Kojic Acid–Tripeptide Amide as a New Tyrosinase Inhibitor Kojic Acid–Tripeptide Amide as a New Tyrosinase Inhibitor Jin-Mi Noh, Seon-Yeong Kwak, Do-Hyun Kim, Yoon-Sik Lee School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea Received 7 November 2006; revised 22 December 2006; accepted 29 December 2006 Published online 8 January 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20670
ABSTRACT: Twenty two kojic acid–tripeptide amides were prepared using a solid-phase Fmoc/tBu strategy with Rink Amide SURE1 resin. To effectively obtain kojic acid–tripeptide amide conjugates, the coupling conditions of kojic acid to the tripeptide on the resin were optimized. The tyrosinase inhibitory activity of kojic acid–tripeptide amides and the effect of the amino acid sequence on the activity were compared with those of kojic acid– tripeptide acids. The stability of kojic acid–tripeptide amides were then compared with those of kojic acid and kojic acid–tripeptides acids. As a consequence, kojic acid-FWY-NH2 proved to be the best compound, with the highest inhibitory activity, which was maintained over different storage times under various temperatures and pHs. # 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 300–307, 2007. Keywords: kojic acid peptide derivatives; tyrosinase inhibitor; whitening agent; stability This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley. com Correspondence to: Yoon-Sik Lee, School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea; e-mail:
[email protected] Contract grant sponsor: Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea. Contract grant number: A050432
C 2007 V
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INTRODUCTION
F
or several decades, the cosmetic industry has been using biologically active ingredients from all imaginable sources. The choice and application of new cosmetic ingredients are influenced by the recent advances in analytical sciences, biochemistry, and other disciplines. Traditional cosmetic ingredients used to be limited to mainly plant extracts, hydrolysates, essential oils, and vitamins, etc; but now, more synthetic molecules are being designed, tested, and finally incorporated into cosmetic final products. Furthermore, human biological processes are being monitored, with active molecules found in human bodies examined and studied in search for better synthetic analogues. Recently, peptides have appeared to be more effective cosmetic ingredients than natural extracts, which contain dozens or even hundreds of individual molecules. Among cosmetics, whitening agents,1 which are popular with Orientals, have maintained constantly high levels of consumption since the late 1980s. While the global market for skin-whitening agents has grown, a clinically proven safe and effective cosmetic formulation remains to be found. Conventionally, whitening has been considered as the eliminating agent of the melanin, which acts as a self-defense molecule of human skin against the exposure to ultraviolet (UV) rays. The step of melanin synthesis in melanocytes is mediated by several enzymes, of which tyrosinase is essential. Tyrosinase2–7 (monophenol or o-diphenol oxygen oxidoreductase), also known as polyphenol oxidase, phenolase, catecholase, and cresolase, is a copper-containing enzyme, which is widespread in nature. This bifunctional enzyme catalyzes two distinct reactions involving molecular oxygen; the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the oxidation of o-diphenols to o-quinones (diphenolase activity). These quinones are highly reactive and spontaneously polymerize to the high molecular weight brown pigment, melanin, which determines the color of mammalian skin and hair. Accordingly, to regulate the pigmenting activity toward tyrosine, the inhibition of tyrosinase is a major strategy in the prevention of hyperpigmentation
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Kojic Acid Tripeptide Amide as a New Tyrosinase Inhibitor
