Identification of secretin diastereoisomers produced during synthesis.
The byproducts P-1 and P-2, which were produced during the synthesis of porcine secretin, were isolated in pure form from the crude secretin by HPLC. These were identified by a combination of amino acid analysis, enzymatic digestion, and isocratic or linear gradient reversed-phase (RP)-HPLC. The amino acid compositions of P1 and P2, determined by amino acid analysis after acid hydrolysis, were found to be the same as those of porcine secretin without distinction between L-and D-amino acids. But, HPLC of their digestive fragments with trypsin and alpha-chymotrypsin differed from that of secretin. The fragments, S7-12 of P-1 and S13-21 of P-2 were determined to be different from the corresponding fragments obtained from secretin by HPLC analysis of their digestive fragments. The amino acid composition of each acid hydrolysate, following digestion with D-amino acid oxidase, was found to have less leucine or alanine content than secretin. The HPLC analysis of the fragments from P-1 and P-2 by tryptic and alpha-chymotryptic digestion showed that they are the same as those from synthetic D-Leu10 secretin or D-Ala17 secretin, respectively. Consequently, P-1 and P-2 are concluded to be the secretin diastereoisomers, D-Leu10 and D-Ala17 secretin, respectively.
Tsuda
T
T
Pharmaceutical Research Laboratories, Eisai Company, Ltd., Ibarki, Japan.
Uchiyama
M
M
Sato
T
T
Yoshino
H
H
Tsuchiya
Y
Y
Ishikawa
S
S
Ohmae
M
M
Watanabe
S
S
Miyake
Y
Y
D000596:Amino Acids / Q000032:analysis; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D003605:D-Amino-Acid Oxidase; D006868:Hydrolysis; D007202:Indicators and Reagents; D012633:Secretin / Q000032:analysis* / Q000138:chemical synthesis / Q000302:isolation & purification; D013237:Stereoisomerism; D014357:Trypsin
D000596:Amino Acids; D007202:Indicators and Reagents; D012633:Secretin; D003605:D-Amino-Acid Oxidase; D002918:Chymotrypsin; D014357:Trypsin