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


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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  • Attard TJ, O’Brien-Simpson N, Reynolds EC (2007) Synthesis of phosphopeptides in the Fmoc mode. Int J Pept Res Ther 13:447–468

    Article  CAS  Google Scholar 

  • Bernatowicz M, Daniels S, Koster H (1989) A comparison of acid labile linkage agents for the synthesis of C-terminal amides. Tetrahedron Lett 30:4645–4648

    Article  CAS  Google Scholar 

  • Brandt M, Gammeltoft S, Jensen KJ (2006) Microwave heating for solid-phase peptide synthesis: general evaluation and application to 15-mer phosphopeptides. Int J Pept Res Ther 12:349–357

    Article  CAS  Google Scholar 

  • Burke TR Jr, Lee K (2003) Phosphotyrosyl mimetics in the development of signal transduction inhibitors. Acc Chem Res 36:426–433

    Article  PubMed  CAS  Google Scholar 

  • Fields GB (1994) Methods for removing the Fmoc group. In: Pennington MW, Dunn BN (eds) Peptide synthesis protocols. Humana Press, Totowa

    Google Scholar 

  • Flick MB, Sapi E, Perrotta PL, Maher MG, Halaban R, Carter D, Kacinski BM (1997) Recognition of activated CSF-1 receptor in breast carcinomas by a tyrosine 723 phosphospecific antibody. Oncogene 14:2553–2561

    Article  PubMed  CAS  Google Scholar 

  • Garcia-Martin F, Quintanar-Audelo M, Garcia-Ramos Y, Cruz LJ, Gravel C, Furic R, Cote S, Tulla-Puche J, Albericio F (2006) ChemMatrix, a poly(ethylene glycol)-based support for the solid-phase synthesis of complex peptides. J Comb Chem 8:213–220

    Article  PubMed  CAS  Google Scholar 

  • George A, Hao J (2005) Role of phosphophoryn in dentin mineralization. Cells Tissues Organs 181:232–240

    Article  PubMed  CAS  Google Scholar 

  • Lacombe JM, Andriamanampisoa F, Pavia AA (1990) Solid-phase synthesis of peptides containing phosphoserine using phosphate tert.-butyl protecting group. Int J Pept Protein Res 36:275–280

    PubMed  CAS  Google Scholar 

  • Lukszo J, Patterson D, Albericio F, Kates SA (1996) 3-(1-Piperidinyl)alanine formation during the preparation of C-terminal cysteine peptides with the Fmoc/t-Bu strategy. Lett Pept Sci 3:157–166

    Article  CAS  Google Scholar 

  • Muntane G, Dalfo E, Martinez A, Ferrer I (2008) Phosphorylation of tau and a-synuclein in synaptic-enriched fractions of the frontal cortex in Alzheimer’s disease, and in Parkinson’s disease and related a-synucleinopathies. Neuroscience 152:913–923

    Article  PubMed  CAS  Google Scholar 

  • O’brien-Simpson NM, Attard TJ, Loganathan A, Huq NL, Cross KJ, Riley PF, Reynolds EC (2007) Synthesis and characterization of a multiphosphorylated phosphophoryn repeat motif; H-[Asp-(Ser(P))2]3-Asp-OH. Int J Pept Res Ther 13:469–478

    Article  CAS  Google Scholar 

  • Ottinger EA, Shekels LL, Bernlohr DA, Barany G (1993) Synthesis of phosphotyrosine-containing peptides and their use as substrates for protein tyrosine phosphatases. Biochemistry (Mosc) 32:4354–4361

    Article  CAS  Google Scholar 

  • Otvos L Jr, Elekes I, Lee VM (1989) Solid-phase synthesis of phosphopeptides. Int J Pept Protein Res 34:129–133

    PubMed  CAS  Google Scholar 

  • Perich JW, Ede NJ, Eagle S, Bray AM (1999) Synthesis of phosphopeptides by the Multipin method: evaluation of coupling methods for the incorporation of Fmoc-Tyr(PO3Bzl, H)-OH, Fmoc-Ser(PO3Bzl, H)-OH and Fmoc-Thr(PO3Bzl, H)-OH. Lett Pept Sci 6:91–97

    CAS  Google Scholar 

  • Pospisilik JA, Knauf C, Joza N, Benit P, Orthofer M, Cani PD, Ebersberger I, Nakashima T, Sarao R, Neely G, Esterbauer H, Kozlov A, Kahn CR, Kroemer G, Rustin P, Burcelin R, Penninger JM (2007) Targeted deletion of AIF decreases mitochondrial oxidative phosphorylation and protects from obesity and diabetes. Cell 131:476–491

    Article  PubMed  CAS  Google Scholar 

  • Reynolds EC (1999) Anticariogenic casein phosphopeptides. Protein Pept Lett 6:295–303

    CAS  Google Scholar 

  • Songyang Z, Shoelson SE, Mcglade J, Olivier P, Pawson T, Bustelo XR, Barbacid M, Sabe H, Hanafusa H, Yi T et al (1994) Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB–2, HCP, SHC, Syk, and Vav. Mol Cell Biol 14:2777–2785

    PubMed  CAS  Google Scholar 

  • Vorherr T, Bannwarth W (1995) Phospho-serine and phospho-threonine building blocks for the synthesis of phosphorylated peptides by the Fmoc solid phase strategy. Bioorg Med Chem Lett 5:2661–2664

    Article  CAS  Google Scholar 

  • Wakamiya T, Saruta K, Yasuoka J, Kusumoto S (1994) An efficient procedure for solid-phase synthesis of phosphopeptides by the Fmoc strategy. Chem Lett 1099–1102

  • Wakamiya T, Nishida T, Togashi R, Saruta K, Yasuoka J-I, Kusumoto S (1996) Preparations of Na-Fmoc-O-[(benzyloxy)hydroxyphosphinyl] b-hydroxy a-amino acid derivatives. Bull Chem Soc Jpn 69:465–468

    Article  CAS  Google Scholar 

  • Waksman G, Shoelson SE, Pant N, Cowburn D, Kuriyan J (1993) Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms. Cell 72:779–790

    Article  PubMed  CAS  Google Scholar 

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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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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).

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