because of the enzymatic oxidation. Some of tyrosinase inhibitors; hydroquinine,8–11 azelaic acid,12,13 electron-rich phenols,14 corticosteroids,15 resinoids,16,17 and albutin, have been utilized in the field of cosmetics. However, these agents do have some limitations, such as relatively high toxicity and insufficient skin penetration ability. Kojic acid18–23 has been used in many countries as a skinwhitening agent, because of its high inhibitory activities toward melanin synthesis.18 However, its use in cosmetics has been limited because of the skin irritation caused due to its cytotoxicity and instability on storage. Accordingly, many kojic acid derivatives have been synthesized to try and improve the properties by modification of the C-7 hydroxyl group into an ester,24 hydroxyphenyl ether,25 glycoside,26 and amide derivatives.27 In our previous study, it was found that some kojic acid– tripeptide free acids were more stable and had powerful inhibitory activities toward tyrosinase compared with kojic acid itself.28 Many bioactive peptide hormones, such as oxytocin,29 gastrin,30 and calcitonin,31 possess a C-terminal amide group for their full biological potency.32 These facts prompted us to further investigate whether kojic acid– tripeptide coupled with a C-terminal amide (kojic acid– tripeptide amide) might be a more powerful inhibitor toward tyrosinase, with higher storage stability for use in cosmetics. In this study, 22 kojic acid–tripeptide amides were prepared using solid-phase parallel peptide synthesis,33 employing Fmoc-Rink amide AM SURE1 resin.34 To this end, kojic acid was activated with 1,10 -carbonyldiimidazole (CDI) and coupled to the N-terminal of the tripeptides on the resin. After cleavage of the kojic acid–tripeptide derivatives from the resin,35 the desired kojic acid–tripeptide amides were obtained, with both high yields and purity. The tyrosinase inhibitory activity and stability of the newly prepared kojic
acid–tripeptide amides were then measured and compared with those of kojic acid and kojic acid–tripeptide free acids.
EXPERIMENTAL PROCEDURES Materials 2-Chlorotrityl chloride (CTC) (100–200 mesh, 0.9–1.1 mmol/g) resin, aminomethyl surface-layered polystyrene (AM SURE1) (100– 200 mesh, 0.76 mmol/g) resin, Libra tubes1 (15ml, 5 ml) and Fmoc-l-amino acids were purchased from BeadTech. (Seoul, Korea). Kojic acid was purchased from TCI Organic Chemicals (Tokyo, Japan). 1,10 -Carbonyldiimidazole (CDI), tyrosinase, L-tyrosine, diisopropylethylamine (DIPEA), 1,2-ethane-dithiol (EDT), thioanisole, and ninhydrin were purchased from Sigma (St.Louis, MO). FmocRink linker, benzotriazol-1-yl-oxytris(dimethylamino)-phosphoniumhexafluorophosphate (BOP), o-benzotriazole-N,N,N0 ,N0 -tetramethyluroniumhexafluorophosphate (HBTU), and 1-hydroxybenzotriazole (HOBt) were purchased from GL Bio-Chem (Shanghai, China). N-Methyl-2-pyrrolidone (NMP) was purchased from Junsei Chemicals (Tokyo, Japan). Piperidine and dichloromethane (DCM) were purchased from Dae-Jung Chemicals (Siheung, Korea). Tetrahydrofuran (THF) was purchased from SamChun Chemicals (Pyongtack, Korea). N,N-Dimethyl-formamide (DMF) was purchased from Mallinckrodt Backer. Trifluoroacetic acid (TFA) was purchased from Acros Organics (NJ), and phenol was purchased from DC Chemical (Seoul, Korea). All solvents were of reagent grade and used without further purification.36
Synthesis of Kojic Acid-7-Imidazolide, Compound 2 Kojic acid 1 (5 g, 35 mmol) was dissolved in THF (90 ml) and DMF (10 ml). After stirring for 1 h, with N2 purging, CDI (5.1 g, 0.9 equiv.) in THF (50 ml) was added. A white solid powder was formed, and the mixture stirred for 24 h at room temperature. The resulting white solids 2 were filtered, washed with THF and dried in vacuo to give white solids, with a yield of 70%. The structure of compound 2 was confirmed from the NMR spectra, which were recorded on a JNM-LA300 spectrometer (Jeol) in deuterated solvents, and referenced to TMS (d scale). 1H NMR (300 MHz,
SCHEME 1 Selective activation of the hydroxyl group at position 7 of kojic acid.
