Photoirradiation products of flavin derivatives, and the effects of photooxidation on guanine

Article abstract of DOI:10.1016/j.bmcl.2009.01.112

Photoirradiation in the presence of riboflavin led to guanine oxidation and the formation of imidazolone. Meanwhile, riboflavin itself was degraded by ultraviolet light A (UV-A) and visible light (VIS) radiation, and the end product was lumichrome. VIS radiation in the presence of riboflavin oxidized guanine similarly to UV-A radiation. Although UV-A radiation with lumichrome oxidized guanine, VIS radiation with lumichrome did not. Thus, UV-A radiation with riboflavin can oxidize guanine even if riboflavin is degraded to lumichrome. In contrast, following VIS radiation degradation of riboflavin to lumichrome, VIS radiation with riboflavin is hardly capable of oxidizing guanine. The consequences of riboflavin degradation and guanine photooxidation can be extended to flavin mononucleotide and flavin adenine dinucleotide. In addition, we report advanced synthesis; carboxymethylflavin was obtained by oxidation of formylmethylflavin with chlorite and hydrogen peroxide; lumichrome was obtained by heating of formylmethylflavin in 50% AcOH; lumiflavin was obtained by incubation of formylmethylflavin in 2 M NaOH, followed by isolation by step-by-step concentration.

Full text of DOI:10.1016/j.bmcl.2009.01.112

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2070–2074
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Photoirradiation products of flavin derivatives, and the effects
of photooxidation on guanine
*
Katsuhito Kino , Teruhiko Kobayashi, Eiji Arima, Rie Komori, Takanobu Kobayashi, Hiroshi Miyazawa
Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa 769-2193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Photoirradiation in the presence of riboflavin led to guanine oxidation and the formation of imidazolone.
Meanwhile, riboflavin itself was degraded by ultraviolet light A (UV-A) and visible light (VIS) radiation,
and the end product was lumichrome. VIS radiation in the presence of riboflavin oxidized guanine sim-
ilarly to UV-A radiation. Although UV-A radiation with lumichrome oxidized guanine, VIS radiation with
lumichrome did not. Thus, UV-A radiation with riboflavin can oxidize guanine even if riboflavin is
degraded to lumichrome. In contrast, following VIS radiation degradation of riboflavin to lumichrome,
VIS radiation with riboflavin is hardly capable of oxidizing guanine. The consequences of riboflavin deg-
radation and guanine photooxidation can be extended to flavin mononucleotide and flavin adenine dinu-
cleotide. In addition, we report advanced synthesis; carboxymethylflavin was obtained by oxidation of
formylmethylflavin with chlorite and hydrogen peroxide; lumichrome was obtained by heating of form-
ylmethylflavin in 50% AcOH; lumiflavin was obtained by incubation of formylmethylflavin in 2 M NaOH,
followed by isolation by step-by-step concentration.
Received 8 October 2008
Revised 6 January 2009
Accepted 21 January 2009
Available online 14 February 2009
Keywords:
Guanine oxidation
Flavin
UV-A radiation
VIS radiation
Ó 2009 Elsevier Ltd. All rights reserved.
Guanine is highly susceptible to oxidative stress in the DNA, as
it has the lowest redox potential among the four DNA bases. Ribo-
flavin (vitamin B2) (RF) (Fig. 1A) is a photosensitizer that causes
electron transfer reactions1,2 and generates singlet oxygen.3 Ultra-
violet light A (UV-A) radiation with RF oxidizes guanine, and imi-
were found to generate LC from RF, FMF, CMF, HEF, flavin adenine
dinucleotide (FAD) and flavin mononucleotide (FMN) under near-
physiological conditions, such as pH 8. In addition, we found that
the photochemical effects of LC on guanine oxidation are different
from those of RF, FAD and FMN.
dazolone (Iz) is formed as
a
guanine oxidative product
We first prepared FMF, HEF, CMF, LC, and LF from RF. The reac-
tion of RF with periodate ion led to FMF, and HEF was obtained by
reduction with sodium borohydride.14,15 We applied the oxidation
method16 to the preparation of CMF; FMF was oxidized by chlorite
and hydrogen peroxide (Scheme 1).17 The procedure reported here
is a simple method relative to the previous synthesis of CMF.18,19
LC was obtained by incubation of FMF in a hot acidic solution
(Scheme 1).20 This procedure is an easier and simpler method for
large-scale synthesis of LC than previous methods: construction
of isoalloxazine,21 chromatographic separation of RF photolysis,22
and hydrolysis of FMF by alkali.23,24
Although it was previously reported that treatment with 2 M
sodium hydroxide resulted in the conversion of FMF to LF,25 our
HPLC analysis of the reaction showed that the obtained LF con-
tained a small amount of LC.26 By trial and error, pure LF was ob-
tained by preferential removal of LC in a concentrated acidic
solution (Scheme 1).27
(Fig. 1B),1,4 in addition to spirohydantoin and other products.5 Iz
may be responsible for DNA mutations, which might cause G:C
to C:G transversions.4,6–8 Thus, RF contained in our cells, especially
basal cells and dermis, may be involved in the photooxidation of
DNA, despite the fact that RF is degraded by light. Therefore, it is
quite important to understand the photochemical effects of the
degradation products of RF. Previously, RF degradation to lumi-
chrome (LC), lumiflavin (LF), formylmethylflavin (FMF) and carb-
oxymethylflavin (CMF) (Fig. 1A) by light was reviewed.9 In
addition, FMF and hydroxyethylflavin (HEF) (Fig. 1A) degradation
to LC and LF by light were reported,10,11 and the mechanism of
photo-degradation from RF was described.12,13 However, little is
known about the quantitative analysis of photo-degradation prod-
ucts of the flavin derivatives under identical conditions. In addi-
tion, it has not yet been reported whether these degradation
products from photoirradiated RF can oxidize guanine and
whether blue visible light (VIS) instead of UV-A can oxidize guan-
ine with flavin derivatives. In this study, UV-A or VIS radiation
Figure 2 shows HPLC analysis of the degradation of flavin deriv-
atives by UV-A or VIS radiation. UV-A radiation at 366 nm de-
graded RF to LC as a major product, and FMF was formed as a
minor product at 5 min in the reaction, and then degraded
(Fig. 2A, open symbols). Moreover, the photolysis of FMF lead to
* Corresponding author. Tel.: +81 87 894 5111; fax: +81 87 894 0181.
E-mail address: kkino@kph.bunri-u.ac.jp (K. Kino).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.112

K. Kino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2070–2074
2071
Figure 1. The structures of (A) flavin derivatives and (B) deoxynucleoside.
LC, and FMF was more rapidly degraded than RF (Fig. 2B, open
symbols). Hence, it was confirmed that FMF is an intermediate
product in the photolysis of RF to LC.10,25 In addition, CMF was rap-
idly degraded, but HEF was slowly degraded (Fig. 2C and D, open
symbols). RF, FMN, FAD and HEF have 2’-hydroxyl group, and re-
duced FMF arises from the flavins (step 2 in Scheme 2), followed
by air-oxidation (step 3 in Scheme 2).9 In the pathway leading to
reduced FMF, the hydrogen abstraction reaction (step 1 in Scheme
2) is likely to be slow and rate-limiting. In contrast, McBride et al.
suggested that reduced FMF could readily undergo cyclization
(step 4 in Scheme 2).10,11 Additionally, this cyclic reduced form
can lead to the formation of LC (step 5 in Scheme 2). Therefore,
the degradation rates of FMF and CMF, which have 2’-carbonyl
group, were faster than those of the flavins which have 2’-hydroxyl
group. These results are consistent with the previously reported
mechanism.12,13
excited state. FMF, CMF and HEF were also degraded to LC by VIS
radiation (Fig. 2B–D, closed symbols). Like UV-A radiation, VIS radi-
ation rapidly degraded FMF and CMF, and slowly degraded RF and
HEF (Fig. 2A–D, closed symbols). In addition, LF was not further de-
graded by UV-A or VIS radiation.
Moreover, FMN and FAD were also degraded to LC by UV-A or
VIS radiation (Fig. 2E and F). In addition, the degradation rate of
FMN was similar to that of RF, whereas FAD was slowly degraded.
The difference between the FMN and FAD degradation rate is likely
dependent on whether adenine nucleotide exists.
Under all conditions used in Figure 2, the quantity of detected
LF was less than 2%, and CMF and HEF intermediates were not de-
tected at all. Therefore, UV-A or VIS radiation was concluded to
convert from RF, FAD, and FMN to LC at least via FMF.
Figure 3 shows HPLC analysis of deoxyguanosine irradiated by
UV-A or VIS radiation with RF, LC, LF, FMN or FAD. UV-A radiation
at 366 nm with RF oxidized guanine and generated Iz30 (Fig. 3A),
and this result is compatible with the previous finding.4 VIS radia-
tion, as well as UV-A radiation, with RF can oxidize guanine
(Fig. 3A). UV-A radiation with LC can oxidize guanine, but VIS radi-
Since RF has two absorption maxima at 360–380 nm and 440–
450 nm,13,28,29 VIS radiation at 440 nm, as well as UV-A radiation at
366 nm, degraded RF to LC (Fig. 2A, closed symbols). This result
confirmed that RF excited at 366 nm or 440 nm falls to the lowest
Scheme 1. Synthesis of CMF, LC and LF.

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K. Kino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2070–2074
Figure 2. Photodegradation of flavin derivatives by UV-A or VIS radiation. Each 23
buffer (pH 8.0) was irradiated by UV-A light at 366 nm (open symbols) or VIS light at 440 nm (closed symbols). The samples were analyzed by HPLC using a 5C4-MS column
(Nakalai Tesque, 5
m, 150 Â 4.6 mm, elution with a solvent mixture of water, 0–30% CH3CN/30 min at a flow rate of 1.0 ml/min) in panel A–D. In panel E and F, the samples
were analyzed by HPLC using a 5C18-MS column (Nakalai Tesque, 5
lM of (A) RF, (B) FMF, (C) CMF, (D) HEF, (E) FAD or (F) FMN in 9 mM sodium phosphate
l
l
m, 150 Â 4.6 mm, elution with a solvent mixture of 10 mM TEAA, 0–30% CH3CN/30 min at a flow rate of
1.0 ml/min). The amount of flavin derivatives was monitored at 366 nm absorbance. In the UV-A radiation experiments, squares indicate the amount of the starting materials,
and diamonds indicate that of LC. In panel A and F, circles indicate the amount of intermediate FMF. (G–I) HPLC analysis of reaction mixtures. RF (G) was irradiated for 5 min
by UV-A light at 366 nm (H) or VIS light at 440 nm (I).
Scheme 2. Suggested photodegradation mechanism of RF, FMN, FAD, HEF, and FMF.
ation at 440 nm with LC was hardly capable of oxidizing guanine
(Fig. 3B). In contrast to RF, LC can hardly absorb light at
440 nm.13,28,31 LF has the same flavin chromophore as RF, and
the absorption of LF is almost the same as that of RF.13,29 Hence,

