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Amino Acids and Peptides
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
241, 595–598 (1997)
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Amino Acids and Peptides XXXII: A Bifunctional Poly(Ethylene Glycol) Hybrid of Fibronectin-Related Peptides Mitsuko Maeda, Yasuhiro Izuno, Koichi Kawasaki,1 Yoshihisa Kaneda,* Yu Mu,* Yasuo Tsutsumi,* Kenneth W. Lem,† and Tadanori Mayumi* Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe 651-21, Japan; *Faculty of Pharmaceutical Sciences, Osaka University, Yamadaoka 1-6, Suita 565, Japan; and †School of Pharmacy, University of California San Francisco, 3333 California Street, San Francisco, California 94118
Received October 28, 1997
An amino acid type poly(ethylene glycol) (aaPEG) was prepared and its application to a drug carrier was examined. The peptides, Arg-Gly-Asp (RGD) and GluIle-Leu-Asp-Val (EILDV) which were reported as active fragments of Fibronectin (a cell adhesion protein), were conjugated with aaPEG (molecular weight, 10,000). The hybrid, RGD-aaPEG-EILDV, was prepared by a combination of the solid-phase method and the solution method. Antiadhesive activity of the peptides was not lost by its hybrid formation with the large aaPEG molecule. A mixture of RGD (0.43 mmol) and EILDV (0.43 mmol) did not demonstrate an antiadhesive effect, but the hybrid containing 0.43 mmol of each peptide did exhibit this effect. q 1997 Academic Press Key Words: fibronectin; poly(ethylene glycol); poly(ethylene glycol) hybrid; peptide hybrid; drug carrier; antiadhesive effect.
Since poly(ethylene glycol) (PEG) has a low toxicity, a low immunogenicity, and good solubility in both aqueous and organic solvents, it appears to be a promising drug-carrier. Consequently, PEG hybrids have become the focus of attention in studies on drug delivery systems, and the term ‘‘pegylation’’ has become popular. Many studies on the pegylation of proteins (such as PEG-insulin,1a) PEG-asparginase1b), PEG-urokinase1c) and PEG-tumor necrosis factor a1d) have been reported, but few of them report on the pegylation of small peptides. The reason for this lack of reporting is because it was assumed that the modification of a small bioactive peptide with such a big molecule as PEG would result in a loss of activity of the small peptide. However, we 1 To whom reprint requests should be addressed. Fax: 81-78-9745689. E-mail: [email protected].
were successful in forming PEG hybrids of small peptides [such as Arg-Gly-Asp (RGD) derived from fibronectin2) and Tyr-Ile-Gly-Ser-Arg (YIGSR) derived from laminin3)] that resulted in the potentiation of activity of the parent peptides.4) The two hydroxyl groups situated at each terminal of commercially available PEG are the functional groups that can attach to drugs. Since PEGs with one hydroxyl group and one alkyl ether group at each terminal are also available, PEG hybrids can be produced with either one or two same drugs attached to PEG. We prepared biologically active PEG hybrids of laminin- and fibronectin-related peptides by coupling a peptide with an amino-PEG (aPEG).5) The resulting hybrid consisted of the same 2 peptides and 1 aaPEG as described above. Recently, Lu and Felix6) reported that multiple pegylated peptides and site-directed pegylated peptides exhibit enhanced biological activity. We planned to prepare a multi-functional PEG-peptide hybrid (such as --W-PEG-X-PEG-Y-PEG-Z. W, X, Y, Z : different functional peptides) by conjugating different bioactive peptides with PEGs. As a first step for preparation of a multi-functional peptide-PEG conjugate, we prepared a bifunctional peptide-PEG hybrid [X-PEG-Y (X,Y:different bioactive peptides)] using an amino acid type PEG (aaPEG) which was prepared from aminopropyl-PEG [poly(oxyethylene)dipropylamine, apPEG, average molecular weight 10,000]. Fibronectin is a cell adhesion protein consisting of A and B peptide chains (Fig. 1).7) Each chain contains the Arg-Gly-Asp (RGD) sequence2) which was found to be the critical sequence for cell attachment activity. In addition to this RGD sequence, a Glu-Ile-Leu-Asp-Val (EILDV) sequence8) was found to be another requisite sequence in the IIICS domain of the A chain for cell attachment. Our method included the preparation of a peptide-PEG hybrid containing these two functional sequences.
