retention time 3.8 min (Fig. 4) This finding confirmed that Н2Ptr takes
part in forming dimers during Н4Bip photooxidation.
HPLC-MS/MS also proved that irradiation of H4Bip causes predominantly
forming the dimers of dihydropterin. On a Synergi Polar column (0.05M ammonium
formate, pH 6.2, 0 - 60% acetonitrile gradient, the length of registered wave was 245
nm) we managed to divide the dimers of dihydropterin and the dimers of
dihydrobiopterin. The mass spectrum of the photooxidation products contained the

11
dominant signal МН+/z = 331, that corresponds to mass of the dimer (Н2Ptr)2 – 330
Da, and the small signal МН+/z=479, that corresponds to mass of the dimer (Н2Bip)2
– 478 Da. The signal of molecular ion of the dimer (Н2Ptr)2 was almost a thousand
times higher than the signal of (Н2Bip)2. It worth mentioning here that the dimers
(Н2Ptr)2 remained intact after they had being kept in the fridge for 24 hours (~ +6oC),
while the dimers (Н2Bip)2 – disappeared.
When the total exposure time was 20 minutes and there was an alternation of
dark intervals (1.5 min) and light intervals (0.5 min), the yield of dimers (on the
initial tetrahydropterins in calculation) reached 96 % from Н4Bip and 100 % from
Н4Ptr. When irradiation process was not interrupted for 20 minutes this values were
equal to 64 % and 76 %, respectively. Therefore, in the case of alternation of dark
and light intervals H4Bip converted into dimers almost quantitatively. It is possible
that in dark the formation of an intermediate intermolecular complex (qH2Ptr-Н2Ptr),
in which isomerization of quinonoid to 5,8-dihydropterin and further
photodimerization of dihydropterins, will take place. The key point of the
dimerization is photoexcitation of the intermolecular complexes (qH2Ptr-Н2Ptr)
generated in dark. Hence, it is important to alternate dark and light intervals. We can
assume that dark interval must be equal to or exceed the halftime (t1/2) of qH2Bip
(1.5 min).
Data on irradiation of Н2Bip support our assumption that intermediate
molecular complexes (qH2Ptr-Н2Ptr) take part in the process of photodimerization.
UV (300-380 nm or 320 nm) irradiation of Н2Bip in the presence of oxygen did not
cause the formation of dimers. In this case, only the oxidation took place and Bip
and Н2Xap were formed. We observed the dynamics of forming the two latter
compounds by means of evolving UV absorption spectra, HPLC and HPLC/MS. It
should be mentioned here that at the initial stage of irradiation an insignificant
amount of dimers, probably, was formed (difference absorption spectra of the first
minute of irradiation did prove this fact). This seems to be connected with a
possibility of occurring disproportionation reaction Н2Bip  H4Bip + Bip. Then a
small amount of dimers was formed from H4Bip. Meanwhile, in general, the
oxidation of Н2Bip to Bip and Н2Xap and to a series of other oxidized derivatives
was the main point. From this, we can conclude that the intermediate quinonoid
dihydro form of pterin is the necessary participant in the process leading to dimers.
The possible formation of intermolecular donor-acceptor complexes between
Н2Ptr and qH2Ptr derives from the following statements:
(1) Between two tautomeric pterin forms created by means of lactam-lactim
tautomerism (N=C-OH  NH-C=O), at pH 7 the lactam form prevails; but the
lactim form has more nucleophilic charachteristics and, thus, it should be more

12
effective in the formation of complex with electrophilic quinonoid. UV irradiation
may promote the formation of such complexes, since the lactim form seems to be
more active under irradiation [27].
(2) By means of circular dichroism (CD), it was demonstrated that intermediate
intermolecular complexes (qH2Ptr-H2Ptr) appear to participate in photodimerization
of dihydropterins. The absorption spectrum of H4Bip in the 200-250 nm region has
a brightly pronounced band with maximum of 221 nm (Fig. 5b); in the CD spectrum
this maximum is absent but a positive band at 228 nm and a negative band with a
maximum of about 205 are observed (Fig.5a). Apparently, the absorption band at
221nm is a superposition of these two bands. As H4Bip photooxidation process
proceeds, both bands in the CD spectrum disappear synchronously. On the
photoformation of dimers can be judged by the appearance of absorption bands of
dimers at 243 and 275 nm in the difference absorption spectrum (Fig. 5b, inset). The
dimers with an absorption maximum of 243 nm do not give significant CD signals,
that is, they have a very weak optical activity. Intermolecular complexes (qH2Ptr-
Н2Ptr) formed as an intermediate product do not contribute to CD signals, since they
do not possess optical activity. This is due to the fact that the chiral center is present
in qH2Bip, but in qH2Ptr it is absent. We believe that dihydropterin dimers are mainly
formed.
(3) After H4Bip solution was being irradiated, the effective quantum yield of dimers
formation was 0.8. This is evidence that within the intermolecular complex two
dihydropterin compounds are optimally space oriented for photoreaction of
dimerization.
As the formation of intermolecular complex is a biomolecular reaction, the
formation of the intermediate complex seems to be the limiting stage of the whole
process. Substituents that are located near the reaction center usually affect the
kinetics of biomolecular reaction. In our case, the side C6-radical in Н2Bip and
qH2Bip may probably act as such the substituent. This fact might be one of the
reasons why these are mainly the dimers of dihydropterin (Н2Ptr)2 that are formed
during the H4Bip photooxidation.
Based on B3LYP/6-31G(d,p) method we compared Gibbs free energies of the
azacyclobutane H2Ptr dimers. We also compared Gibbs free energies of cyclobutane
dimers proposed by M. Vignoni and co-authors [25,26]. Azacyclobutane dimer (cis
isomer) is > 230 kJ mol-1 more stable than the most stable cyclobutane dimer (anti
(h,t) isomer) (Fig. 6) Also, the cis-form of the azacyclobutane dimer is 1.6 kJ mol-1
more stable than the trans-form. Although the cis and the trans-form population is
almost equal (65% and 35%, respectively, according to Boltzmann distribution), we
conclude that the most stable H2Ptr dimer is the azacyclobutane cis isomer.

