Detection and minimization of H-phosphonate side reaction during phosphopeptide synthesis by a post-assembly global phosphorylation strategy |
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Authors: | Qinghong Xu, Elizabeth A. Ottinger, Nuria A. Solé George Barany |
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Affiliation: | (1) Department of Chemistry, University of Minnesota, 207 Pleasant Street S.E., 55455 Minneapolis, MN, U.S.A.;(2) Present address: The Rockefeller University, 1230 York Avenue, 10021 New York, NY, U.S.A.;(3) Present address: Joslin Diabetes Center, One Joslin Place, 02215 Boston, MA, U.S.A.;(4) Present address: Quality Controlled Biochemicals, Inc., 3 Avenue D, 01748 Hopkinton, MA, U.S.A. |
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Abstract: | Summary In the course of solid-phase synthesis of phosphopeptides by a post-assembly global phosphorylation strategy, the corresponding H-phosphonate peptides form as byproducts. We describe model studies to investigate this side reaction as a function of reaction conditions, and use this information to develop conditions that minimize the problem, i.e., use of dibenzyl N,N-di-isopropyl phosphoramidite for phosphitylation, followed immediately by oxidation with anhydrous tert-butyl hydroperoxide in dry tetrahydrofuran under argon, and final acidolytic cleavage.This work was taken in part from the Ph.D. Theses of E.A. Ottinger (1994) and Q. Xu (1996), University of Minnesota, Minneapolis, MN, U.S.A. Preliminary presentations of portions of this work were made at the Twenty-Second European Peptide Symposium, Interlaken, Switzerland, September 13–19, 1992, see Ref. 1, at the 14th American Peptide Symposium, Columbus, OH, U.S.A., June 18–23, 1995, and at the Fourth International Symposium on Solid Phase Synthesis & Combinatorial Chemical Libraries, Edinburgh, Scotland, U.K., September 12–16, 1995, see Ref. 2. The title side reaction was first discussed for tyrosine (see Refs 1 and 3), but all of the mechanism studies discussed herein are for serine and threonine.Amino acid symbols denote the l-configuration, and abbreviations for amino acids and peptides follow rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977]. |
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Keywords: | Post-assembly phosphorylation Phosphoramidite chemistry H-phosphonate byproduct Phosphopeptides Gel-phase nuclear magnetic resonance Solid-phase peptide synthesis |
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