Polymer Journal, Vol. 24, No. 5, pp 465---472 (1992) Liquid-Phase Synthesis and Cell Attachment Activity of Bioactive Pentapeptide YIGSR Yoshiaki HIRANO, Motoya OKUNO, Toshio HAYASHI,* and Akio NAKAJIMAt Department of Applied Chemistry, Osaka Institute of Technology, Ohmiya, Asahi-ku, Osaka 535, Japan *Research Center for Engineering, Kyoto University, Kawahara-cho, Sakyo-ku, Kyoto 606, Japan (Received October 3, 1991) ABSTRACT: Pentapeptide Tyr-Ile-Gly-Ser-Arg (YIGSR), an amino acid sequence existing in the cell attachment domain of the Bl chain of laminin, was synthesized by an improved liquid­ phase procedure. Bioactivity of YIGSR as cell recognition determinant was investigated using L-929 fibroblast cell and A431 epidermoid cell. The YIGSR pentapeptide was immobilized to ethylene-acrylic acid copolymer (PEA) film, and the number of cells attached to the immobilized film was counted. In addition, the cell-attachment activity of YIGSR toward the cells was also evaluated by the cell inhibition test. These two tests led to a conclusion that YIGSR acts as a cell determinant toward L-929 and A431 cells. KEY WORDS Tyr-Ile-Gly-Ser-Arg (YIGSR) Pentapeptide / Liquid-Phase Peptide Synthesis I Cell Attachment Activity I YIGSR-Immobilized Ethylene-Acrylic Acid Copolymer/ L-929 Fibroblast Cell/ A431 Epidermoid Cell/ Laminin, the major non-collagenous glyco­ cell-spreading and chemotaxis. In addition, protein distributing only in the basement Iwamoto and co-workers7 have demonstrated membrane, an extracellular matrix between that the synthetic pentapeptide YIGSR in­ epithelial cell and stroma, has various bio­ hibited the formation of metastasis by logical activities, such as adhesion, migration, malignant melanoma cells. In 1989, Murata differentiation, and growth, of cell. 1 •2 Laminin and co-workers10 reported that synthetic is composed of three chains designated as A, sequential polypeptide poly(YIGSR) drasti­ B I, and B2, which are arranged in a cross­ cally inhibited the metastatic formation of shaped structure. 3 These three chains of B16--BLI6 melanoma cells. laminin have been cloned and sequenced, and The aim of this work is to establish a several active sites in the molecule have been method to synthesize YIGSR pentapeptide in identified. 4 - 6 Iwamoto et al. 7 and Graf et al. 8 •9 satisfactory yields by an improved liquid­ suggested that the peptide sequence Cys-Asp­ phase procedure, and to elucidate the cell­ Pro-Gly-Tyr-Ile-Gly-Ser-Arg (CDPGYIGSR, attachment activity of the pentapeptide and in one letter amino acid code) in the B I chain of pentapeptide7immobilized polymer films. concerns with cell-attachment activity, and, The reason why we use the liquid-phase in particular, pentapeptide sequence YIGSR procedure for peptide synthesis is that we can plays important role for cell-attachment, obtain much more amounts of pentapeptide t To whom correspondence should be addressed. 465 Y. HIRANO eta/. in one cycle compared with the use of (Peptide Institute, Inc.) were used as deblock­ automatic peptide synthesizer. The polymer ing agents. Laminin sample used for coating used for immobilization is an ethylene-acrylic of cell culture dishes was purchased from Iwaki acid copolymer (PEA) containing 8.9mol% Glass, Co. All the solvents and chemicals used (20 wt%) acrylic acid. The cells used are were of reagent grade. fibroblast L-929 cell originating in mouse, and epidermoid A431 cell originating in human. Synthesis of Tyr-Ile-Gly-Ser-Arg ( YIGSR) by Liquid-Phase Procedure EXPERIMENTAL NG-Tosyi-L-arginine methyl ester (NH2- Arg(Tos)-0Me) was synthesized11 from Boc­ Materials Arg(Tos)-OH, and Boc-Tyr-0Su12 was from t-Butoxycarbonyl-L-tyrosine (Boc-Tyr), t­ Boc-Tyr-O H. Scheme of synthesis of YIGSR butoxycarbonyl-0-benzyl-L-serine (Boc­ by liquid-phase procedure is shown in Figure Ser(Bzl)), glycine methyl ester hydrochloride 1. Fragment condensation method was used. (Giy-OMe · HCl), L-isoleucine methyl ester First, Boc-Tyr-OSu and NHz-IIe-OMe were (Ile-OMe · HCl), were purchased from Koku­ coupled by N-succinimide procedure, secondly, san Chemical Works, Ltd., and t-butoxy­ Boc-Tyr-IIe-NHNH2 was coupled with NH2- carbonyl-N0-tosyl-L-arginine (Boc-Arg(Tos)) Gly-0Me by azide procedure. On one hand, was from Peptide Institute, Inc. Dicyclohexyl­ Boc-Ser(Bzl) was condensed with NH2-Arg­ carbodiimide (DCC) used as a dehydration (Tos)-0Me by DCC procedure. Finally, Boc­ agent, and N-hydroxysuccinimide (HOSu) Tyr-IIe-Gly-NHNH2 was coupled with NH2- were purchased from Peptide Institute, Inc. Ser(Bzl)-Arg(Tos)-0Me