Manabu Kitazawa, Keilj Iwasaki, Yukiko Ishitsuka, Kumi Arakane
*Ajinooto Co., Inc. AminoScience Laboratories Suzuki-cho 1-1 Kawasaki-ku, Kawasaki 210-8581, Giappone
**Kosé Corporation, Fundamental Research Laboratories Azusawa 1-18-4, Itabashi-ku, Tokyo 174-0051, Giappone

Abstract
We designed a novel antioxidant whit a n iron sequestering capacity to suppress UV-introduced skin photoaging.The principe of the molecular design is to mimic the structures and functions of iron sequestering proteins, such as transferrin, by conjuating a vitamin whit an amino acid. Thus, N-(4-pyridoxylmethylene)- L-serine (PYSer),a stabilized conjugate molecule of pyridoxal (vitamin B6 aldehyde form) and L-serine, was prepared. PYSer formed a 2:1 complex whit iron- induced hydroxyl radical generation, was found by means of ESR spin trapping and salicylate hydroxylation assays. The redox potential of PYSer-Fe3+/ PYSer-Fe2+ showed superior stabilization of the Fe3+ active catalytic species, rather than the Fe2+ species, for hydroxyl radical generation. In the hairless mice photoaging study, PYSer suppressed UVB-induce wrinkle formation and dermal hypertropy. These results indicate that the conjgation of vitamin B6 whit an amino acid is a promissing approach for the molecular desing of antioxidants whit iron sequestering capacity.

Introduction
Solar ultraviolet (UV) light contributers to skin photodamage, such as skin cancer, photoaging, photosentisizasion, and other light-related skin pathologies [1,2]. It is known that reactive oxygen species (ROS) are deeply involved in the UV-induced photodamage. Cutaneous iron catalyzesthe production of ROS, especially hydroxyl radicals, via the Haber-Weiss reaction (Fig.1) [3,4]. In living sysem, the iron is trasported and stored by iron-binding proteis in such as trasferrin and ferrin. However, free iron is released inside cells under oxidative stress. UVA radiation induces the release of iron in human primary skin fibroblast [5]. The enhanced production of metalloproteases and lipid peroxidation in dermal fibroblas irradiated whit UVB is suppresed by desferrioxamine (DFO), a strong iron chelator [6].these results indicate that the iron is involved in the oxidative stress caused by both UVA and UVB. Therefore, it is important to design antioxidants whit an iron sequestering capacity. Several researchers have been investigatin iron chelators for the reduction of UV- induced oxidative stress. Bissett et al. are pioneers in this field.They found that UV radiation increased the cutaneuos iron content, and an iron chelator, dipyridiylamine,suppressed the UV-induced winkle formation in hairless mice [7] . We have noticed that conjugates of amino acids whit salicylaldehyde have structural similarity to iron sequestering proteins, such as transferrin [8]. We proved that conjugates of amino acids whit naphtylaldehyde or salycilaldehyde suppressed iron-iduced hyroxyl radical generation and reduced the UV-induced oxidative stress by sequestring the catalytically active iron chelators was reporter [11].

FIG. 1
Generalized machanism for the iron-catalyzed Haber-Weiss reaction and lipid (L-H) peroxidation in cells and tissues, as shown in ref. (4).

To create on ideal cosmetic oxidant that is not only functional but also provides cosmetic usefulness, we applied the molecular design to conjugate a vitamin whit an amino acid. We have noticed that vitamin B6 and its derivatives are used as cosmetic ingredients to treat rough skin. Vitamin B6 is a coenzime related to amino acid metabolism, and naturally occurring conjugated form of pyridoxal-5-phospate (one form of vitamin B6) and amino acid are known. These molecules have structural similarity to the amino acid iron chelator we have designed. As metabolic intermediates, the conjugatedforms are not chemically stable in cosmetic formulation because of their Shift base stucture. We have applied the hydrogenation of the Shift base to create a stable conjugate of vitamin B6 and an amino acid. Thus, N-(4-pyridoxylmenthylene)-L-serine (PYSer), a stabilized conjugate molecule of pyridoxal (vitamin B6 aldehyde form) whit L-serine, was prepared. Here we demonstrate the antioxidative and the anti-photoaging capacities of this novel molecule.