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MATERIALS AND METHODS The apPEG was purchased from Wako Pure Chemical Industries, Ltd., and p-Methyl-benzhydrylamine resin, Rink resin, and coupling reagents from Watanabe Chemical Industries, Ltd. Amino acid compositions of acid hydrolysates were determined with a Kyowa K202SN amino acid analyzer. Synthetic hybrids were hydrolyzed in 6N HCl at 1107C for 48 h. The RP-HPLC was conducted with a Waters 600 on a YMC Pack AQ-ODS-5 column using gradient systems of CH3CN/H2O containing 0.1 % TFA. t-Butoxycabonyl-aaPEG (Boc-aaPEG-OH). aaPEG was prepared from apPEG by succinylation with succinic anhydride (equal molar to that of apPEG). The resultant aaPEG was purified by a Sephadex G-25 column and a AG1x4 column. The t-Butoxycarbonyl-aaPEG (Boc-aaPEG-OH) was obtained from a reaction of aaPEG with di-tbutyl dicarbonate. The product was purified via Sephadex G-25 column chromatography. Yield 45% (calculated from apPEG), fluffy powder after lyophilization from water. Rf 0.44 (CHCl3/MeOH/water; 8/3/1. lower phase), H1-NMR (CDCl3) d 1.47(t-butyl). Boc-Gly-Asp(OcHx)-OPac. Na-t-Butoxycarbonylglycine(Boc-GlyOH) and a-phenacyl-b-cyclohexyl-aspartate (H-Asp(OcHx)-OPac) were coupled by the benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) method.9) This product was purified by silica gel chromatography using a mixture of CHCl3/MeOH as an eluent. Yield 25%, mp 1007C, Rf 0.79 (ethyl acetate/benzene, 24 01.97 (cÅ1.0,MeOH). Amino acid ratio in an acid hydroly1/1), [a]D sate: Gly 1.00, Asp 1.02 (average recovery 97%). Anal. Calcd. for C25H33N2O8:C,61.33; H,6.81; N,5.72. Found: C,61.15; H,6.97;N,5.61. Boc-Arg(Tos)-Gly-Asp(OcHx)-OPac. Boc-Gly-Asp(OcHx)-OPac was treated with trifluoroacetic acid(TFA) to remove the Boc group. The resultant H-Gly-Asp(OcHx)-OPac was coupled with Na-Boc-NGtosylarginine [Boc-Arg(Tos)-OH] by the BOP method. The product was purified by silica gel column chromatography using an ethyl acetate/n-hexane mixture as an eluent. Yield 73%, mp 84-927C, Rf 24 0.87 (ethyl acetate/benzene, 1/1), [a]D 01.17 (cÅ1.0, MeOH). Amino Acid ratios in an acid hydrolysate: Arg 0.95, Gly 1.00, Asp 0.95 (average recovery 91%). Anal. Calcd. for C38H51N6O11Sr1/2H2O: C,56.41; H,6.49; N,10.39. Found: C,56.51; H,6.77; N,10.54. Boc-Arg(Tos)-Gly-Asp(OcHx)-OH. Boc-Arg(Tos)-Gly-Asp(OcHx)OPac was treated with zinc powder in 90% AcOH for 2 h at 07C and 2 h at room temperature. Zinc was removed by filtration and the solvent was evaporated off. The product was extracted with ethyl acetate and purified by precipitation from ethyl acetate/petroleum ether. Yield 70%, mp 68-757C, Rf 0.78 (ethyl acetate/benzene, 1/1), 24 [a]D 01.57 (cÅ1.0, MeOH). Amino acid ratios in an acid hydrolysate: Arg 0.94, Gly 1.00, Asp 0.94 (average recovery 88%). Anal. Calcd. for C30H45N6O9SrH2O: C,53.96; H,7.09; N,12.56. Found: C,53.77; H, 7.18; N,12.29. RGD-aaPEG-EILDV. Na -Fluorenylmethyloxycarbonylvaline (Fmoc-Val-OH), Fmoc-b-cyclohexyl-aspartate (Fmoc-Asp(OcHx)-OH), Fmoc-Leu-OH and Fmoc-Ile-OH was introduced on Rink resin in a stepwise manner by the diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) method.10) Removal of the Fmoc group was performed by 20% piperidine/DMF treatment. Na-t-butoxycarbonyl-
g-cyclohexyl glutamate[Boc-Glu(OcHx)-OH] was introduced on the peptide-resin and the resultant Boc-Glu(OcHx)-Ile-Leu-Asp(OcHx)Val-resin was treated with TFA to generate the pentapeptide, HGlu(OcHx)-Ile-Leu-Asp(OcHx)-Val-NH2 . The product exhibited 90% purity by HPLC, and it was used for the next reaction without purification. The pentapeptide was coupled with Boc-aaPEG-OH by the BOP method to produce the Boc-aaPEG-pentapeptide. The Boc group was subsequently removed by TFA treatment to give H-aaPEG-Glu(OcHx)-Ile-Leu-Asp(OcHx)-Val-NH2 . Boc-Arg(Tos)-Gly-Asp(OcHx)OH was coupled with the H-aaPEG-pentapeptide to give BocArg(Tos)-Gly-Asp(OcHx)-aaPEG-Glu(OcHx)-Ile-Leu-Asp(OcHx)-ValNH2 . The product was treated with HF to remove all protecting groups and purified by RP-HPLC to give RGD-aaPEG-EILDV. Amino acid ratios in an acid hydrolysate: Arg 0.87; Gly 1.00; Asp 2.04; Glu 1.00; Ile 0.96; Leu 1.01; Val 1.04. Peptide content calculated from an average recovery of each amino acid by an amino acid analysis of an acid hydrolysate: 0.09 mmol/g. Antiadhesive assay. 96 well plates were coated with fibronectin in PBS(-) (3 mg/ml) at room temperature for 2 h. After blocking with PBS(-) and supplemented with 1% heat-denatured (807C for 5 min) BSA for 60 min, pre-diluted samples in MEM containing 0.05% BSA (50 ml/well) were added to a 96-well microplate. This assay was studied in tetraplicate wells. B16-BL6 melanoma (5 1 104 cells) were admixed in wells, and incubated at 377C for 1 h under 5% CO2/air. Thereafter, adherent cells were fixed with 10% formalin in a neutral buffer. The fixed cells were then stained with 0.05% methylene blue in PBS(-). Following a washing with water, 200 ml of 0.3N HCl was added to each well, and the absorbance at 655 nm was measured.
