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Identification and Suppression of β-Elimination Byproducts Arising from the Use of Fmoc-Ser(PO3Bzl,H)-OH in Peptide Synthesis

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Abstract

The formation of 3-(1-piperidinyl)alanyl-containing peptides via phosphoryl β-elimination was identified from the application of Fmoc-Ser(PO3Bzl,H)-OH in peptide synthesis as shown by RP-HPLC, ES-MS and 31P-NMR analysis. An Nα-deprotection study using the model substrates, Fmoc-Xxx(PO3Bzl,H)-Val-Glu(OtBu)-Resin (Xxx = Ser, Thr or Tyr) demonstrated that piperidine-mediated phosphoryl β-elimination occurred in the N-terminal Ser(PO3Bzl,H) residue at a ratio of 7% to the target phosphopeptide, and that this side reaction did not occur in the corresponding Thr(PO3Bzl,H)- or Tyr(PO3Bzl,H)- residues. The generation of 3-(1-piperidinyl)alanyl-peptides was also shown to be enhanced by the use of microwave radiation during Fmoc deprotection. An examination of alternative bases for the minimization of byproduct formation showed that cyclohexylamine, morpholine, piperazine and DBU gave complete suppression of β-elimination, with a 50% cyclohexylamine/DCM (v/v) deprotection protocol providing the crude peptide of highest purity. Piperidine-induced β-elimination was found only to occur in Ser(PO3Bzl,H) residues that were in the N-terminal position, since the addition of the next residue in the sequence rendered the phosphoseryl residue stable to multiple piperidine treatments. The application of the alternative Nα-deprotection protocol using 50% cyclohexylamine/DCM (v/v) is therefore recommended for deprotection of the Fmoc group from the Fmoc-Ser(PO3Bzl,H) residue, with particular benefit anticipated for the synthesis of multiphosphoseryl peptides.

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Acknowledgments

This work was supported by the National Health and Medical Research Council grant 454475 and The CRC for Oral Health Science. We would like to thank Dr. Chris Barlow for the provision of the FT-ICR mass spectral data.

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  1. Cooperative Research Center for Oral Health Science, Melbourne Dental School, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, 720 Swanston Street, Melbourne, VIC, 3010, Australia

    Troy J. Attard, Neil M. O’Brien-Simpson & Eric C. Reynolds

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  1. Troy J. Attard
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  2. Neil M. O’Brien-Simpson
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  3. Eric C. Reynolds
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Correspondence to Eric C. Reynolds.

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Attard, T.J., O’Brien-Simpson, N.M. & Reynolds, E.C. Identification and Suppression of β-Elimination Byproducts Arising from the Use of Fmoc-Ser(PO3Bzl,H)-OH in Peptide Synthesis. Int J Pept Res Ther 15, 69–79 (2009). https://doi.org/10.1007/s10989-008-9165-9

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