| 16 mg |
|
Example 4 N-Me-Tyr1-D-Ala2-Asp3-Ala4-Ile5-Phe6-Thr7-Gln8-Ser9-Tyr10-Arg11-Orn12-Val13-Leu14-Abu15-Gln16-Leu17-Ser18-Ala19-Arg20-Orn21-Leu22-Leu23-Gln24-Asp25-Ile26-Nle27-Asp28-Arg29-NH-CH2-CH3 (Peptide 28420) N-Me-Tyr1, D-Ala2, Gln8, Orn12, Abu15, Orn21, Nle27, Asp28]hGHRH(1-29)NH-CH2-CH3. The synthesis is conducted in a stepwise manner using manual solid phase peptide synthesis equipment. Briefly, 3-[(Ethyl-Fmoc-amino)methyl]-indol-1-yl]-acetyl AM resin (Nova Biochem, La Jolla, Calif.) (610 mg, 0.50 mmol) is deprotected with 20% piperidine in DMF for 5 and 15 minutes and washed according to the protocol described in Table 3. The solution of Fmoc-Arg(Pbf)-OH (975 mg, 1.5 mmol) in DMF is shaken with the washed resin and DIC (235 muL, 1.5 mmol) in a manual solid phase peptide synthesis apparatus for 1 hour. After washing the resin three times with DMF, the coupling reaction was repeated as described above. After the repeated coupling and after the completion of the reaction is proved by negative ninhydrin test, the deprotection and neutralization protocols described in Table 3 are performed in order to remove the Fmoc protecting group and prepare the peptide-resin for coupling of the next amino acid. The synthesis is continued and the peptide chain is built stepwise by coupling the following protected amino acids in the indicated order on the resin to obtain the desired peptide sequence: Fmoc-Asp(OBut)-OH, Fmoc-Nle-OH, Fmoc-Ile-OH, Fmoc-Asp(OBut)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Abu-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-AspfOBuVOH, Fmoc-D-Ala-OH, Fmoc-N-Me-Tyr(tBu)-OH. These protected amino acid residues (also commonly available from Novabiochem, Advanced Chemtech, Bachem, and Peptides International) are represented above according to a well accepted convention. The suitable protecting group for the side chain functional group of particular amino acids appears in parentheses. The OH groups in the above formulae indicate that the carboxy-terminus of each residue is free. The protected amino acids (1.5 mmol each) are coupled with DIC (235 muL, 1.5 mmol) with the exceptions of Fmoc-Asn(Trt)-OH and Fmoc-Gln(Trt)-OH which are coupled with HBTU reagent. In order to cleave the peptide from the resin and deprotect it, a portion of 250 mg of the dried peptide resin is stirred with 2.5 mL of cleavage cocktail (94% TFA, 3% H2O, 1.5% m-cresol, and 1.5% phenol) at room temperature for 3 hours. To induce peptide precipitation, the cleavage mixture is added dropwise to cold (preferably -20 C.) ether. The precipitated material is collected by filtration or centrifugation and is washed three times with cold ether. The cleaved and deprotected peptide is dissolved in 50% acetic acid and separated from the resin by filtration. After dilution with water and lyophilization, 110 mg crude product is obtained. The crude peptide is checked by analytical HPLC using a Hewlett-Packard Model HP-1090 liquid chromatograph equipped with a Supelco Discovery HS C18 reversed-phase column (2.1 mm×5 cm, packed with C18 silica gel, 300 pore size, 3 mum particle size) (Supelco, Bellefonte, Pa.). Linear gradient elution (e.g., 40-70% B) is used with a solvent system consisting of (A) 0.1% aqueous TFA and (B) 0.1% TFA in 70% aqueous MeCN, and the flow rate is 0.2 mL/min. Purification is performed on a Beckman System Gold HPLC system (Beckman Coulter, Inc., Brea, Calif.) equipped with 127P solvent Module; UV-VIS Detector, model 166P; Computer workstation with CPU Monitor and printer, and 32-Karat software, version 3.0. 110 mg of crude peptide is dissolved in AcOH/H2O, stirred, filtered and applied on an XBridge Prep OBD reversed phase column (4.6×250 mm, packed with C18 silica gel, 300 A pore size, 5 mum particle size) (Waters Co., Milford, Mass.). The column is eluted with a solvent system described above in a linear gradient mode (e.g., 40-60% B in 120 min); flow rate 12 mL/min. The eluent is monitored at 220 nm, and fractions are examined by analytical HPLC. Fractions with purity higher than 95% are pooled and lyophilized to give 16 mg pure product. The analytical HPLC is carried out on a Supelco Discovery C18 reversed-phase column described above using isocratic elution with a solvent system described above with a flow rate of 0.2 mL/min. The peaks are monitored at 220 and 280 nm. The product is judged to be substantially (>95%) pure by analytical HPLC. Molecular mass is checked by electrospray mass spectrometry, and the expected amino acid composition is confirmed by amino acid analysis. |
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