Peptides.Vol. 13, pp. 653-662, 1992
0196-9781/92 $5.00 + .00 Copyright© 1992PergamonPress Ltd.
Printed in the USA.
Bombyxin-II and Its Disulfide Bond Isomers: Synthesis and Activity KOJI NAGATA,* KAZUNORI MARUYAMA,* HIROMICHI NAGASAWA,* IKUMI URUSHIBATA,* A K I R A ISOGAI,* H I R O N O R I ISHIZAKII" A N D A K I N O R I S U Z U K I *l
*Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, and ?Department of Biology, School of Science, Nagoya University, Chikusa-ku, Nagoya 464, Japan Received 17 D e c e m b e r 1991 NAGATA, K., K. MARUYAMA, H. NAGASAWA,I. URUSHIBATA, A. ISOGAI, H. ISHIZAKI AND A. SUZUKI. BombyxinH and its disulfidebondisomers:Synthesisand activity. PEPTIDES 13(4) 653-662, 1992.--Bombyxin-lI, an insulin superfamily peptide of the silkmoth Bombyx mori, and its disulfide bond isomers have been synthesized by two ways of stepwise, semiregioselective disulfide bond formation. The disulfide bond CysA2°-CysB22or CysAT-Cysal° was formed first, and then the two other disulfide bonds were formed by iodine oxidation. The conditions for the iodine oxidation were improved to suppress oxidative degradation of unprotected Trp residues. With these conditions, bombyxin-II was synthesized in high yields (26% and 32%). Its disulfide bond isomers were also obtained. Specific activity of the products indicates that the disulfide bond CysA2°-CysB22is important to the bombyxin activity.
Bombyx mori Iodine oxidation
Bombyxin Disulfidebond formation Disulfidebond isomer Peptidesynthesis Prothoracicotropic activity
BOMBYXIN is a brain secretory peptide of the silkmoth Bombyx mori. Its amino acid sequence shows considerable homology with vertebrate insulin family peptides (Fig. 1) (6,15,17,22). Bombyxin acts on the prothoracic glands of the saturniid moth Samia cynthia ricini (5) to stimulate the synthesis and release of ecdysone, a steroid required for insect growth and metamorphosis (13). But the physiological function of bombyxin in Bombyx still remains obscure. The bombyxin molecule is a beterodimer consisting of A- and B-chains, which are linked together by disulfide bonds in exactly the same way as in the insulin molecule (16). The primary structure of bombyxin has been determined completely for bombyxin-II (15,16) and -IV (11) (Fig. 1), and partially for bombyxin-I, -III, and -V (7,14). The amount of natural bombyxin obtainable is very limited: 150 #g from 650,000 adult Bombyx heads (13-15,17). In order to investigate the biological aspects and the three-dimensional molecular structure of bombyxin, a large amount of sample should be required. We have been making efforts to develop an efficient methods for the synthesis of bombyxin. Nagasawa et al. first reported the synthesis of bombyxin-II by means of the spontaneous air oxidation of the A- and B-chains with free thiols at all the Cys residues in 4% yield (16). Maruyama et al. then reported the synthesis of bombyxin-IV by means of a stepwise, semi-regioselective formation of the disulfide bonds in 8% yield (12), in which the removal of S-Acm groups and concomitant formation of the two other disulfide bonds was attempted by
Requests for reprints should be addressed to A. Suzuki.
653
Insulin superfamily peptide
iodine oxidation (8,9). During the iodine oxidation considerable side reactions occurred, and the yield lowered. By improving the iodine oxidation step, we have accomplished synthesis of bombyxin-II in high yields by two ways of semi-regioselective disulfide bond formation. Disulfide bond isomers of bombyxin-II were also obtained as a result of the strategies of semi-regioselective disulfide bond formation. We describe here the synthesis, chemical characterization, and relationship between helix content and biological activity ofbombyxin-II and its disulfide bond isomers. METHOD
Materials Protected amino acids were purchased from Kokusan Chemical Works, Ltd., Novabiochem AG and Applied Biosysterns Inc. Boc-Cys(MBzl)-OCH2-Pam resin (0.71 mmol/g) and Boc-Asp(OBzl)-OCH2-Pam resin (0.74 mmol/g, 0.73 mmol/g) were from Applied Biosystems Inc., and Fmoc-Cys(Acm)-OCH2phenoxymethyl resin (0.57 mmol/g) was from Kokusan Chemical Works, Ltd. Dithiothreitol (DTT), 2,2'-dithiodipyridine (DTDP), and sodium iodoacetate were from Nacalai Tesque, Inc. Pyroglutamate aminopeptidase (5-oxoproryl-peptide hydrolase, EC 3.4.19.3) was from Boehringer Mannheim GmbH, and thermolysin (EC 3.4.24.4) from Seikagaku Kogyo Co., Ltd.
654 A- chain Bombyxin- I I Bombyxin- IV Human Insulin B -chain Bombyxin- I I Bombyxin- IV Human Insulin
NAGATA ET AL. The following reaction conditions were tried.