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SCHEME 2 Solid-phase kojic acid–tripeptide amides synthesis. Reagents and condition; (a) 20% piperidine in NMP, 30 min (b) Fmoc-AA1-OH (2 equiv.), BOP (2 equiv.), HOBt (2 equiv.), DIPEA (4 equiv.), and NMP, 2 h (c) [repeat (a) and (b) twice] (d) KA-imidazolide, HOBt (2 equiv.), NMP, 2 h (e) reagent K. [TFA/thioanisole/phenol/water/EDT (82.5/5/5/5/2.5 v/v)], 1 h, and diethyl ether precipitation.
DMSO-d6, d): 9.34 (1H, s, OH), 8.33 (1H, s, N CH¼ ¼N), 8.13 (1H, s, CH O), 7.67 (1H, s, imidazole), 7.10 (1H, s, imidazole), 6.66 (1H, s, CH C¼ ¼O), 5.30 (2H, s, CH2 O).
Fmoc Quantitation37 A known quantity of Fmoc-group containing resin (30 mg) was suspended in 3.0 ml of 20% (v/v) piperidine/DMF. The mixture was shaken in a shaking incubator at 258C for 50 min. Part of the above mixture (0.10 ml) was withdrawn and diluted to 10.0 ml with DMF. The number of Fmoc groups on the resin was quantified by the absorbance at 290 nm.
Solid-Phase Synthesis of Kojic Acid-Tripeptide Amides The Fmoc-Rink amide linker (2 equiv.) was anchored onto the AM SURE1 resin (0.76 mmol/g), with HBTU (2 equiv.), HOBt (2 equiv.) and DIPEA (4 equiv.) in NMP at 308C for 3 h. The resins were filtered, washed with NMP and DCM, and dried in vacuo. The loading levels of the linker onto the resins were 0.50–0.67 mmol/g resin, which was determined using a Fmoc titration. After deprotection of the Fmoc groups with 20% piperidine in NMP for 30 min, Table I The Effect of Additives for Coupling of KA-Imidazolide to Resin Bound Tripeptide
Additives pKa Coupling time (h)
HOBt HOSu 3.47 2
3.59 3
Acetic No acid Pentafluorophenol Additive 4.75 5.5
5.5 18
24
N-Fmoc-amino acid (2 equiv.), in NMP, was quantitatively introduced to the resin using the general protocol of the BOP-mediated solid phase Fmoc/tBu strategy. Two further Fmoc-amino acids were then coupled in series to the amino acid-loaded resin, using the Table II
Yield and Purity of the Selected KA-Tripeptide Amides
KA-tripeptide Amides KA-YGG KA-YIG KA-YYG KA-YSG KA-YMG KA-YRG KA-YQG KA-YHG KA-YNG KA-YDG KA-KGG KA-FRY KA-FYY KA-FWY KA-KRY KA-KKY KA-YGT KA-YGE KA-YGW KA-YGF KA-YGH KA-YGD a
Yield (%)
Purity (%)a
46 64 75 59 64 78 70 76 34 70 70 72 38 36 50 50 40 65 50 47 64 36
91 92 87 88 83 86 81 82 84 93 80 82 91 95 87 92 91 93 98 94 94 95
Determined by HPLC.
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FIGURE 1 Comparison of KA-tripeptide free acids and KA-tripeptide amides on tyrosinase inhibitory activities; the degree of inhibition was measured with 20 lM of each KA-derivatives solution, 2 units/ll of mushroom tyrosinase, 0.3 mg/ml of tyrosine.(blue: KA-tripeptide amides; red:KA-tripeptides acids).
