1. Solid Phase Synthesis of Mimosine Tetra Pep Tides (JAFC 2)

ARTICLE pubs.acs.org/JAFC Solid-Phase Synthesis of Mimosine Tetrapeptides and Their Inhibitory Activities on Neuraminidase and Tyrosinase Atul Upadhyay,† Jamnian Chompoo,† Nozomi Taira,§ Masakazu Fukuta,§ Shinichi Gima,X and Shinkichi Tawata*,§ † Department of Bioscience and Biotechnology, The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan § Department of Bioscience and Biotechnology, Faculty of Agriculture, and XInstrumental Research Center, University of the Ryukyus, Senbaru 1, Nishihara-cho, Okinawa 903-0213, Japan ABSTRACT: Neuraminidase is a rational target for influenza inhibition, and the search for neuraminidase inhibitors has been intensified. Mimosine, a nonprotein amino acid, was for the first time identified as a neuraminidase inhibitor with an IC50 of 9.8 ( 0.2 μM. It was found that mimosine had slow, time-dependent competitive inhibition against the neuraminidase. Furthermore, a small library of mimosine tetrapeptides (MÀA1ÀA2ÀA3) was synthesized by solid-phase synthesis and was assayed to evaluate their neuraminidase and tyrosinase inhibitory properties. Most of the tetrapeptides showed better activities than mimosine. MimosineÀ FFY was the best compound, and it exhibited 50% neuraminidase inhibition at a low micromolar range of 1.8 ( 0.2 μM, whereas for tyrosinase inhibition, it had an IC50 of 18.3 ( 0.5 μM. The kinetic studies showed that all of the synthesized peptides inhibited neuraminidase noncompetitively with Ki values ranging from 1.9 -to 7.2 μM. These results suggest that mimosine could be used as a source of bioactive compounds and may have possibilities in the design of drugs as neuraminidase and tyrosinase inhibitors. KEYWORDS: mimosine, mimosine tetrapeptides, neuraminidase, tyrosinase ’ INTRODUCTION Neuraminidase, an enzyme present in the influenza virus, is involved in the release of progeny virus from infected cells, by cleaving sugars that bind the mature viral particles.1 Specifically, neuraminidase cleaves the α-ketosidic bond that links a terminal neuraminic acid residue to the adjacent oligosaccharide moiety. Neuraminidase is therefore essential for the movement of the virus at the site of infection in the respiratory tract.2,3 On the other hand, tyrosinase is a copper-containing enzyme that catalyzes the ortho-hydroxylation of monophenols to catechols and their subsequent oxidation to o-quinones. In mammals, tyrosinase is responsible not only for browning of hair and skin pigmentation4 but also for skin anomalies such as hypo- or hyperpigmentation.5 Furthermore, tyrosinase may play a role in cancer and neurodegenerative diseases, such as Parkinson’s disease.6 As both enzymes are quite a significant target in the field of medicine, the development of neuraminidase and/or tyrosinase inhibitors has received much attention. Mimosine [β-[N-(3-hydroxy-4-oxypyridyl)]-α-aminopropionic acid] is a nonprotein amino acid containing an alanine side chain bound to the nitrogen atom of a pyridine ring. It is found in several tropical and subtropical plants of genera Leucaena and Mimosa. Mimosine has been confirmed to be responsible for the allelopathy of the plant.7À9 The therapeutic roles of mimosine include antiproliferative and antifibrotic,10 antitumor,11 and antiviral.12,13 In addition, the anti-tyrosinase activity of mimosine has been studied.14,15 Peptides have been found to inhibit neuraminidase16 and tyrosinase.17 We had previously isolated three compounds from Alpinia zerumbet rhizomes and studied the probable mechanism of neuraminidase inhibition.18 In this study, the neuraminidase r 2011 American Chemical Society inhibitory activity of mimosine was identified, and hence mimosine peptides were