RESULTS AND DISCUSSIONS Boc-aaPEG-OH was prepared from apPEG by succinylation with equimolar succinic anhydride, followed by the introduction of the Boc group with di-t-butyldicarbonate as shown in Fig. 2. The hybrid was prepared by a combination of the solid-phase method and the solution methods as shown in Fig. 3. First, we tried to prepare RGD-aaPEG-EILDV by the solid-phase method. H-Glu(OcHx)-Ile-Leu-Asp(OcHx)-Val-( p-methylbenzhydryl resin) was prepared by the Na-Boc strategy and was coupled with BocaaPEG-OH. Neither the DIC/HOBt, BOP nor O-(7azabenzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HATU)11) methods were successful in introducing Boc-aaPEG-OH onto the peptide-resin (EILDV-resin). We speculated that Boc-aaPEG-OH (molecular weight, 10,000) was too large to react with the bulky peptide-resin, EILDV-resin. Therefore, we synthesized the hybrid, RGD-aaPEG-EILDV, by combining the solid-phase method with the solution method as shown
FIG. 1. Structure of the fibronectin A chain. 596
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FIG. 2. Preparation of Boc-aaPEG-OH.
in Fig. 3. H-Ile-Leu-Asp(OcHx)-Val-[Rink resin] was prepared by employing the Na-Fmoc strategy. Boc-Glu(OcHx)-OH was introduced onto the peptide resin, and the product was treated with TFA to cleave the peptide portion from the resin. The product, H-Glu(OcHx)-IleLeu-Asp(OcHx)-Val-NH2 , was coupled with Boc-aaPEGOH by the BOP method in a solution to produce BocaaPEG - Glu(OcHx) - Ile - Leu - Asp(OcHx) - Val - NH2 . Although the coupling reaction of Boc-aaPEG-OH with the peptide-resin failed as described above, we were successful in coupling them by using the solution method. The Boc group on the product was removed by TFA treatment to yield H-aaPEG-Glu(OcHx)-Ile-Leu-Asp(OcHx)Val-NH2 . The protected N-terminal tripeptide was prepared by the solution method. The a-carboxyl group of Asp was protected by the phenacyl group (-OPac), and the syn-
thetic protected tripeptide (Boc-Arg(Tos)-Gly-Asp(OcHx)-OPac) was treated with Zinc to remove the Pac group. The resulting tripeptide was coupled with the HaaPEG-Glu(OcHx)-Ile-Leu-Asp(OcHx)-Val-NH2 by the BOP method to give Boc-Arg(Tos)-Gly-Asp(OcHx)aaPEG-Glu(OcHx)-Ile-Leu-Asp(OcHx)-Val-NH2 . Subsequently, all protecting groups of the peptide were removed by HF treatment and the product, RGDaaPEG-EILDV, was purified by RP-HPLC. The peptide content of the hybrid calculated from an average recovery of amino acids in an acid hydrolysate was 0.09 mmol/g. Lu and Felix12) suggested that a partial degradation of PEG might occur by HF treatment so we studied the stability of PEG by subjecting it to HF treatment and reported it elsewhere.13) We proved that HF treatment at 07C for 1 h is applicable for deprotection of PEGcontaining material. Antiadhesive activity of the hybrid and related peptides is shown in Fig. 4. RGD (0.36 mg, 0.86 mmol)/ml) did not show this activity, but EILDV (0.54 mg, 0.86 mmol) did. A mixture of RGD (0.18 mg, 0.43 mmol) and EILDV (0.27 mg, 0.43 mmol) also exhibited this activity. The hybrid, RGD-aaPEG-EILDV, at a concentration of 4.7 mg (0.43 mmol)/ml was approximately equivalent to 0.54 mg (0.86 mmol)/ml of EILDV from an in vitro assay. The amount of the hybrid amounted to just half of the EILDV in terms of molar ratio. It is interesting to note that the hybrid formation with such
FIG. 3. Synthetic scheme of RGD-aaPEG-EILDV. DIC, diisopropylcarbodiimide. cHx, cyclohexyl. TFA, trifluoroacetic acid. BOP, benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate. Pac, phenacyl. 597
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FIG. 4. Antiadhesive activity of RGD-aaPEG-EILDV and its related peptides on fibronectin. B16-BL6 melanoma cells and peptides were admixed in fibronectin-coated wells, and incubated at 377C for 1 h under 5% CO2/air. The adherent cells were fixed and stained with 0.05% methylene blue in PBS(-). After dissolving the stained cells, the absorbance of each well at 655 nm was measured. The assay was studied in tetraplicate wells. Each value represents mean { S.E.