K. Kino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2070–2074
2073
Figure 3. Photooxidation of deoxyguanosine under UV-A or VIS radiation. Deoxyguanosine (100
lM) with each 20
lM of (A) RF, (B) LC, (C) LF, (D) FAD or (E) FMN in 20 mM
sodium phosphate buffer (pH 8.0) was irradiated by UV-A light at 366 nm (open symbols) or VIS light at 440 nm (closed symbols). The samples were analyzed by HPLC using a
5C4-MS column (Nakalai Tesque, 5
l
m, 150 Â 4.6 mm, elution with a solvent mixture of water, 0–7% CH3CN/20 min at a flow rate of 1.0 ml/min) and the amount of dG
(square) and dIz (diamond) was monitored at 254 nm absorbance. (F–H) HPLC analysis of reaction mixtures. Deoxyguanosine with LC (F) was irradiated for 60 min by UV-A
light at 366 nm (G) or VIS light at 440 nm (H).
the rate of guanine oxidation by LF was similar to that by RF
(Fig. 3C). The fluorescence lifetime of LC is shorter than that of
RF and almost the same as that of LF.13 The lifetime of the trip-
let-excited LC is shorter than that of LF.13 Thus the oxidation rate
of guanine greatly depends not only on the molar absorption coef-
ficients but also on the lifetimes of the singlet-excited and triplet-
excited states; VIS radiation with LC was not capable of oxidizing
guanine, and UV-A radiation with LC oxidized guanine more slowly
than that with RF or LF.
Moreover, UV-A or VIS radiation in the presence of FAD and FMN
can oxidize guanine (Fig. 3D and E). Notably, the photoreactivity of
guanine in the presence of FMN was almost the same as RF, while
the rate of guanine photooxidation with FAD was slower than that
with RF. These different results are likely to depend on whether ade-
nine nucleotide exists, similar to the results in Figure 2.
generate Iz. Conversely, once RF, FAD and FMN are degraded
completely to LC by VIS radiation, guanine is hardly oxidized.
RF is a vitamin, and FMN and FAD are natural constituents of
living organisms. Since UV-A and VIS are easy to transmit to ba-
sal cells and dermis, the effects of UV-A and VIS radiation in the
presence of flavin derivatives are not negligible. We speculate
that guanine oxidation by UV-A radiation in the presence of fla-
vin derivatives is sustained, but that by VIS radiation is not sus-
tained. Guanine oxidation causes point mutation and telomere
shortening, and relates to cancer and senescence.32 Thus, the dif-
ferent photoreactivity of UV-A and VIS is expected to affect the
ease and persistence of such phenomena as the cytotoxicity of
RF activation by UV or VIS radiation.33,34 In addition, RF photo-
sensitized inactivation of phage.35 The selection of light wave-
length for photoirradiation can decide the duration of oxidation
and cytotoxicity of tumour cells, and our results seems to be
available to improve laser therapies.
In summary, even though RF, FAD and FMN were degraded to
LC by UV-A radiation, UV-A radiation can oxidize guanine and

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K. Kino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2070–2074
19. Yagi, K.; Matsuoka, Y. J. Biochem. 1960, 48, 93–100.
Acknowledgment
20. The synthesis of LC: Prepurified FMF (2.16 g, 7.59 mmol) was dissolved in 50%
acetic acid aqueous solution (220 ml), and then was heated at 80 °C for 3 h.
During heating, the solution had a color varying from initially yellow to finally
black. The black solution was cooled to room temperature on standing, and a
green precipitate was isolated by filtration, washed with water and methanol,
and dried. The product LC was prepared in 61% yield (1.12 g, 4.64 mmol).
21. Seng, F.; Ley, K. Angew. Chem., Int. Ed. 1972, 11, 1010–1011.
This work was supported by grants from Tokushima Bunri
University.
References and notes
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23. Song, P.-S.; Smith, E. C.; Metzler, D. E. J. Am. Chem. Soc. 1965, 87, 4181–4184.
24. By the procedure based on Ref. 23, LC was obtained at 51% yield.
25. Smith, E. C.; Metzler, D. E. J. Am. Chem. Soc. 1963, 85, 3285–3288.
26. The synthesis of impure LF: As well as the procedure in Ref. 17, FMF was used
without isolation. While stirring, sodium hydroxide (16 g) was added to the
suspension (200 ml) containing FMF. The suspension immediately changed to
a dark wine red solution, and was stirred for 0.5 h at room temperature. The
impure precipitation formed by addition of conc. HCl (60 ml) was removed by
filtration. Then conc. NH3 was added to the filtrate until the orange solution
changed to a dark green suspension. The precipitate (1.09 g) was obtained, but
the obtained LF was impure. The ratio of LF and LC was 86:14.
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conc. HCl (200 ml), and then the solution was concentrated to half of its
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17. The synthesis of CMF: As well as the procedure in Ref. 14, FMF was generated
from RF (3.0 g, 8.0 mmol). Without isolation of FMF, NaClO2 (6.0 g) was added
to the suspension containing FMF, and then 6.9% H2O2 aqueous solution
(13 ml) was added dropwise. The suspension was vigorously stirred at room
temperature for 1.5 h in the dark. The reaction was monitored by TLC (CH2Cl2/
MeOH, 4:1), which showed the absence of FMF. Conc. HCl (1 ml) was added,
and the solid was isolated from the suspension by filtration, washed with cold
water, and dried. The product CMF was prepared in 96% total yield (2.3 g,
7.7 mmol) as an orange solid.
31. The molar absorption coefficients (L molÀ1 cmÀ1) (H2O) were as follows: RF,
e
366 = 9800 and e440 = 12000; LC, e366 = 6500 and e440 = 160.
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