synthesized to assess their neuraminidase and tyrosinase inhibitory activities. We also report the inhibition kinetics of mimosine and tetrapeptides against the neuraminidase. This is the first report on the synthesis of peptides from mimosine and the investigation of their therapeutic potentials. ’ MATERIALS AND METHODS Chemicals. The Fmoc amino acids were bought from Hipep Laboratories (Kyoto, Japan), whereas Fmoc-OSu [N-(9-fluorenylmethoxycarbonyloxy)succinimide] was obtained from Merck, Darmstadt, Germany. N,N0 -Diisopropylethylamine (DIEA), 1-hydroxy-1H-benzotriazole (HOBt), and N,N0 -diisopropylcarbodiimide (DIC) were bought from Wako, Osaka, Japan, whereas HBTU was acquired from Nova Biochem, China. All other reagents used were of analytical grade and were obtained from Wako. Isolation of Mimosine from Leucaena. One kilogram of Leucaena leaves was boiled in 5 L of water for 10 min. The cooled extract was filtered, and the filtrate was added to the cation-exchange resin (2 kg). The extractÀresin mixture was shaken overnight at room temperature in a shaker (Shaking Baths SB-20, AS ONE, Osaka, Japan). The cation-exchange resin was then washed with 80% ethanol (5 L) and further with distilled water several times to remove impurities other than mimosine. Finally, the resin was eluted with 4 N NH4OH (6 L) to obtain mimosine. The eluate was vacuum concentrated, and the crude mimosine was further dissolved in NaOH solution. The pH of the solution was then adjusted to 4.5À5.0 by 6 N HCl and kept overnight in the Received: September 2, 2011 Revised: October 31, 2011 Accepted: November 2, 2011 Published: November 02, 2011 12858 dx.doi.org/10.1021/jf203494t | J. Agric. Food Chem. 2011, 59, 12858–12863 Neuraminidase Inhibitory Assay.6 mmol) and DIC (1.8 ( 0. The purified compounds were identified using LC-MS (ESIÀ): m/z [M À H]+ 693.1. M-FGY.108 g).9 ( 0. For kintetic studies.2 e 13. MO).1021/jf203494t |J. 59. Solid-Phase Synthesis of Mimosine Tetrapeptides. Preparation of Fmoc-Mimosine. b Different letters in the same column indicate the existence of significant difference (Tukey test. further purification was achieved using HPLC. 12858–12863 . and the fluorescence was measured using an MTP-880 fluorescence meter (Corona Electric.8 78.2 ( 0.5 ( 0.2) 80. and then it was incubated overnight at room temperature.2 a 7. 9-fluorenemethanol and 9-methylenefluorene. 12859 dx.2 ( 0.2 a 6.1. and the reaction mixtures were stirred for 17 h (see Figure 1A). and the major peaks were collected using a Cadenza CD-C18 (20  100 mm.2 ( 0. 554.5) noncompetitive (2. All of the crude peptides obtained were white solids. Fifty microliters of enzyme solution was added to 20 μL of an inhibitor mixed with 80 μL of acetate buffer in a microplate.9 ( 0. The resin was filtered and washed with TFA.4 ( 0.8 ( 0. M-FFY. Louis.5 g of Fmoc-OSu.org/10.4 ( 0.6) noncompetitive (7. The reaction mixture was further stirred for 1 h (see Figure 1B).1 mM in 50 mM sodium acetate buffer (pH 5.0 ( 0. The resins were filtered. The neuraminidase inhibition was done as reported previously. After deprotection of Fmoc with 25% piperidine (reagent a) in DMF for 30 min.3 g b inhibition type (Ki. The obtained precipitate was filtered out.2 c 7. respectively. 670. M-QGY.5) at a flow rate of 5 mL/min. 0.2. Following this.5:8. Scheme of mimosine tetrapeptide synthesis: attachment of Wang resin to Fmoc-amino acid (A).2 minÀ1) noncompetitive (2. The pH of the water fraction was lowered to 4 using 6 N HCl in an ice bath. washed with dichloromethane. Similarly. was used as the enzyme source.6 66. M-WGY.2 d 36. Bacterial neuraminidase from Clostridium perfringenes (Sigma). 