Biosci. Biotechnol. Biochem., 71 (10), 2596–2599, 2007
Note
Reaction of Retinol with Peroxynitrite Rie S UZUKI,1; * Aditya K ULKARNI,2; * Yuya Y OMODA,1 Hirokazu K AWAGISHI,1 Yukimasa T ERADA,3 Takashi M AOKA,4 and Hideo E TOH1; y 1
Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan 3 Center of Computers and Technology, Meijo University, Shiogamaguchi, Tempaku, Nagoya 468-8502, Japan 4 Research Institute for Production Development, 15 Shimogamo-Morimoto-cho, Sakyou-ku, Kyoto 606-0805, Japan 2
Received June 4, 2007; Accepted July 18, 2007; Online Publication, October 7, 2007 [doi:10.1271/bbb.70349]
The reactivity of retinol with peroxynitrite, which is a strong oxidant and has been reported to induce several biological damages, was investigated. 13-cis-14-nitroretinol (1), 13-trans-14-nitroretinol (2), 13-apo- -carotenone (3), retinal (4), 11,14-epoxyretinol (5), and 11,15epoxyretinol (6) were identified as reaction products of retinol with peroxynitrite. From these results, it was observed that retinol can undergo a nitration reaction with peroxynitrite. Furthermore, the formation mechanisms of 1, 2, and 3 from retinol with peroxynitrite are discussed. Key words:
retinol; peroxynitrite; antioxidative compounds
Retinol, a circulating form of vitamin A, is a fatsoluble molecule that plays an important role in the visual cycle and is essential for the normal growth of bones, reproduction, and embryonic development and in the differentiation of epithelial tissues. It is generated from either -carotene or retinyl esters and is stored as retinyl esters in the liver. Retinols are sensitive to oxidation.1–4) The extended polyene structure of retinol readily reacts with the peroxyl radical and is thought to inhibit lipid peroxydation by trapping lipid-peroxy radicals.5) Peroxynitrite is a strong oxidant and has the potential to cause several forms of biological damage. Peroxynitrite and its decomposition products induce DNA strand scission, protein modification by nitration and hydroxylation, and membrane lipid peroxydation in LDL,6,7) but the peroxynitrite reaction mechanism of retinol, which probably figures in the human body, is not yet understood. In our recent efforts, we isolated for the first time nitro carotenoids from the reaction of astaxanthin and -carotene with peroxynitrite.8) In our continuous efforts in this field, we investigated the reactivity of retinol with peroxynitrite and attempted to identify its reaction products after scavenging with * y
peroxynitrite. Here we discuss the formation mechanisms of the above reaction products. All-trans-retinol (150 mg) was dissolved in 100 ml of THF (final concentration, 4.8 mM). To this, 200 ml of TFA (final concentration, 0.2%) was added, followed by 10 ml of peroxynitrite, which was prepared according to a method described in the literature9) (final concentration 4.5 mM), and allowed to react for 2 min. To the above mixture, 200 ml of chloroform and 200 ml of H2 O were added, so as to separate the reaction products into organic and aqueous phases. The entire procedure was performed two times. The organic layer was then dehydrated using anhydrous Na2 SO4 and concentrated. This organic concentrate was subjected to HPLC analysis with a semiprepatative C30 column (Develosil C30-UG-5 20 250 mm, Nomura Chemical Co., Ltd., Seto, Japan) with 65% MeCN as the mobile phase. For more specific separation, an analytical C30 column was used (Develosil C30-UG-5 4:6 250 mm, Nomura Chemical Co., Ltd., Seto, Japan) with 65% MeCN and seven fractions were obtained, as shown in Fig. 1. Compounds 1, 2, 3, 4, 5, and 6 were identified as retinal (3.8%) (4), 13-apo--carotenone (3.6%) (3),10) 13-cis-14-nitroretinol (3.1%) (1), 13-trans-14-nitroretinol (2.1%) (2), 11,15-epoxyretinol (1.5%) (6),11,12) and 11,14-epoxyretinol (1.3%) (5), by MS, UV–VIS, 1 H and 13 C NMR data and by comparison with authentic samples (Fig. 2). Among these, 13-cis-14-nitroretinol and 13-trans-14-nitroretinol13) were identified for the first time as major products of this reaction. In order to determine the stereochemistry of 1 and 2, NOESY studies were performed. NOE interaction was observed between 20-CH3 and 14-H of 1, indicating cis isomerism, but no NOE interaction was observed between 20CH3 and 14-H in case of 2, confirming it to be a trans isomer of nitroretinol. Compounds 5 and 6 were identified as mixtures, since they showed the same
The two authors contributed equally to this work. To whom correspondence should be addressed. Tel/Fax: +81-54-238-4884; E-mail:
[email protected]
Reaction of Retinol with Peroxynitrite
2597
Fig. 1. HPLC Chromatogram of Peroxynitrite Reaction of Retinol. HPLC conditions: C30, Develosil C30-UG-5 4:6 250 mm, 65% MeCN, flow rate; 1 ml/min, column temperature, 40 C, detection range, 200–600 nm.
20 15
13
OH
14
Retinol 20
20
13
13
H
14
12
14
12
H
NO2
13-trans-14-nitroretinol (2)
13-cis-14-nitroretinol (1) 20 11
10
H
13
O
O
12
H
13-apo-β -carotenone (3)
retinal (4) 15
15 14
O
O
OH
13
10
20
12
OH 14
11
11 10
NO2
11,14-epoxyretinol (5)
13 12
20
11,15-epoxyretinol (6) O
7
8
1
(H3C)3C
C(CH3)3
2
6
3
5 4 9
O
O 4'
1' 2'
3'
BHT-THF adduct Fig. 2. Structures of Peroxynitrite Reaction Products of Retinol.
2598
R. S UZUKI et al. 20 15
13 14
Nitronium radical pathway
OH
Dioxetane pathway Retinol
15
CHO
20
Retinal
14
15
13 15
O
COOH
O
Retinoic acid
14
NO2 (ONOOH*)
13, 14-dioxetane retinol
20
O 13
OH
14
C
11
15
O
14
10
13
O 12
NO2 20
3 13 14
NO2 1
20 13
NO2 14
2 Fig. 3. Reaction Mechanism.
retention time in HPLC chromatograms (Fig. 1). The peak at a retention time of 34 min corresponded to the reaction product between THF and BHT. THF contained a trace amount of BHT as a stabilizer, which might undergo a radical reaction in presence of peroxynitrite to form the adduct. The taller peak at a retention time of 55 min was of unreacted retinol. 13-cis-14-nitroretinol (1) UV max (CH3 CN): 427 nm; high resolution (HR) FAB-MS calculated for C19 H27 O2 N + Hþ = 301.2042, found = 301.2038; yield 3.1% 1 H-NMR (CDCl3 ): 0.97 (6H, s, 16, 17-CH3 ), 1.41 (2H, m, 2-CH2 ), 1.56 (2H, m, 3-CH2 ), 1.66 (3H, s, 18-CH3 ), 1.97 (3H, s, 19-CH3 ), 1.98 (2H, m, 4-CH2 ), 2.03 (3H, s, 20-CH3 ), 6.14 (1H, d, J ¼ 16 Hz, 8-CH), 6.24 (1H, d, J ¼ 12 Hz, 10-CH), 6.33 (1H, d, J ¼ 16 Hz, 7-CH), 6.85 (1H, s, 14-CH), 7.17 (1H, dd, J ¼ 12 Hz, 15 Hz, 11-CH), 7.69 (1H, d, J ¼ 15 Hz, 12-CH). 13-trans-14-nitroretinol (2) UV max (CH3 CN): 419 nm; high resolution (HR) FAB-MS calculated for C19 H27 O2 N + Hþ = 301.2042, found = 301.2042; yield 2.1%. 1 H-NMR (CDCl3 ): 1.04 (6H, s, 16,17 CH3 ), 1.48 (2H, m, 2-CH2 ), 1.58 (2H, m, 3-CH2 ), 1.72 (3H, s, 18-CH3 ), 2.04 (3H, s, 19-CH3 ), 2.05 (2H, m, 4-CH2 ), 2.45 (3H, s, 20-CH3 ), 6.18 (1H, d, J ¼ 17 Hz, 8-CH), 6.32 (1H, d, J ¼ 11 Hz, 10-CH), 6.38 (1H, d, J ¼ 17 Hz,
7-CH), 7.13 (1H, s, 14-CH), 7.23 (1H, dd, J ¼ 11 Hz, 15 Hz, 11-CH), 6.22 (1H, d, J ¼ 15 Hz, 12-CH). BHT-THF adduct 1 H-NMR (CDCl3 ): 1.21 (6H, s, 7,8-(CH3 )6 ), 1.33 (3H, s, 9-CH3 ), 6.46 (1H, d, 3 or 5CH), 6.61 (1H, d, 5 or 3-CH), 1.60 (1H, m, 20 -CH), 1.71 (1H, m, 30 -CH), 3.84 (1H, q, J ¼ 15 Hz, 8 Hz, 40 -CH), 3.90 (1H, sp, J ¼ 4 Hz, 8 Hz, 40 -CH), 5.35 (1H, d, J ¼ 8 Hz, 10 -CH). 13 C-NMR (CDCl3 ): 23.9 (9-C), 24.1 (30 C), 29.1 (20 -C), 29.4 (7 and 8-(C)3 ), 34.7 (7 and 8-C), 67.5 (40 -C), 78.0 (4-C), 107.2 (10 -C), 129.1 (5-C), 140.5, 141.2 (3 and 5-C), 147.2, 147.6 (2 and 6-C), 186.5 (1-C). From the results of the present investigation, we summarized the possible reaction mechanisms of peroxynitrite with retinol (Fig. 3) and proposed formation pathways for 13-cis (1) and 13-trans-nitroretinol (2) (nitronium radical pathway), as follows: Retinoic acid, a possible intermediate, was formed from retinol via retinal (4) by an oxidation reaction. Then retinoic acid was reacted with the ( NO2 ) or (ONOOH ) radical at the C-14 position to yield 13-cis- and 13-trans-14-nitroretinol as major products. We did not detect retinoic acid as an intermediate product of this reaction, but we carried out a reaction between retinoic acid and peroxynitrite separately, which gave us the expected reaction products 13-cis (1) and 13-trans-14-nitroretinol (2).
Reaction of Retinol with Peroxynitrite
The steric energy of 13-cis-14-nitroretinol (1) and 13trans-14-nitroretinol (2) was calculated by the ab initio molecular orbital method.14) The calculation revealed that the cis isomer was 1.8070 kJ mol1 more stable than the trans isomer, and this agreed quantitatively with the yield of these isomers (3.1%, for 1 and 2.1%, for 2). For the formation of 13-apo--carotenone (3), we propose the Dioxetane pathway as follows: Retinols oxidize to 13, 14-dioxetane of retinol, a possible intermediate, by the oxygen radical, which might form during the reaction of peroxynitrite with retinol. Then oxidative cleavage between C-13 and C-14 of 13, 14dioxetane retinol results in the formation of 13-apo-carotenone (3) as one of the main products. In conclusion, we have for the first time isolated and characterized nitroretinols 1 and 2 from the reaction products of peroxynitrite with retinal, and, we propose the formation mechanism. These results indicate that retinol caught the peroxynitrite or nitrogen dioxide radical ( NO2 ) in its molecule to form nitroretinols. This information should be of value in the future for understanding certain complex in vivo reaction patterns of retinol and its oxidation products. Efforts are underway to elucidate the structure of intermediates and products by way of understanding the complete mechanism of scavenging peroxynitrite.