~I~DE~SRP~SVDV~LS~ ~4~DE~IQP~TLDV~A~ aIS~3~sI~s~ Y ~
0196-9781/92 $5.00 + .00 Copyright© 1992PergamonPress Ltd.
Printed in the USA.
Bombyxin-II and Its Disulfide Bond Isomers: Synthesis and Activity KOJI NAGATA,* KAZUNORI MARUYAMA,* HIROMICHI NAGASAWA,* IKUMI URUSHIBATA,* A K I R A ISOGAI,* H I R O N O R I ISHIZAKII" A N D A K I N O R I S U Z U K I *l
*Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, and ?Department of Biology, School of Science, Nagoya University, Chikusa-ku, Nagoya 464, Japan Received 17 D e c e m b e r 1991 NAGATA, K., K. MARUYAMA, H. NAGASAWA,I. URUSHIBATA, A. ISOGAI, H. ISHIZAKI AND A. SUZUKI. BombyxinH and its disulfidebondisomers:Synthesisand activity. PEPTIDES 13(4) 653-662, 1992.--Bombyxin-lI, an insulin superfamily peptide of the silkmoth Bombyx mori, and its disulfide bond isomers have been synthesized by two ways of stepwise, semiregioselective disulfide bond formation. The disulfide bond CysA2°-CysB22or CysAT-Cysal° was formed first, and then the two other disulfide bonds were formed by iodine oxidation. The conditions for the iodine oxidation were improved to suppress oxidative degradation of unprotected Trp residues. With these conditions, bombyxin-II was synthesized in high yields (26% and 32%). Its disulfide bond isomers were also obtained. Specific activity of the products indicates that the disulfide bond CysA2°-CysB22is important to the bombyxin activity.
Bombyx mori Iodine oxidation
Bombyxin Disulfidebond formation Disulfidebond isomer Peptidesynthesis Prothoracicotropic activity
BOMBYXIN is a brain secretory peptide of the silkmoth Bombyx mori. Its amino acid sequence shows considerable homology with vertebrate insulin family peptides (Fig. 1) (6,15,17,22). Bombyxin acts on the prothoracic glands of the saturniid moth Samia cynthia ricini (5) to stimulate the synthesis and release of ecdysone, a steroid required for insect growth and metamorphosis (13). But the physiological function of bombyxin in Bombyx still remains obscure. The bombyxin molecule is a beterodimer consisting of A- and B-chains, which are linked together by disulfide bonds in exactly the same way as in the insulin molecule (16). The primary structure of bombyxin has been determined completely for bombyxin-II (15,16) and -IV (11) (Fig. 1), and partially for bombyxin-I, -III, and -V (7,14). The amount of natural bombyxin obtainable is very limited: 150 #g from 650,000 adult Bombyx heads (13-15,17). In order to investigate the biological aspects and the three-dimensional molecular structure of bombyxin, a large amount of sample should be required. We have been making efforts to develop an efficient methods for the synthesis of bombyxin. Nagasawa et al. first reported the synthesis of bombyxin-II by means of the spontaneous air oxidation of the A- and B-chains with free thiols at all the Cys residues in 4% yield (16). Maruyama et al. then reported the synthesis of bombyxin-IV by means of a stepwise, semi-regioselective formation of the disulfide bonds in 8% yield (12), in which the removal of S-Acm groups and concomitant formation of the two other disulfide bonds was attempted by
Requests for reprints should be addressed to A. Suzuki.
653
Insulin superfamily peptide
iodine oxidation (8,9). During the iodine oxidation considerable side reactions occurred, and the yield lowered. By improving the iodine oxidation step, we have accomplished synthesis of bombyxin-II in high yields by two ways of semi-regioselective disulfide bond formation. Disulfide bond isomers of bombyxin-II were also obtained as a result of the strategies of semi-regioselective disulfide bond formation. We describe here the synthesis, chemical characterization, and relationship between helix content and biological activity ofbombyxin-II and its disulfide bond isomers. METHOD
Materials Protected amino acids were purchased from Kokusan Chemical Works, Ltd., Novabiochem AG and Applied Biosysterns Inc. Boc-Cys(MBzl)-OCH2-Pam resin (0.71 mmol/g) and Boc-Asp(OBzl)-OCH2-Pam resin (0.74 mmol/g, 0.73 mmol/g) were from Applied Biosystems Inc., and Fmoc-Cys(Acm)-OCH2phenoxymethyl resin (0.57 mmol/g) was from Kokusan Chemical Works, Ltd. Dithiothreitol (DTT), 2,2'-dithiodipyridine (DTDP), and sodium iodoacetate were from Nacalai Tesque, Inc. Pyroglutamate aminopeptidase (5-oxoproryl-peptide hydrolase, EC 3.4.19.3) was from Boehringer Mannheim GmbH, and thermolysin (EC 3.4.24.4) from Seikagaku Kogyo Co., Ltd.
654 A- chain Bombyxin- I I Bombyxin- IV Human Insulin B -chain Bombyxin- I I Bombyxin- IV Human Insulin
NAGATA ET AL. The following reaction conditions were tried.
~I~DE~SRP~SVDV~LS~ ~4~DE~IQP~TLDV~A~ aIS~3~sI~s~ Y ~