saki, Y., Wada, H., Sako, M., Tsujino, G., and Inada, Y. (1986) Jpn. J. Cancer Res.(Gann) 77, 1264–1270; (c) Sakuragawa, N., Shimizu, K., Kondo, K., Kondo, S., and Miwa, M. (1986) Thromb. Res. 41, 627–635; (d) Tsutsumi, Y., Kihara, T., Yamamoto, S., Kubo, K., Nakagawa, S., Miyake, M., Horisawa, Y., Kanamori, T., Ikegami, H., and Mayumi, T. (1994) Jpn. J. Cancer Res. 85, 9–12.
a large molecule as aaPEG (molecular weight, 10,000) did not result in a loss of activity of the parent peptide. Furthermore, although the mixture of RGD (0.43 mmol) and EILDV (0.43 mmol) failed to show the activity, the hybrid containing 0.43 mmol of each peptide did. The antiadhesive effect of the parent peptides might be enhanced by the hybrid formation with aaPEG. Our objective was to prepare multifunctional PEGpeptide hybrids, and, our first step of preparing a bifunctional PEG-peptide hybrid was successful. Although any remarkable potentiation of the activity of the parent peptides was not observed in this experiment, we proved that preparing a multifunctional PEG-peptide hybrid using aaPEG is possible. Generally, bioactive proteins possess some functional groups (an active site, a binding site, etc.) which co-operate when exhibiting biological activity. There is a good possibility that a multifunctional PEG-peptide hybrid(W-aaPEG-X-aaPEG-Y-aaPEG-Z, whereof W, X, Y and Z are functional peptides) can be prepared as a protein model.
2. Pierschbacher, M. D., and Ruoslahti, E. (1984) Nature 309, 30–33. 3. Iwamoto, Y., Robey, F. A., Graf, J., Sasaki, M., Kleinman, H. K., Yamada, Y., and Martin, G. R. (1987) Science, 238, 1132–1134. 4. (a) Kawasaki, K., Namikawa, M., Murakami, T., Mizuta, T., Iwai, Y., Hama, T., and Mayumi, T. (1991) Biochem. Biophys. Res. Commun. 174, 1159–1162; (b) Kawasaki, K., Namikawa, M., Yamashiro, Y., Hama, T., and Mayumi, T. (1991) Chem. Pharm. Bull. 39, 3373–3375. 5. Pillai, V. N. R., and Mutter, M.(1980) J. Org. Chem. 45, 5364– 5370. 6. (a)Lu, Y., and Felix, A. M. (1994) Reactive Polymer, 22, 221–229. (b) Felix, M., Lu, Y., and Campbell, R. T. (1995) Int. J. Peptide Protein Res. 46, 253–264. 7. Hynes, R., and Yamada, K. (1982) J. Cell. Biol. 95, 369–377. 8. Humphries, M. J., Mervic, M., Yamada, K. M., and Komoriya, A. (1988) J. Cell Biol. 107, 384a. 9. Castro, B., Dormoy, J. R., Evin, G., and Selve, C. (1975) Tetrahedron Lett. No.14; 1219–1222.
ACKNOWLEDGMENTS This work was supported in part by Grand-in-Aid from the Japanese Ministry of Education, Science and Culture and by a grant from The Health Science Fund of Kobe Gakuin University.
10. Hudson, D. (1988) J. Org. Chem. 53, 617–624. 11. Carpino, L. A. (1993) J. Am. Chem. Soc. 115, 4397–4398. 12. Lu, Y., and Felix, A. M. (1993) Peptide Res. 6, 140–146.
REFERENCES 1. (a) Ehrat, M., and Luisi, P.L. (1983) Biopolymer 22, 569–573; (b) Yoshimoto, T., Nishimura, H., Saito, Y., Sakurai, K., Kami-
13. Maeda, M., Izuno, Y., Kawasaki, K., Kaneda, Y., Mu, Y., Tsutsumi, Y., Nakagawa, S., and Mayumi, T. (1997) Chem. Pharm. Bull., in press.