300 mL of Na2CO3 solution (0. and then finally dried under vacuum. and 531.3 f 25. the next amino acids (Fmoc-AA2-OH) were coupled to the resin mixture solution (Fmoc-amino acid/HOBt/ HBTU/DIEA = 4:3:3.8 ( 0.1 ( 1. The obtained solution (450 mL) was filtered and washed with ARTICLE Figure 1. Japan).6 mmol) follwed by 10 min of stirring. elongation of amino acid chain to form Fmoc-mimosine tetrapeptides (B).1) noncompetitive (2.2. After final coupling with mimosine.1 ( 0.Journal of Agricultural and Food Chemistry refrigerator for crystallization. before the enzyme inhibition assays were performed.0). To this solution was added 12. and removal of Fmoc group to obtain desired mimosine tetrapeptides (C).5 42. Mimosine was identified by LC-MS (ESIÀ): m/z [M À H]+ 197.6:8) (reagent b). was used as the substrate.3 e yield (mg) a IC50 (μM) 9. 603.2) noncompetitive (1.3 c 9.0 ( 0. and the collected filtrate was precipitated with icecold diethyl ether. 2011. we Table 1.2 ( 0.1 ( 0.2 b 9. respectively.1 b 5.7 71. and the crystallized Fmoc-mimosine was filtered and dried under vacuum (7. St.1. the resin was agitated gently with 95% trifluoroacetic acid (TFA) (reagent k) for 1 h (see Figure 1C).2 c 9. and their yields are given in Table 1. IC50 Values of Mimosine and Mimosine Tetrapeptides neuraminidase inhibitor mimosine M-FFY M-FYY M-FWY M-VGY M-QGY M-WGY M-FGY M-HGY a tyrosinase IC50b (μM) 44. isopropyl alcohol.1) noncompetitive (2. 564.2 65.387 g (0.8 ( 0. μM) competitive (Km = 39. All of the inhibitors were dissolved in methanol and diluted to the appropriate concentrations in methanol. 4-methylumbelliferyl-1α-D-N-acetylneuramic acid sodium salt hydrate (Sigma.2) noncompetitive (2.1.1 e 11.1 U/mL in the acetate buffer. The crystals were filtered and further dried under vacuum to obtain 5.2 e 1.2 d 9.5 g of Na2CO3 were dissolved in 75 mL of distilled water containing 75 mL of dioxane. Reaction was started by adding 50 μL of substrate. 654.19 Briefly.1% TFA/ CH3CN (1.5 85. The uncoupled Fmoc-AA2-OH was capped with the acetyl group using a mixture of HOBt/acetic acid/DIEA/DMF (0. However. Agric.3 ( 0.4 66.3 c 16. 5 g of mimosine and 5.4 f 5.1 ( 0. 545. ethyl acetate (150 mL) to remove unreacted Fmoc-OSu and the byproducts. 3 μm) column with 0.2 a 2. and dried under vacuum to obtain the desired mimosine tetrapeptide.2 and [M + H]+ 199.6 mmol) in 5 mL of dimethylacetoamide was prepared by adding HOBt (1. To prepare Fmoc-mimosine.1 for M-FWY. and M-HGY. M-VGY. M-FYY.1 noncompetitive (2. A solution mixture of Fmoc-amino acid (Fmoc-AA1-OH) (1.2.01).8 ( 0.3 ( 0.54%) of mimosine with a purity of >95%.doi.7 ( 0.9 ( 0. This mixture was added to a swollen Wang resin (1 g) in DMF. p = 0. Food Chem. Coupling completeness was determined by ninhydrin test.2. The excitation and emission wavelengths were set at 360 and 450 nm. washed with diethyl ether (three times).1 M) was added and the mixture was further stirred in a magnetic stirrer (300 rpm) for 5 h at 25 °C. 0.4 Isolated pure crude yield.3 ( 0.6 ( 0.8:19:9:400) (20 mL/g resin). and methanol.7( 0. Fmoc-AA3-OH was coupled with the dipeptides to form tripeptides. 21 We further investigated the inhibitory mechanisms of mimosine at its IC50 concentration. all calculations were performed in Excel.1 ( 1. The IC50 value of mimosine against neuraminidase was 9. All statistical analyses were performed using SPSS version 16. and 20 μM. which is the same mode of inhibition against tyrosinase. 15. To obtain the effect of enzyme concentrations. when the effect of enzyme concentration was probed. Food Chem. (B) effect of enzyme concentration on neuraminidase inhibition [a typical plot of residual activity of neuraminidase at various concentrations (0À0. and the means were separated using Tukey’s HSD range test at p = 0. the data were analyzed by one-way ANOVA. Effect of preincubation time on hydrolysis of substrate by neuraminidase: (A) time-dependent inhibition of neuraminidase in the presence of 5 μM mimosine [(inset) decrease in slopes as a function of time].12-labdadiene-15. Agric. 