3)
4)
5)
6)
7)
8)
9)
10)
Acknowledgments This work was supported by the program Cooperation of Innovative Technology and Advanced Research in the Evolutional Area (Metropolitan Regions) of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References 1)
2)
Jungalwala, F. B., and Cama, H. R., Preparation and properties of 5,6-monoepoxyvitamin A acetate, 5,6monoepoxyvitamin A alcohol, 5,6-monoepoxyvitamin A aldehyde and their corresponding 5,8-monoepoxy (furanoid) compounds. Biochem. J., 95, 17–26 (1965). Takashima, Y., Nakajima, T., Tanaka, S., Washitake, M., Anmo, T., and Matsumaru, H., Stability of retinol analogs. IX. Stability of vitamin A acetate in aqueous ethanolic solutions and quantitative analysis of its decomposition products by high performance liquid chromatography. Chem. Pharm. Bull., 27, 1553–1563 (1979).
11)
12)
13)
14)
2599
Ikeda, A., Iwahashi, H., Negoro, Y., and Kido, R., Detection of radical species in mixtures of some retinoids with hematin by using the ESR spin-trapping technique. J. Biochem., 101, 743–749 (1987). Oyler, A. R., Motto, M. G., Naldi, R. E., Facchine, K. L., Hamburg, P. F., Burinsky, D. J., Dunphy, R., and Cotter, M. L., Characterization of autoxidation products of retinoic acid. Tetrahedron, 45, 7679–7694 (1989). Samokyszyn, V. M., and Marnett, L. J., Inhibition of liver microsomal lipid peroxidation by 13-cis-retinoic acid. Free Rad. Biol. Med., 8, 491–496 (1990). King, P. A., Anderson, V. E., Edwards, J. O., Gustafson, G., Plumb, R. C., and Suggs, J. W., A stable solid that generates hydroxyl radical upon dissolution in aqueous solutions: reaction with proteins and nucleic acid. J. Am. Chem. Soc., 114, 5430–5432 (1992). Radi, R., Beckman, J. S., Bush, K. M., and Freeman, B. A., Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch. Biochem. Biophys., 288, 481–487 (1991). Yoshioka, R., Hayakawa, T., Ishizuka, K., Kulkarni, A., Terada, Y., Maoka, T., and Etoh, H., Nitration reactions of astaxanthin and -carotene by peroxynitrite. Tetrahedron Lett., 47, 3637–3640 (2006). Niwa, T., Doi, U., Kato, Y., and Osawa, T., Antioxidative properties of phenolic antioxidants isolated from corn steep liquor. J. Agric. Food Chem., 49, 177– 182 (2001). Laurent, A., Prat, V., Valla, A., Andriamialisoa, Z., Giraud, M., Labia, R., and Potier, P., Syntheses of new 9- and 13-methylene isomers of retinal. Tetrahedron Lett., 41, 7221–7224 (2000). Yamauchi, R., Miyake, N., Kato, K., and Ueno, Y., Peroxyl-radical reaction of retinyl acetate in solution. Biosci. Biotechnol. Biochem., 56, 1529–1532 (1992). Welch, S. C., Gruber, J. M., and Prakasa Rao, A. S. C., Epoxidation of retinol and the structure of chromogen 574. Tetrahedron, 36, 1179–1182 (1980). Panzella, L., Manini, P., Crescenzi, O., Napolitano, A., and d’Ischia, M., Nitrite-induced nitration pathways of retinoic acid, 5,6-epoxyretinoic acid, and their esters under mildly acidic conditions: toward a reappraisal of retinoids as scavengers of reactive nitrogen species. Chem. Res. Toxicol., 16, 502–511 (2003). Ab initio calculations were carried out using the Gaussian 03 program (Gaussian, Wallingford, CT, 2004). The starting coordinates were generated by inspection of the molecular model, and then they were fully optimized using RHF/6-31G (D). The calculations were carried out using a Fujitsu Primepower HPC2500 super computer at the Information Technology Center of Nagoya University.