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241, 595–598 (1997)
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Amino Acids and Peptides XXXII: A Bifunctional Poly(Ethylene Glycol) Hybrid of Fibronectin-Related Peptides Mitsuko Maeda, Yasuhiro Izuno, Koichi Kawasaki,1 Yoshihisa Kaneda,* Yu Mu,* Yasuo Tsutsumi,* Kenneth W. Lem,† and Tadanori Mayumi* Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe 651-21, Japan; *Faculty of Pharmaceutical Sciences, Osaka University, Yamadaoka 1-6, Suita 565, Japan; and †School of Pharmacy, University of California San Francisco, 3333 California Street, San Francisco, California 94118
Received October 28, 1997
An amino acid type poly(ethylene glycol) (aaPEG) was prepared and its application to a drug carrier was examined. The peptides, Arg-Gly-Asp (RGD) and GluIle-Leu-Asp-Val (EILDV) which were reported as active fragments of Fibronectin (a cell adhesion protein), were conjugated with aaPEG (molecular weight, 10,000). The hybrid, RGD-aaPEG-EILDV, was prepared by a combination of the solid-phase method and the solution method. Antiadhesive activity of the peptides was not lost by its hybrid formation with the large aaPEG molecule. A mixture of RGD (0.43 mmol) and EILDV (0.43 mmol) did not demonstrate an antiadhesive effect, but the hybrid containing 0.43 mmol of each peptide did exhibit this effect. q 1997 Academic Press Key Words: fibronectin; poly(ethylene glycol); poly(ethylene glycol) hybrid; peptide hybrid; drug carrier; antiadhesive effect.
Since poly(ethylene glycol) (PEG) has a low toxicity, a low immunogenicity, and good solubility in both aqueous and organic solvents, it appears to be a promising drug-carrier. Consequently, PEG hybrids have become the focus of attention in studies on drug delivery systems, and the term ‘‘pegylation’’ has become popular. Many studies on the pegylation of proteins (such as PEG-insulin,1a) PEG-asparginase1b), PEG-urokinase1c) and PEG-tumor necrosis factor a1d) have been reported, but few of them report on the pegylation of small peptides. The reason for this lack of reporting is because it was assumed that the modification of a small bioactive peptide with such a big molecule as PEG would result in a loss of activity of the small peptide. However, we 1 To whom reprint requests should be addressed. Fax: 81-78-9745689. E-mail: [email protected].
were successful in forming PEG hybrids of small peptides [such as Arg-Gly-Asp (RGD) derived from fibronectin2) and Tyr-Ile-Gly-Ser-Arg (YIGSR) derived from laminin3)] that resulted in the potentiation of activity of the parent peptides.4) The two hydroxyl groups situated at each terminal of commercially available PEG are the functional groups that can attach to drugs. Since PEGs with one hydroxyl group and one alkyl ether group at each terminal are also available, PEG hybrids can be produced with either one or two same drugs attached to PEG. We prepared biologically active PEG hybrids of laminin- and fibronectin-related peptides by coupling a peptide with an amino-PEG (aPEG).5) The resulting hybrid consisted of the same 2 peptides and 1 aaPEG as described above. Recently, Lu and Felix6) reported that multiple pegylated peptides and site-directed pegylated peptides exhibit enhanced biological activity. We planned to prepare a multi-functional PEG-peptide hybrid (such as --W-PEG-X-PEG-Y-PEG-Z. W, X, Y, Z : different functional peptides) by conjugating different bioactive peptides with PEGs. As a first step for preparation of a multi-functional peptide-PEG conjugate, we prepared a bifunctional peptide-PEG hybrid [X-PEG-Y (X,Y:different bioactive peptides)] using an amino acid type PEG (aaPEG) which was prepared from aminopropyl-PEG [poly(oxyethylene)dipropylamine, apPEG, average molecular weight 10,000]. Fibronectin is a cell adhesion protein consisting of A and B peptide chains (Fig. 1).7) Each chain contains the Arg-Gly-Asp (RGD) sequence2) which was found to be the critical sequence for cell attachment activity. In addition to this RGD sequence, a Glu-Ile-Leu-Asp-Val (EILDV) sequence8) was found to be another requisite sequence in the IIICS domain of the A chain for cell attachment. Our method included the preparation of a peptide-PEG hybrid containing these two functional sequences.