20 μL of sample. Effect of mimosine on neuraminidase inhibition: LineweaverÀ Burk plot in the presence of mimosine at concentrations of 0. 12858–12863 .5. Microsoft Office 2007.16dial. and 36. 59.6-dehydrokawain. samples were dissolved in methanol to make different concentrations in micromolar.22 This led us to develop mimosine 12860 dx.Journal of Agricultural and Food Chemistry ARTICLE Figure 2. and kinetic studies revealed mimosine to inhibit the enzyme competitively (Km = 39. pH 6. 5.85 mM L-tyrosine solution to each well. We explored the effect of preincubation time on the inhibition of the hydrolysis of neuraminic acid. respectively. Furthermore. Because the decrease in residual activity was observed with increasing preincubation time.6 μM. which is also a slow and timedependent inhibitor of neuraminidase. For the time-dependent studies. we used different enzyme concentrations over a range of inhibitor concentrations. thereby indicating a reduction in both initial and steadystate velocity (see Figure 3A).18 The kinetic studies revealed that mimosine inhibited neuraminidase competitively (see Figure 2).doi. reaction was initiated by adding 20 μL of 0. The data represent the mean ( the standard deviation (SD) of six results. increasing the preincubation time of mimosine also led to a decrease in the slope.0 for Windows Vista. we first identified neuraminidase inhibition by mimosine.4 U/mL) in the presence of mimosine at 5 μM]. Moreover.org/10. All of the data were analyzed using Microsoft Excel Office. For significance analysis. The IC50 value indicated that mimosine is a better neuraminidase inhibitor than our previously isolated compounds. The 96-well plate was set up in the following order: 120 μL of phosphate buffer (20 mM. Statistical Analysis.2 μM. Synthesis of Mimosine Tetrapeptides and Their Neuraminidase Inhibitions.20 Briefly. and 20 μL of mushroom tyrosinase (500 units/mL in 20 mM buffer). used a time-driven protocol with initial velocity recorded over a range of substrate concentrations for different inhibitor concentrations.6. The IC50 value was determined graphically as the concentration of each sample required to give 50% inhibition activity. Ko-FFY. which had 50% inhibitory activities at 25. 2011. namely. it was found that the residual activity of neuraminidase increased with time at fixed substrate concentration (see Figure 3B). It has been reported that kojic acid (Ko) tripeptides (Ko-FWY. dihydro-5. Figure 3. To search for neuraminidase inhibitors from plant-based sources.2 minÀ1) (Table 1). mimosine emerged as a slow-binding inhibitor at low concentrations (see Figure 3A). For kinetic studies. The percentage of tyrosinase inhibition was calculated as % tyrosinase inhibition ¼ ½ðA À BÞ À ðC À Dފ=ðA À BÞ Â 100 where A and B are the absorbances of control (methanol) and test samples with enzyme.8 ( 0. After incubation at 25 °C for 15 min. ’ RESULTS AND DISCUSSION Neuraminidase Inhibitory Activity by Mimosine. and 8(17). and the slopes of lines obtained were plotted against preincubation time. The inhibitory effects of mimosine and peptides on tyrosinase enzyme were assayed as described previously. The enzyme activity was determined by measuring the absorbance at 470 nm using the microplate reader. 2007. The inhibition was calculated using % inhibition ¼ ½1 À ðS À S0 Þ=ðC À C0 ފ  100 where S and C represent relative fluorescence units (RFU) for sample and control after reaction time and S0 and C0 are RFU at zero time. 24. All of the experiments were conducted in triplicates and repeated twice. Neuraminidase inhibition was assayed as described in the text. Tyrosinase Inhibition.1021/jf203494t |J.6-dehydorkawain.8).01. 