Discussion
PYSer supressed iron-iduced hydroxyl radical generation and UV-induced wrikle formation. Since the compound forms stable complexes whit Fe3+ and inhibits iron-induced hydroxyl radical generetion, it is espected to suppres free radical reactions by sequestering the catalytic iron in the body.
We have confirmed the formation of 2:1 complexes of PYSer to Fe3+. The compound has three possible binding sites to Fe3+: the hydroxyl group in the amino acid residue. Therefore, it is expected that all six binding sites to Fe3+ would be occupied whit two ligand molecules. Thus, the iron complex no longer catalyzes the Haber-Weiss reaction because of the absence of the catalytic site.Hence, PYSer,at double the molar quantity of iron ion,inhibited the radical generation. The isufficient inhibition in the presence of PYSer at less than twice the molar quantity of iron ion is due to the incomplete occupation of the iron binding site.
In our analysis of the PYSer-Fe3+ complex, we estimated the logk values to be 21 to 22. These values are larger than those of iron proteins (logk = 16-18) [16] but are much smaller than that of EDTA (logk = 25) [14]. The moderate affinity of the PYSer coumpond for iron ions should circumvent the undesirable side-effects.
These suitable properties of PYSer as an antioxidant are thougt to be derived from the similarity of its iron-binding mechanism to that of iron proteins. In a preliminary study of the redox potential of PYSer -Fe3+/PYSer-Fe2+, we estimated the value to be -34mV vs. NHE. This low redox potential of PYSer -Fe3+/PYSer-Fe2+ , as compared whit that of EDTA-Fe3+/EDTA-Fe2+(+120mV vs . NHE) [15], promotes the oxidation of Fe2+ to Fe3+, whitc is less reactive whit H2O2 in the Haber-Weiss reaction. The antioxidation mechanism of PYSer is suggested to be the stabilization of Fe3+, to which Fe2+is rapidly converted by auto-oxidation [17].
In hairless mice irradiated whit UVB, as a model of human photoaging [18,19],PYSer delayed wrinkle formation and suppressed in the increase in cutaneous iron content in UVB-exposed mice [7]. We believe that the meccanism of the photoprotection by PYSer is also througt chelation of iron ion, which is involved in the suppression of catalytic ROS generation. It has been reported that dermal thickness as well as epidermal thickness increased whit time of UVB exposure in the suppression of catalytic ROS generation. It has been reported that dermal thickness as well as epidermal thickness increased whit time of UVB exposure in the photoaging model [20]. The PYSer treatment significantly suppressed the increase dermal thickness as compared whit the vehicle treatment. It is known that cronic exposure of murine skin to UVB radiation results in alterations of dermal extracellular martix components, such as collagen, elastin, and glycosaminoglycans (GAGs), as well as wrinkle formation on the surface of the skin [19,21,22]. The suppression of UVB-induced dermal hypertrophy by PYSer may be related to the protection from dermal demage. Although EDTA is a commonly user iron chelator, it is qite ineffetive for protection against the UVB-induced wrinkle formation and the increasein skin thickness. EDTA enhanced hydroxyl radical formation in the salicylate hydroxylation assay. This enhancement would be due to insufficient occupation of the iron-binding site [23]. In addition, the EDTA-iron complex is more reactive whit H2O2 in the Haber-Weiss reaction, because of its higt redox potential as compared with that of the PYSer-iron complex, as described previously. Therefore, the ineffectiveness of EDTA for photodamage protection may be due to the absence ofa suppressive effect on iron-iduced ROS formation.
In summary, PYSer showed a protective effect on photoaging ih hairless mice. The meccanism of the photoprotection seems to be througt the suppression of hydroxyl radical generation, as shown in an in vitro assay. Rrecently , we have found further preventive effects of PYSer against photoaging related to ROS. Namely, PYSer reduced singlet oxygen generation in a photosensitive reaction and melanogenesis in melanoma cells [24]. PYSer is prepared by the conjugation of two natural compounds: L-serine and vitamin B6, whitc are a natural moisturizing amino acid and an essential coenzyme, respectively. The conjugated forms is analogous to that found in amino acid metabolism. On the other hand, The molecules reported for the prevention of hydroxil radical generation are all synthetic chelators, for which sefety and adverse effect are great concerns. As result, PYSer is a novel and promising antioxidant for cosmetic application to prevent chronic photoaging.

Acknowledgments
The authors thank Prof. Lester Packer, of the University of California at Berkeley, and Prof. Barry Halliwell, of National University of Singapore, for helpful discussions regarding our study.