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MATERIALS AND METHODS The apPEG was purchased from Wako Pure Chemical Industries, Ltd., and p-Methyl-benzhydrylamine resin, Rink resin, and coupling reagents from Watanabe Chemical Industries, Ltd. Amino acid compositions of acid hydrolysates were determined with a Kyowa K202SN amino acid analyzer. Synthetic hybrids were hydrolyzed in 6N HCl at 1107C for 48 h. The RP-HPLC was conducted with a Waters 600 on a YMC Pack AQ-ODS-5 column using gradient systems of CH3CN/H2O containing 0.1 % TFA. t-Butoxycabonyl-aaPEG (Boc-aaPEG-OH). aaPEG was prepared from apPEG by succinylation with succinic anhydride (equal molar to that of apPEG). The resultant aaPEG was purified by a Sephadex G-25 column and a AG1x4 column. The t-Butoxycarbonyl-aaPEG (Boc-aaPEG-OH) was obtained from a reaction of aaPEG with di-tbutyl dicarbonate. The product was purified via Sephadex G-25 column chromatography. Yield 45% (calculated from apPEG), fluffy powder after lyophilization from water. Rf 0.44 (CHCl3/MeOH/water; 8/3/1. lower phase), H1-NMR (CDCl3) d 1.47(t-butyl). Boc-Gly-Asp(OcHx)-OPac. Na-t-Butoxycarbonylglycine(Boc-GlyOH) and a-phenacyl-b-cyclohexyl-aspartate (H-Asp(OcHx)-OPac) were coupled by the benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) method.9) This product was purified by silica gel chromatography using a mixture of CHCl3/MeOH as an eluent. Yield 25%, mp 1007C, Rf 0.79 (ethyl acetate/benzene, 24 01.97 (cÅ1.0,MeOH). Amino acid ratio in an acid hydroly1/1), [a]D sate: Gly 1.00, Asp 1.02 (average recovery 97%). Anal. Calcd. for C25H33N2O8:C,61.33; H,6.81; N,5.72. Found: C,61.15; H,6.97;N,5.61. Boc-Arg(Tos)-Gly-Asp(OcHx)-OPac. Boc-Gly-Asp(OcHx)-OPac was treated with trifluoroacetic acid(TFA) to remove the Boc group. The resultant H-Gly-Asp(OcHx)-OPac was coupled with Na-Boc-NGtosylarginine [Boc-Arg(Tos)-OH] by the BOP method. The product was purified by silica gel column chromatography using an ethyl acetate/n-hexane mixture as an eluent. Yield 73%, mp 84-927C, Rf 24 0.87 (ethyl acetate/benzene, 1/1), [a]D 01.17 (cÅ1.0, MeOH). Amino Acid ratios in an acid hydrolysate: Arg 0.95, Gly 1.00, Asp 0.95 (average recovery 91%). Anal. Calcd. for C38H51N6O11Sr1/2H2O: C,56.41; H,6.49; N,10.39. Found: C,56.51; H,6.77; N,10.54. Boc-Arg(Tos)-Gly-Asp(OcHx)-OH. Boc-Arg(Tos)-Gly-Asp(OcHx)OPac was treated with zinc powder in 90% AcOH for 2 h at 07C and 2 h at room temperature. Zinc was removed by filtration and the solvent was evaporated off. The product was extracted with ethyl acetate and purified by precipitation from ethyl acetate/petroleum ether. Yield 70%, mp 68-757C, Rf 0.78 (ethyl acetate/benzene, 1/1), 24 [a]D 01.57 (cÅ1.0, MeOH). Amino acid ratios in an acid hydrolysate: Arg 0.94, Gly 1.00, Asp 0.94 (average recovery 88%). Anal. Calcd. for C30H45N6O9SrH2O: C,53.96; H,7.09; N,12.56. Found: C,53.77; H, 7.18; N,12.29. RGD-aaPEG-EILDV. Na -Fluorenylmethyloxycarbonylvaline (Fmoc-Val-OH), Fmoc-b-cyclohexyl-aspartate (Fmoc-Asp(OcHx)-OH), Fmoc-Leu-OH and Fmoc-Ile-OH was introduced on Rink resin in a stepwise manner by the diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) method.10) Removal of the Fmoc group was performed by 20% piperidine/DMF treatment. Na-t-butoxycarbonyl-
g-cyclohexyl glutamate[Boc-Glu(OcHx)-OH] was introduced on the peptide-resin and the resultant Boc-Glu(OcHx)-Ile-Leu-Asp(OcHx)Val-resin was treated with TFA to generate the pentapeptide, HGlu(OcHx)-Ile-Leu-Asp(OcHx)-Val-NH2 . The product exhibited 90% purity by HPLC, and it was used for the next reaction without purification. The pentapeptide was coupled with Boc-aaPEG-OH by the BOP method to produce the Boc-aaPEG-pentapeptide. The Boc group was subsequently removed by TFA treatment to give H-aaPEG-Glu(OcHx)-Ile-Leu-Asp(OcHx)-Val-NH2 . Boc-Arg(Tos)-Gly-Asp(OcHx)OH was coupled with the H-aaPEG-pentapeptide to give BocArg(Tos)-Gly-Asp(OcHx)-aaPEG-Glu(OcHx)-Ile-Leu-Asp(OcHx)-ValNH2 . The product was treated with HF to remove all protecting groups and purified by RP-HPLC to give RGD-aaPEG-EILDV. Amino acid ratios in an acid hydrolysate: Arg 0.87; Gly 1.00; Asp 2.04; Glu 1.00; Ile 0.96; Leu 1.01; Val 1.04. Peptide content calculated from an average recovery of each amino acid by an amino acid analysis of an acid hydrolysate: 0.09 mmol/g. Antiadhesive assay. 96 well plates were coated with fibronectin in PBS(-) (3 mg/ml) at room temperature for 2 h. After blocking with PBS(-) and supplemented with 1% heat-denatured (807C for 5 min) BSA for 60 min, pre-diluted samples in MEM containing 0.05% BSA (50 ml/well) were added to a 96-well microplate. This assay was studied in tetraplicate wells. B16-BL6 melanoma (5 1 104 cells) were admixed in wells, and incubated at 377C for 1 h under 5% CO2/air. Thereafter, adherent cells were fixed with 10% formalin in a neutral buffer. The fixed cells were then stained with 0.05% methylene blue in PBS(-). Following a washing with water, 200 ml of 0.3N HCl was added to each well, and the absorbance at 655 nm was measured.