10. we obtained progress curves for 600 s at several preincubation times using 10 μM mimosine. Ko-FYY) have inhibited tyrosinase enzyme significantly. This result indicates that mimosine is more like the drug Tamiflu. whereas C and D are the absorbances of control and test samples without enzyme. respectively. M-FGY (G). and the dipeptides formed were further coupled with the next amino acid to form tripeptides. The synthesized compounds had significantly better inhibitions than mimosine (Table 1). phenylalanine (F). We synthesized a series of mimosine peptides.1021/jf203494t |J. glycine (G). To synthesize mimosine tetrapeptides. When the neuraminidase inhibitions by individual amino acids (30 μM) were probed. tetrapeptides using several amino acid combinations in order to develop a potent neuraminidase inhibitor. and M-FFY had 5 times more potency than mimosine. we applied solid-phase synthesis using Fmoc chemistry principles. M-FYY (B). However.Journal of Agricultural and Food Chemistry ARTICLE Figure 4. valine (V). which was achieved by attaching the Fmoc group of Fmoc-OSu to mimosine. 59. mimosine needs to be first converted to Fmoc-mimosine. 12861 dx. The first coupling was done with Y. Agric. to synthesize mimosine tetrapeptides. glutamine (Q).org/10.doi. M-QGY (E). Food Chem. and M-HGY (H) on neruaminidase inhibition: LineweaverÀBurk plot in the presence of 0À2 μM mimosine tetrapeptides. Although mimosine had competitive inhibitions. and histidine (H). using tyrosine (Y). tryptophan (W). 12858–12863 . M-FFY (C). The IC50 values of tetrapeptides were in low micromolar ranges. 2011. M-VGY (D). The low IC50 and Ki values of M-FFY make it a potent compound against neuraminidase. Effect of M-FWY (A). To the Fmoc-mimosine were coupled previously formed tripeptides to obtain the desired mimosine tetrapeptides. mimosine tetrapeptides exhibited noncompetitive inhibitions (see Figure 4). the activities identified were very poor (Table 2). M-WGY (F). Saeed. Tyrosinase Inhibition by Mimosine and Tetrapeptides. The results of this study certainly widen the use of solid-phase peptide synthesis in the development of potent neuraminidase inhibitors. N. Aagulska. Rani. 381. Khan. Drug Discovery 2007. 2006. Chem. J. Mol. Dermatol. Seasonal Changes of Mimosine Contents in Leucaena Leaves. 89–97. Nat. Li. Byron. Acta 1998.2 ( 3. The inhibition of tyrosinase enzyme by mimosine and tetrapeptides is shown in Table 1.. The results also showed that mimosine and most of the tetrapeptides are better inhibitors against neuraminidase enzyme.. Slowinska. 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It was found that the leaves had the highest amount of mimosine during the summer period of JulyÀSeptember. Med.. Engl. D. M. (6) Cavalieri. Hanuske.5 ( 2.23 It seems that the temperature of the environment may have an impact on mimosine content and that the higher temperatures favor more mimosine . T. These results suggest that peptides synthesized from mimosine may have applicability in the search for neuraminidase and tyrosinase inhibitors.1 7. 26..24 and. 353. 8. E. H.-M. Kobamoto. G. T. we are also synthesized mimosine di. Cancer 1973. 2000. F. Kuwano. Hanauske. S. I..doi.. E.. N. Nat. A.. G.and tripeptides to develop neuraminidase and/or tyrosinase inhibitors. Tawata. It was found that mimosine inhibited tyrosine activity with IC50 of 44. 2011. 1996. Mimosine has been associated with the allelopathy of the plant. therefore. Salgado. Agric. Tyrosinase and related proteins in mammalian pigmentation.4 μM. 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