Synthesis of dl-[2-13C]leucine and it use in the preparation of [3-dl-[2-13C]leucine]oxytocin and [8-dl-[2-13C]leucine]oxytocin: Preparative separation of diastereoisomeric peptides by partition chromatography and high pressure liquid chromatography |
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Authors: | V. Viswanatha B. Larsen V.J. Hruby |
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Affiliation: | University of Arizona, Tucson, AZ 85721, U.S.A. |
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Abstract: | dl-[2-13C]Leucine was prepared by condensing the sodium salt of ethyl acetamido-[2-13C]cyanoacetate with isobutylbromide in hexamethylphosphoroustriamide followed by acid hydrolysis. N-Boc-dl-[2-13C]Leucine was prepared and incorporated into [8-dl-[2-13C]leucine]oxytocin by total synthesis. The 13C-labeled hormone derivative [8-[2-13C]leucine]oxytocin was separated from its 8-position diastereoisomer by partition chromatography. The specifically 13C-labeled peptide hormone diastereoisomeric analog [3-dl-[2-13C]leucine]oxytocin also was prepared by solid phase peptide synthesis. No suitable solvent system for partition chromatography separation of the latter diastereoisomeric peptide mixture could be found. However an excellent preparative separation of the diastereoisomers could be obtained by reverse phase high pressure liquid chromatography on a partisil 10 M9 ODS column using the solvent system 0.05 M ammonium acetate (pH 4.0), acetonitrile (81:19, ) to give pure [3-(2-13C]leucine]oxytocin and [3-D-(2-13C]leucine]oxytocin. An excellent separation of [8[2-13C]leucine]oxytocin and the corresponding 8-D-leucine diastereoisomer derivative could also be accomplished by high pressure liquid chromatography. |
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