RESULTS AND DISCUSSIONS Boc-aaPEG-OH was prepared from apPEG by succinylation with equimolar succinic anhydride, followed by the introduction of the Boc group with di-t-butyldicarbonate as shown in Fig. 2. The hybrid was prepared by a combination of the solid-phase method and the solution methods as shown in Fig. 3. First, we tried to prepare RGD-aaPEG-EILDV by the solid-phase method. H-Glu(OcHx)-Ile-Leu-Asp(OcHx)-Val-( p-methylbenzhydryl resin) was prepared by the Na-Boc strategy and was coupled with BocaaPEG-OH. Neither the DIC/HOBt, BOP nor O-(7azabenzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HATU)11) methods were successful in introducing Boc-aaPEG-OH onto the peptide-resin (EILDV-resin). We speculated that Boc-aaPEG-OH (molecular weight, 10,000) was too large to react with the bulky peptide-resin, EILDV-resin. Therefore, we synthesized the hybrid, RGD-aaPEG-EILDV, by combining the solid-phase method with the solution method as shown
FIG. 1. Structure of the fibronectin A chain. 596
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FIG. 2. Preparation of Boc-aaPEG-OH.
in Fig. 3. H-Ile-Leu-Asp(OcHx)-Val-[Rink resin] was prepared by employing the Na-Fmoc strategy. Boc-Glu(OcHx)-OH was introduced onto the peptide resin, and the product was treated with TFA to cleave the peptide portion from the resin. The product, H-Glu(OcHx)-IleLeu-Asp(OcHx)-Val-NH2 , was coupled with Boc-aaPEGOH by the BOP method in a solution to produce BocaaPEG - Glu(OcHx) - Ile - Leu - Asp(OcHx) - Val - NH2 . Although the coupling reaction of Boc-aaPEG-OH with the peptide-resin failed as described above, we were successful in coupling them by using the solution method. The Boc group on the product was removed by TFA treatment to yield H-aaPEG-Glu(OcHx)-Ile-Leu-Asp(OcHx)Val-NH2 . The protected N-terminal tripeptide was prepared by the solution method. The a-carboxyl group of Asp was protected by the phenacyl group (-OPac), and the syn-
thetic protected tripeptide (Boc-Arg(Tos)-Gly-Asp(OcHx)-OPac) was treated with Zinc to remove the Pac group. The resulting tripeptide was coupled with the HaaPEG-Glu(OcHx)-Ile-Leu-Asp(OcHx)-Val-NH2 by the BOP method to give Boc-Arg(Tos)-Gly-Asp(OcHx)aaPEG-Glu(OcHx)-Ile-Leu-Asp(OcHx)-Val-NH2 . Subsequently, all protecting groups of the peptide were removed by HF treatment and the product, RGDaaPEG-EILDV, was purified by RP-HPLC. The peptide content of the hybrid calculated from an average recovery of amino acids in an acid hydrolysate was 0.09 mmol/g. Lu and Felix12) suggested that a partial degradation of PEG might occur by HF treatment so we studied the stability of PEG by subjecting it to HF treatment and reported it elsewhere.13) We proved that HF treatment at 07C for 1 h is applicable for deprotection of PEGcontaining material. Antiadhesive activity of the hybrid and related peptides is shown in Fig. 4. RGD (0.36 mg, 0.86 mmol)/ml) did not show this activity, but EILDV (0.54 mg, 0.86 mmol) did. A mixture of RGD (0.18 mg, 0.43 mmol) and EILDV (0.27 mg, 0.43 mmol) also exhibited this activity. The hybrid, RGD-aaPEG-EILDV, at a concentration of 4.7 mg (0.43 mmol)/ml was approximately equivalent to 0.54 mg (0.86 mmol)/ml of EILDV from an in vitro assay. The amount of the hybrid amounted to just half of the EILDV in terms of molar ratio. It is interesting to note that the hybrid formation with such
FIG. 3. Synthetic scheme of RGD-aaPEG-EILDV. DIC, diisopropylcarbodiimide. cHx, cyclohexyl. TFA, trifluoroacetic acid. BOP, benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate. Pac, phenacyl. 597
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FIG. 4. Antiadhesive activity of RGD-aaPEG-EILDV and its related peptides on fibronectin. B16-BL6 melanoma cells and peptides were admixed in fibronectin-coated wells, and incubated at 377C for 1 h under 5% CO2/air. The adherent cells were fixed and stained with 0.05% methylene blue in PBS(-). After dissolving the stained cells, the absorbance of each well at 655 nm was measured. The assay was studied in tetraplicate wells. Each value represents mean { S.E.
