title: Site-specific PEGylation for high-yield preparation of Lys(21)-amine PEGylated growth hormone-releasing factor (GRF) (1-29) using a GRF(1-29) derivative FMOC-protected at Tyr(1) and Lys(12).
abstract: PEGylation has been viewed as an effective means of overcoming the therapeutic restriction of growth hormone-releasing factor (1-29) (GRF(1-29)) due to its short biological lifetime caused by severe proteolysis and rapid glomerular filtration. Of three isomers according to the PEGylation sites (Tyr1, Lys12, or Lys21), PEGylated GRF(1-29) at Lys21-amine (Lys21-PEG-GRF(1-29)) was shown to have the highest bioactivity. In this report, we propose a unique two-step site-specific PEGylation method capable of producing only Lys21-PEG-GRF(1-29) with a single composition in high yield using a GRF(1-29) derivative protected at Tyr1 and Lys12 and remained available at Lys21 (FMOC1,12-GRF(1-29)). The first step of this reaction involved the PEG attachment to FMOC1,12-GRF(1-29), and the second step involved the removal of FMOC moieties. This PEGylation process was optimized at the following conditions: 0.2-0.3% (v/v) triethylamine concentration, 5.0-6.0-fold molar amount of PEG, reaction temperature of 25-45 degrees C, and reaction time of 30 min. Under these conditions, the maximum yield of Lys21-PEG-GRF(1-29) produced was ca. approximately 95%, 6.3-fold higher than that by nonspecific PEGylation at pH 8.5. Significantly, this site-specific Lys21-PEG-GRF(1-29) was found to have greatly increased resistance to rat plasma, liver, and kidney homogenates, with 7.0-, 25.4-, and 16.4-fold longer half-lives vs GRF(1-29), respectively. Furthermore, 125I-Lys21-PEG-GRF(1-29) displayed significantly reduced liver and kidney distributions and extended blood presence vs 125I-GRF(1-29) in rats. Due to these benefits, Lys21-PEG-GRF(1-29) displayed an enhanced initial growth hormone release in vivo despite having 15% remaining activity in vitro. This devised PEGylation method using an FMOC-protection/deprotection strategy would provide great usefulness for PEGylating bioactive peptides in terms of improved biological potency, elevated production yield, and a uniform composition.
authors.affiliation: Drug Targeting Laboratory, College of Pharmacy, SungKyunKwan University, 300 Chonchon-dong, Jangan-ku, Suwon City 440-746, Korea.
authors.forename: Yu Seok
authors.initials: YS
authors.lastname: Youn
authors.forename: Kang Choon
authors.initials: KC
authors.lastname: Lee
mesh_terms: D000588:Amines / Q000737:chemistry*; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D005449:Fluorenes / Q000737:chemistry*; D013006:Growth Hormone / Q000378:metabolism; D013007:Growth Hormone-Releasing Hormone / Q000138:chemical synthesis* / Q000493:pharmacokinetics / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008239:Lysine / Q000737:chemistry*; D008297:Male; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry*; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D052683:Somatotrophs; D014018:Tissue Distribution
chemical_list: D000588:Amines; D000596:Amino Acids; D005449:Fluorenes; D007457:Iodine Radioisotopes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D010446:Peptide Fragments; D011092:Polyethylene Glycols; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone; D008239:Lysine