same protocol. After removing Fmoc group, compound 2 (2 equiv.) was then added to the resin bound tripeptide with HOBt, and the mixture was shaken for 2 h Each reaction step was monitored using the Kaiser’s Ninhydrin Test.38,39 Finally, the resin was treated with reagent K [TFA/thioanisole/phenol/water/EDT (82.5/5/5/5/2.5 v/v)] for 60 min at room temperature, and the resin was filtered. The crude peptide in the filtrate was concentrated under light vacuum, and precipitated with cold diethyl ether, to yield a white powder. The powder was further washed with diethyl ether, and dried in vacuo, to give the desired kojic acid–tripeptide amide. The crude peptide was analyzed by high performance liquid chromatography (HPLC, YoungLin Autochro 2000), using the following conditions: Waters lBondapak C18 reverse phase column (125 A˚, 10 lm, 3.9 3 150 mm2); gradient elution with A: 0.1% TFA/water, B: 0.1% TFA/ acetonitrile; from 10 to 90% B over 50 min; flow: 1 ml/min; detection: UV, 220 nm, with the HPLC purity expressed as peak height%. The peptide was further characterized by matrix-assisted laser disorption ionization time of flight (MALDI-TOF) mass spectroscopy (Bruker, Germany), under delayed extraction conditions, operating with a pulsed N2 laser at 337 nm.
Stability Test of Kojic Acid and Kojic Aicd-Tripeptide Derivatives The stability of the kojic acid–tripeptide derivatives was tested using the mushroom tyrosinase inhibition assay. Firstly, the stability test, as a function of the storage time, was evaluated by measuring the tyrosinase inhibition by the test substrate, which was stored as a solid at room temperature for up to 2 months. The stability test as a function of temperature was measured by the addition of 20 lM of test substance as an aqueous solution in dil. water, with storage at 0, 25, and 508C for 48 h. The stability test, as a function of pH, was performed using the mushroom tyrosinase inhibition assay, with 20 lM of test substance, with storage in 0.1M phosphate buffer solutions of different pH for 24 h.
RESULTS AND DISCUSSION Optimizing the Synthetic Conditions for Kojic Acid–Tripeptide Amides For the coupling of kojic acid (KA) to the N-terminal of the peptides via a urethane bond, KA was activated by CDI,
Mushroom Tyrosinase Inhibition Assay40 Five hundred microlitres of 0.1M phosphate buffer (pH 6.8), 500 ll of L-tyrosine solution (0.3 mg/ml in water), 50 ll of tyrosinase solution (2 units/ll in 0.1M phosphate buffer, pH 6.8), and 400 ll of water were mixed in an effendorf tube (1.5 ml), and 50 lM inhibitor was then added. The solution was stirred at 378C for 15 min, and immediately cooled in an ice bath. After standing in the ice bath for 10 min, the UV absorbance of the solution was measured at 475 nm. The same solution, but without the test substance, was also prepared, with the UV absorbance also measured at 475 nm. The inhibition percent was calculated using the following formula: [(A B)/A] 3 100 (A, absorbance of control solution; B, absorbance of test substance solution). The IC50 value was calculated by varying the concentration of the test substance.
Biopolymers (Peptide Science) DOI 10.1002/bip
FIGURE 2 Positional effect of the amino acid at the KA + 1 position on the tyrosinase inhibitory activity.
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The Positional Effect of Amino Acid of Kojic Acid–Tripeptides on Tyrosinase Inhibitory Activity
FIGURE 3 Positional effect of the amino acid at the KA + 2 position on the tyrosinase inhibitory activity.
yielding KA-imidazolide 2 (Scheme 1). To prevent side reactions, such as oxidation of the kojic acid moiety or decomposition of the desired product, the reaction was performed at room temperature. To improve the solubility of kojic acid in THF, the minimum amount of DMF was added into the reaction mixture as a cosolvent. As the reaction proceeded, the solubility changed, with the desired product precipitated in a 78% yield (Scheme 1). The resin-bound tripeptides were synthesized separately using the general solid-phase Fmoc/ tBu strategy (Scheme 2). When the activated kojic acid was coupled to the N-terminal of tripeptide on the resin, a long reaction time of at least 1 day was required (Scheme 2d),28 and the purity of the final product was greatly reduced. Therefore, catalysts which could minimize the coupling time of KA-imidazolide, 2, onto the resin-bound tripeptides were screened. Four catalysts were tested, including HOBt, HOSu, acetic acid, and pentafluorophenol, which could donate a proton to the leaving group, imidazole. As expected, the reaction was accelerated by 1.5– 12 times (Table I). As the pKa value of the catalyst became lower, the reaction accelerated. From this, HOBt proved to be the most effective catalyst for the coupling of KA-imidazolide to the resin bound tripeptide. It is believed the HOBt acted as a general acid catalyst to the leaving group during another activated KA, KA-HOBt ester formation.
Table III
Tyrosinase Inhibition of Selected KA-Tripeptide Derivativesa
KA-tripeptide acids KA KA-FWY KA-FHY KA-FRY a
To discover the effect of the peptide structure coupled to KA on the tyrosinase inhibitory activity, KA-amino acid, KAdipeptide, and KA-tripeptide libraries have previously been prepared and screened. In our previous work, KA-tripeptide free acids were reported to show better inhibitory activities than KA-amino acids and KA-dipeptides. Based upon this previous study with KA-tripeptides free acids, 22 kojic acid– tripeptide amides were chosen and screened (Table II). According to the general method described above, all the kojic acid amides were prepared, with overall yields between 40 and 80%, and purities exceeding 80%, as confirmed by HPLC. Firstly, the tyrosinase inhibitory activities of the KA-tripeptide amides were evaluated and compared with those of KA and KA-tripeptide free acids.41 Most of the synthesized KA-tripeptide amides showed similar inhibitory activities to their acid forms, with all showing enhanced inhibitory activities than KA itself (Figure 1). In particular, KA-tripeptide, with Phe at the KA + 1 position, showed the best inhibitory activity. To examine the effect of amino acids at the KA + 1 position on the inhibitory activity, the KA + 2 and KA + 3 positions were fixed as Arg and Tyr, respectively, and the inhibitory activities toward tyrosinase measured (Figure 2). When hydrophobic aromatic amino acids, such as Phe, Trp, and Tyr, substituted at the KA + 1 position, the substances showed higher inhibitory activities toward tyrosinase than other substituents. These results support that the residue at the KA + 1 position might play a key role in the tyrosinase inhibitory activity. Following these results, the hydrophobic side chains at the KA + 1 position were confirmed as potentially contributing to their binding to the hydrophobic pocket near the active site of tyrosinase. Secondly, KA + 1 and KA + 3 were fixed as Phe and Tyr, respectively, with the amino acid at the KA + 2 position varied, to examine the effect on the tyrosinase inhibitory activity, most of which showed similarly high inhibitory activities.
Yield (%)
Purity (%)
Inh. (%)
IC50 (lM)
KA-tripeptide amides
Yield (%)
Purity (%)
Inh. (%)
IC50 (lM)
98 99 92
73 94 83
12 92 86 88
94 1.28 4.55 5.92
KA-FWY-NH2 KA-FHY-NH2 KA-FRY-NH2
68 54 62
97 86 81
91 94 92
2.2 2.36 3.59
Measured by mushroom tyrosinase inhibition assay, treated with 20 lM test substance solution.
Biopolymers (Peptide Science) DOI 10.1002/bip
Kojic Acid Tripeptide Amide as a New Tyrosinase Inhibitor
FIGURE 4 The inhibition activity of the selected KA-tripeptide derivatives.
From these results, it was concluded that the amino acid at the KA + 2 position was not so important to the tyrosinase inhibitory activity once the amino acids at KA + 1 and KA + 3 position had been fixed as Phe and Tyr (Figure 3).
305
FIGURE 6 Stability of KA and KA-tripeptide derivatives as a function of storage time at room temperature (The activities of tyrosinase inhibition were compared with those of the original batch).
or amide forms, the resultant substances showed the lowest IC50 values.
Stability of KA-Tripeptide Derivatives Comparison of Tyrosinase Inhibitory Activity of Selected KA-Tripeptide Derivatives From careful analysis of the results, several KA-tripeptide amides which showed the highest inhibitory activities were selected: Phe at the KA + 1 position, Trp, His, and Arg at the KA + 2 position and Tyr at the KA + 3 position. These substituents were chosen, and their inhibitory activities and stability were compared with those of the corresponding KA-tripeptide free acids (Table III). Both of the kojic acid–tripeptide derivatives had higher inhibitory activities than kojic acid itself (Figure 4). Their inhibitory activities toward tyrosinase were almost 100%, which was more than 10 times higher than KA itself. The exhibited IC50 values of the KAtripeptide free acids and KA-tripeptide amides were 20–80 times lower than that of KA itself (Figure 5). When the peptide sequence of FWY was coupled to KA, in either the acid
FIGURE 5 IC50 values of KA-tripeptide derivatives in the mushroom tyrosinase inhibition assay.