by azide procedure, Triethylamine (TEA) used as a neutralization and the product obtained was deblocked by agent, and hydroxybenzotriazole (HOBt) were saponification and TFMSA to obtain H-Tyr­ purchased from Katayama Chemicals, and IIe-Gly-Ser-Arg-OH. Kokusan Chemical Works, respectively. Tri­ Step 1: To 3.48g (1.9x 10-2mol) NH2-Ile- fiuoromethanesulfonic acid (TFMSA) (Kanto 0Me · HCl dissolved in 35 ml tetrahydrofuran Chemicals), and trifiuoroacetic acid (TF A) (THF), equimolar TEA was added. To the Tyr lle Gly Ser Arg Soc --osu NH2- f-OMe Mixed Soc OMe NH2NH2 Tos Soc NHNH2 NH 2- -OMe Soc- OH NH2-L OMe Azide 1/Szl DCC Tos Soc OMe Soc / OMe NH2NH2 Szl TFA Tos 1/ / Soc NHNH2 NH2 OMe Azide 1/Szl Tos Soc / OMe NoOH/MeOH 1/Szl Tos Soc OH TFMSA NH 2 OH Figure 1. Scheme of Tyr-Ile-Gly-Ser-Arg synthesis by liquid phase-procedure. 466 Polym. J., Vol. 24, No. 5, 1992 Synthesis and Cell-Attachment Activity of YIGSR solution obtained, 6.05 g (1.6 x 10-2 mol) Boc­ 5% citric acid and 5% NaHC03 solution. Tyr-OSu was added, and then the mixture kept Purity of the product was checked by TLC. alkaline by TEA was stirred at 4oC for 24h. Further, the extract was washed with NaCl­ After 24 h, the solution was filtered and the saturated water, dried over Na2S04 , and then filtrate was evaporated in high vacuum. The evaporated. Boc-Tyr-Ile-Gly-OMe (2.45 g) ob­ resulting precipitate was extracted with ethyl tained was dissolved in a small amount of acetate, and the extract was washed with 5% MeOH, to which 5 times the molar quantity citric acid and 5% sodium hydrogencarbonate of hydrazine hydrate was added, and the (NaHC03 ). Purity of the product was checked solution was kept at room temperature by using thin-layer chromatography (TLC; overnight. The gelatinous mass formed was Merck) (CHC1 3 : MeOH: H20 = 8: 3: 1). The collected by filtration, and the filtrate was extract was further washed with NaCI­ evaporated. The resulting solid was re­ saturated water, dried over Na2S04 and then crystallized from acetonitrile and vacuum evaporated. Boc-Tyr-Ile-OMe obtained was dried. Thus, Boc-Tyr-Ile-Gly-NHNH 2 (2.14 g; dissolved in small amounts of MeOH, to 0.46 x 10-2 mol) was obtained. which, 5 times the molar quantity of hydrazine Step 3: 2.95 g (1.0 x 10-2 mol) Boc-Ser(Bzl)­ hydrate was added, and the solution was OH dissolved in 50 ml THF was mixed with kept at room temperature overnight. The 1.2 times the molar quantity of DCC and gelatinous mass formed was collected by HOBt under stirring in an ice bath for 30 min. filtration and the filtrate was evaporated. The 4.53 g (1.2 x 10-2 mol) NH2-Arg(Tos)-0Me· resulting solid was recrystallized from acetoni­ HCl was dissolved in 40 ml DMF and trile, and vacuum dried. Thus, Boc-Tyr-Ile­ equimolar TEA was added. The Boc-Ser(Bzl)­ NHNH2 (3.92 g; 0.96 x 10-2 mol) was ob­ OH solution was mixed with the NH2- tained. Arg(Tos)-0Me· HCl solution in a manner as Step 2: At a low temperature cooled down mentioned above, and stirred at 4oc for 24 h. with ice and NaCl, 4N HCl-dioxane (5.8 ml) After 24 h, the Boc-Ser(Bzl)-Arg(Tos)-OMe and isoamylnitrile (1.3 ml) were added succes­ obtained was purified by the same method sively to a solution of 3.92 g (0.96 x 10-2 mol) mentioned in step 2. The resulting solid was Boc-Tyr-Ile-NHNH2 in 30ml dimethylforma­ dissolved in 5 ml anisole. TFA (1 0 ml) was mide (DMF), and the mixture was stirred for added to remove Boc group at a low tem­ 30 min, where the hydrazine test was carried perature cooled down in ice bath for 60 min. out. For hydrazine test, 1% FeC1 3 /2M acetic The solvent was evaporated, and the resulting acid and 1% K 3 [Fe(CN)6] aqueous solution solid was recrystallized from diethylether and mixture (1: 1) was used as a color-producing vacuum dried. Thus, solid NH2 -Ser(Bzl)­ reagent. The solution was neutralized with Arg(Tos)-0Me (2.80g; 0.54x 10- 2 mol) was TEA. 1.44 g (1.2 x 10-2 mol) NH2-Gly-0Me · obtained. HCl was dissolved in 20m! DMF, to which Step 4: At a low temperature cooled with equimolar TEA was added. Now, the Boc­ ice and NaCI, 4N HCl-dioxane (2.8 ml) and Tyr-Ile-NHNH2 solution was mixed with the isoamylnitrile (0. 7 ml) were added successively NH2-Gly-0Me · HCl solution, and stirred at to a solution of 2.14 g (0.46 x 10-2 mol) ooc for 24 h, whereby, the pH of the mixture Boc-Tyr-Ile-Gly-NHNH2 in 30ml DMF, and was kept alkaline. The mixture was neutralized the mixture was stirred for 30 min, while the with acetic acid, filtered and then the filtrate hydrazine test was carried out. The solution was evaporated in high vacuum.