saki, Y., Wada, H., Sako, M., Tsujino, G., and Inada, Y. (1986) Jpn. J. Cancer Res.(Gann) 77, 1264–1270; (c) Sakuragawa, N., Shimizu, K., Kondo, K., Kondo, S., and Miwa, M. (1986) Thromb. Res. 41, 627–635; (d) Tsutsumi, Y., Kihara, T., Yamamoto, S., Kubo, K., Nakagawa, S., Miyake, M., Horisawa, Y., Kanamori, T., Ikegami, H., and Mayumi, T. (1994) Jpn. J. Cancer Res. 85, 9–12.
a large molecule as aaPEG (molecular weight, 10,000) did not result in a loss of activity of the parent peptide. Furthermore, although the mixture of RGD (0.43 mmol) and EILDV (0.43 mmol) failed to show the activity, the hybrid containing 0.43 mmol of each peptide did. The antiadhesive effect of the parent peptides might be enhanced by the hybrid formation with aaPEG. Our objective was to prepare multifunctional PEGpeptide hybrids, and, our first step of preparing a bifunctional PEG-peptide hybrid was successful. Although any remarkable potentiation of the activity of the parent peptides was not observed in this experiment, we proved that preparing a multifunctional PEG-peptide hybrid using aaPEG is possible. Generally, bioactive proteins possess some functional groups (an active site, a binding site, etc.) which co-operate when exhibiting biological activity. There is a good possibility that a multifunctional PEG-peptide hybrid(W-aaPEG-X-aaPEG-Y-aaPEG-Z, whereof W, X, Y and Z are functional peptides) can be prepared as a protein model.
2. Pierschbacher, M. D., and Ruoslahti, E. (1984) Nature 309, 30–33. 3. Iwamoto, Y., Robey, F. A., Graf, J., Sasaki, M., Kleinman, H. K., Yamada, Y., and Martin, G. R. (1987) Science, 238, 1132–1134. 4. (a) Kawasaki, K., Namikawa, M., Murakami, T., Mizuta, T., Iwai, Y., Hama, T., and Mayumi, T. (1991) Biochem. Biophys. Res. Commun. 174, 1159–1162; (b) Kawasaki, K., Namikawa, M., Yamashiro, Y., Hama, T., and Mayumi, T. (1991) Chem. Pharm. Bull. 39, 3373–3375. 5. Pillai, V. N. R., and Mutter, M.(1980) J. Org. Chem. 45, 5364– 5370. 6. (a)Lu, Y., and Felix, A. M. (1994) Reactive Polymer, 22, 221–229. (b) Felix, M., Lu, Y., and Campbell, R. T. (1995) Int. J. Peptide Protein Res. 46, 253–264. 7. Hynes, R., and Yamada, K. (1982) J. Cell. Biol. 95, 369–377. 8. Humphries, M. J., Mervic, M., Yamada, K. M., and Komoriya, A. (1988) J. Cell Biol. 107, 384a. 9. Castro, B., Dormoy, J. R., Evin, G., and Selve, C. (1975) Tetrahedron Lett. No.14; 1219–1222.
ACKNOWLEDGMENTS This work was supported in part by Grand-in-Aid from the Japanese Ministry of Education, Science and Culture and by a grant from The Health Science Fund of Kobe Gakuin University.
10. Hudson, D. (1988) J. Org. Chem. 53, 617–624. 11. Carpino, L. A. (1993) J. Am. Chem. Soc. 115, 4397–4398. 12. Lu, Y., and Felix, A. M. (1993) Peptide Res. 6, 140–146.
REFERENCES 1. (a) Ehrat, M., and Luisi, P.L. (1983) Biopolymer 22, 569–573; (b) Yoshimoto, T., Nishimura, H., Saito, Y., Sakurai, K., Kami-
13. Maeda, M., Izuno, Y., Kawasaki, K., Kaneda, Y., Mu, Y., Tsutsumi, Y., Nakagawa, S., and Mayumi, T. (1997) Chem. Pharm. Bull., in press.
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