Biopolymers (Peptide Science) DOI 10.1002/bip
The stabilities of the KA-tripeptide derivatives were tested as functions of time, temperature, and pH value by measuring the decrease in the tyrosinase inhibitory activity. Firstly, the stability, as a function of storage time, was examined (Figure 6). The KA-tripeptide derivatives were stored in their solid form at room temperature for up to 2 months, with their inhibitory activities then measured, using the Mushroom tyrosinase inhibition assay method, as a function of time. The KA-tripeptide derivatives showed no significant decreases in their inhibitory activities, even after 2 months, whereas KA lost its inhibitory activity about 50% after only 1week. After 1 month, the inhibitory activities of the kojic acid–tripeptide acids tended to decrease; however, that of the kojic acid–tripeptide amides remained intact. The thermal stabilities of the KA-tripeptide derivatives were then measured using the Mushroom tyrosinase inhibi-
FIGURE 7 Thermal stability of KA, KA-tripeptide derivative aqueous solutions (20 lM) at 0, 25, and 508C.
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the stability test results, it was conclude that the KA-tripeptide amides maintained their tyrosinase inhibitory activities better than the free acid form under various storage conditions.
CONCLUSION FIGURE 8 Stability test under acidic conditions. Mushroom tyrosinase inhibition assay was performed with 20 lM of test substance stored in various 0.1M phosphate buffer solutions for 24 h.
tion assay with 20 lM stock solutions in water, and stored at 0, 25, and 58C for 2 days (Figure 7). At 0 and 258C, the inhibitory activities of the kojic acid–tripeptide acids or amides remained unchanged. Therefore, it was concluded that the tyrosinase inhibitory activity of KA-tripeptide derivatives remained reasonably stable when stored in either their solid or solution forms. When the KA-FHY and KA-FRY solutions were heated to 508C, their inhibitory activities decreased. However, the kojic acid–tripeptide amides maintained their inhibitory activities, even at 508C. The KA-tripeptide amides were found to be more stable when heated than the KA-tripeptide acids. Of these, the peptide sequence of FWY, when coupled to KA in either the acid or amide forms, maintained their inhibitory activities relatively well at 0, 25, and 508C. Finally, to investigate the effects of pH on the stability, the Mushroom tyrosinase inhibition assay was performed with 20 lM stock solutions at pH 4.6, 5.8, 6.8, 7.8, and 9.4, after storage for a day at room temperature (Figures 8 and 9). The KA-tripeptide amides maintained their inhibitory activities under both acidic and basic conditions. However, the inhibitory activity of the KA-tripeptide acid dropped slightly under acidic conditions. Moreover, KA-FWY maintained its inhibitory activity under both acidic and basic conditions. From
FIGURE 9 Stability test under basic conditions. Mushroom tyrosinase inhibition assay was performed with 20 lM of test substance stored in various 0.1M phosphate buffer solutions.
A library of kojic acid–tripeptide amides was easily prepared using the general SPPS on the Rink Amide-SURE1 resin, with both high purities and yields. The KA-tripeptide amides were found to exhibit similar inhibitory activities to those of the KA-tripeptide free acids, but superior activities to that of KA itself. Moreover, KA-tripeptide amides were stable under various conditions, and maintain their inhibitory activities relatively longer than other substance. Especially, the peptide sequence of FWY coupled to kojic acid, either in the free acid or amide form, showed the highest inhibitory activity and stability. Further studies on the skin-penetration ability of the kojic acid derivatives are underway. This work was supported by a grant of the Korea Health Z1 R & D Project, Ministry & Welfare, Republic of Korea (A050432).
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