13
4. Conclusion
In dark H4Bip is easily oxidized by molecular oxygen (autoxidation) to H2Bip
and Bip. Simultaneously, elimination of the side-chain radical to form pterin
derivatives (H2Ptr, H2Xap, Ptr) occurs. Similar processes can obviously occur during
the vitiligo disease and a similar set of products was found in vitiligo-depigmented
skin patches. It was shown by MS/MS mass-spectrometry that at pH 7.2 oxidized
pterin derivatives are predominant among oxidation products in aqueous buffer
solutions.
We showed that under UV irradiation light-induced processes took place in
parallel with constantly flowing dark processes. UV accelerated the process of
autoxidation by direct excitation of H4Bip or by excitation of photosensitizers.
Products of the dark and light-dependent degradation of H4Bip (Bip, Ptr, 6-
carboxypterin, 6-formylpterin) can act as the photosensitizers [10,23]. We suppose,
in vivo similar radical processes can lead to the progression of vitiligo pathology.
The main distinguishing characteristic photoreaction of H4Bip under UV
irradiation was the formation of Н2Ptr dimers. Apparently, the dimerization occurs
more readily with Н2Ptr than with Н2Bip. In the case of alternation of dark and light
intervals, H4Bip converted into dimers almost quantitatively. The highly effective
quantum yield 0.8 of dimers formation is obviously associated with the fact that the
photodimerization occurs in the excited intermediate intermolecular complex
(qH2Ptr-Н2Ptr) generated in dark. According to our theoretical calculations,
azacyclobutane dimers (Н2Ptr)2 are predominant over cyclobutane dimers, while the
cis-form of azacyclobutane dimers is the most stable one.
We believe that such formation of dihydropterin dimers may occur in vivo during
the narrowband phototherapy (308 or 311 nm) of vitiligo patients. It is known that
excess of H4Bip and its oxidation products inhibit melanin biosynthesis. A
considerable number of H4Bip oxidation reactions leads to accumulation of
hydrogen peroxide (H2O2) and to oxidative stress in vitiligo [2,3,4,5,28,29].
Furthermore, H2O2 stimulates biosynthesis of excessive H4Bip through the induction
of GTF-cyclohydrolase I [30]. Thus, we have a ‘vicious circle’ here, which includes
excessive H4Bip. This ‘vicious circle’ can be broken by the reaction of
photodimerization, which excludes the H4Bip excess.
Acknowledgements
This study was financially supported by the Basic Research Program N 28 and N22 of the
Presidium of Russian Academy of Sciences and the Russian Foundation for Basic Research
(grant N 11-04-01007a and grant 14-04-31885). This work was funded by the subsidy allocated
to Kazan Federal University for the state assignment in the sphere of scientific activities.
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16
Captions to figures:
Fig. 1. Evolution of the absorption spectra of H4Bip in air-equilibrated 0.1 M
phosphate buffer (pH 7.2) in the course of time. (a) Autoxidation and (b)
Photooxidation under UV irradiation (300-380 nm, 63 mW cm-2). Arrows indicate
the changes observed at different wavelengths. [H4Bip]0 = 1.5 × 10-4M. Insets show
difference spectra (20 min – 0 min).
Fig. 2. HPLC analysis of the products (a) autoxidation and (b) photooxidation
of H4Bip. Partisil SCX ion exchange column. The mobile phase was Ar-saturated 25
mM sodium citrate buffer, pH 2.7, flow rate 0.7 mL min-1.
Fig. 3. Changes in the absorption spectra monitored upon monochromatic
irradiation at 320 nm (11.5 mW cm-2) of (a) H4Ptr (1.75 × 10-4M) and (b) H4Bip
(1.77 × 10-4M) in 0.1 M phosphate buffer, pH 7.2. Arrows indicate the changes
observed at different wavelengths. Insets show the difference spectrum between the
last spectrum (16 or 20 min, accordingly) and the initial one (0 min).
Fig. 4. HPLC analysis of the photooxidation products of (a) H4Bip (1.5 × 10-
4M) and (b) H4Ptr (1.5 × 10-4M). Synergi 4u Polar-RP 80Acolumn. The mobile phase
was 50 mM ammonium-acetate buffer at pH 7.0; 0-40% acetonitrile gradient. Solid
line – registration at 245 nm, dashed line - registration at 330 nm.
Fig. 5. Changes in (a) CD spectra and (b) absorption spectra of H4Bip (0.6 ×
10-4 M) in 0.1 M phosphate buffer, pH 7.2 monitored upon UV irradiation. Arrows
indicate the changes observed at different wavelengths. Solid line – 0 min, Dashed
line - 8min, Dotted line - 16 min, Dash-dotted line - 24 min. Inset show the difference
spectrum (24 min – 0 min).
Fig. 6. Optimized geometries of azacyclobutane and cyclobutane H2Ptr
dimers. Method: B3LYP/6-31G(d,p) – COSMO (water). (To see the color in this
figure, the reader is referred to the web version of this article.)