Facile solid-phase synthesis of sulfated tyrosine-containing peptides: total synthesis of human big gastrin-II and cholecystokinin (CCK)-39.
Chemical synthesis of tyrosine O-sulfated peptides is still a laborious task for peptide chemists because of the intrinsic acid-lability of the sulfate moiety. An efficient cleavage/deprotection procedure without loss of the sulfate is the critical difficulty remaining to be solved for fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase synthesis of sulfated peptides. To overcome the difficulty, TFA-mediated solvolysis rates of a tyrosine O-sulfate [Tyr(SO3H)] residue and two protecting groups, tBu for the hydroxyl group of Ser and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for the guanidino group of Arg, were examined in detail. The desulfation obeyed first-order kinetics with a large entropy (59.6 J.K-1.mol-1) and enthalpy (110.5 kJ.mol-1) of activation. These values substantiated that the desulfation rate of the rigidly solvated Tyr(SO3H) residue was strongly temperature-dependent. By contrast, the SN1-type deprotections were less temperature-dependent and proceeded smoothly in TFA of a high ionizing power. Based on the large rate difference between the desulfation and the SN1-type deprotections in cold TFA, an efficient deprotection protocol for the sulfated peptides was developed. Our synthetic strategy for Tyr(SO3H)-containing peptides with this effective deprotection protocol is as follows: (i) a sulfated peptide chain is directly constructed on 2-chlorotrityl resin with Fmoc-based solid-phase chemistry using Fmoc-Tyr(SO3Na)-OH as a building block; (ii) the protected peptide-resin is treated with 90% aqueous TFA at 0 degree C for an appropriate period of time for the cleavage and deprotection. Human cholecystokinin (CCK)-12, mini gastrin-II (14 residues), and little gastrin-II (17 residues) were synthesized with this method in 26-38% yields without any difficulties. This method was further applied to the stepwise synthesis of human big gastrin-II (34 residues), CCK-33 and -39. Despite the prolonged acid treatment (15-18 h at 0 degree C), the ratios of the desulfated peptides were less than 15%, and the pure sulfated peptides were obtained in around 10% yields.
Kitagawa
K
K
Niigata College of Pharmacy, Kamishin'ei-cho 5-13-2, Niigata 950-2081, Japan. kouki@niigata-pharm.ac.jp
Aida
C
C
Fujiwara
H
H
Yagami
T
T
Futaki
S
S
Kogire
M
M
Ida
J
J
Inoue
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002766:Cholecystokinin / Q000031:analogs & derivatives* / Q000138:chemical synthesis*; D002851:Chromatography, High Pressure Liquid; D005755:Gastrins / Q000138:chemical synthesis*; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D011498:Protein Precursors / Q000138:chemical synthesis*; D051381:Rats; D012697:Serine Endopeptidases / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013431:Sulfates / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*; D014867:Water / Q000737:chemistry
D005755:Gastrins; D007202:Indicators and Reagents; D010455:Peptides; D011498:Protein Precursors; D013431:Sulfates; D014867:Water; C013706:big gastrin; D014443:Tyrosine; C032740:cholecystokinin 39; D002766:Cholecystokinin; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase