Synthesising Difficult Phosphopeptides

Synthesising Difficult Phosphopeptides

Table of Contents

Summary
Introduction
Materials and Methods
     Reagents
     Peptide Synthesis
Results
Conclusion

Summary

Complicated phosphopeptides can be synthesized in a very efficient manner using the Liberty Blue™ automated microwave peptide synthesizer with CarboMAX™ coupling methodology. Using microwave energy is useful for coupling bulky phosphoamino acid derivatives. The use of CarboMAX coupling methodology stabilizes the acid-labile protected phosphate group at high temperatures and this minimizes side reactions. The following phosphopeptides were each synthesized in less than 2 hours using this method: CTEDQY(pS) LVED-NH2 (82% purity), CPSPA(pT)DPSLY-NH2 (73% purity), and CSDGG(pY)MDMSK-NH2 (62% purity).

Introduction

Phosphorylation is a post-translational modification (PTM) achieved by introducing a phosphate group onto the threonine, serine, or tyrosine residues of a peptide or protein.1 Enzymatic phosphorylation controls the function of many proteins and guides various intracellular signal transduction cascades.2 Phosphorylation plays an essential role in biology and hence there has been an increased interest in the synthesis of phosphopeptides. Cell biologists and proteomics researchers are looking to better understand and characterize the phosphoproteome.3 Initially, production of phosphopeptides required synthesizing the entire peptide and a subsequent post-synthetic phosphorylation step. Due to the difficulty of the postsynthesis step, this process often produced impure peptides.4 The introduction of Fmoc-derived, monobenzyl-protected phosphoamino acids including FmocThr(PO(OBzl)OH)-OH, Fmoc-Ser(PO(OBzl)OH)-OH and FmocTyr(PO(OBzl)OH-OH (Figure 1) has drastically enhanced the synthesis process. This allows the automation of SPPS for a variety of phosphopeptides.5

Figure 1. Fmoc-Derived, Monobenzyl-Protected Phosphoamino Acids

The synthesis of phosphopeptides can also be improved by using microwaves. Research has shown that the first deprotection after coupling the phosphoamino acid should be performed at room temperature to avoid beta-elimination of the protected phosphate group. It has also been demonstrated that phospho linkages are acid labile and hence susceptible to removal at high temperature during carbodiimide coupling. CarboMAX methodology uses 0.4 equivalents of DIEA and is powerful for stabilizing acid sensitive linkages including protected phosphate group. A phosphopeptide containing 3 protected phosphate groups has recently been successfully synthesized using CarboMAX. Microwave coupling steps improve direct and subsequent couplings of phosphorylated residues and minimize dephosphorylation and deletion products (CarboMAX).6

Materials and Methods

Reagents

All amino acids were obtained from CEM Corporation (Matthews, NC) and contained the following side chain protecting groups: Asn(Trt), Asp(OMpe), Cys(Trt), Gln(Trt), Glu(OtBu), Lys(Boc), Ser(tBu), Thr(tBu), Tyr(tBu). Fmoc monobenzyl-protected phosphoamino acids were also obtained from CEM Corporation (Matthews, NC): N-α-Fmoc-O-benzyl-Lphosphoserine (pS), N-α-Fmoc-O-benzyl-L-phosphotyrosine (pY), and N-α-Fmoc-O-benzyl-L-phosphothreonine (pT). Oxyma Pure and Rink Amide ProTide™ LL resin were obtained from CEM Corporation (Matthews, NC). N,N’-Diisopropylcarbodiimide (DIC) was purchased from CreoSalus (Louisville, KY). Piperidine was obtained from Alfa Aesar (Ward Hill, MA). Trifluoroacetic acid (TFA), 3,6-dioxa-1,8-octanedithiol (DODT), triisopropylsilane (TIS), N,N-Diisopropylethylamine (DIEA) thioanisole, and acetic acid were obtained from Sigma-Aldrich (St. Louis, MO). Dichloromethane (DCM), N,N-dimethylformamide (DMF), and anhydrous diethyl ether (Et2O) were obtained from VWR (West Chester, PA). LCMS-grade water (H2O), and LCMS-grade acetonitrile (MeCN) were obtained from Fisher Scientific (Waltham, MA).

Peptide Synthesis

The peptides were prepared at 0.1 mmol scale using the CEM Liberty Blue automated microwave peptide synthesizer on Rink Amide ProTide LL resin (0.20 meq/g substitution). Deprotection was performed with piperidine and Oxyma Pure in DMF. Coupling reactions were performed with a 5-fold excess of Fmoc-AA-OH, DIC in DMF and Oxyma Pure/DIEA in DMF (CarboMAX).6 Cleavage was performed with TFA/thioanisole/ TIS/H2O/DODT on the CEM Razor™ parallel peptide cleavage system. Following cleavage, the peptide was precipitated in Et2O and lyophilized overnight.

Results

Microwave-enhanced SPPS of CTEDQY(pS)LVED-NH2 on the Liberty Blue automated microwave peptide synthesizer produced the target peptide with 82% purity (Figure 2). No Tyr deletions were observed. Microwave-enhanced SPPS of CPSPA(pT)DPSLY-NH2 produced the target peptide in 73% purity (Figure 3). Truncation of amino acids 7–11 was observed in a small quantity. Microwave-enhanced SPPS of CSDGG(pY) MDMSK-NH2 produced the target peptide in 62% purity (Figure 4). There was minimal aspartimide formation and methionine oxidation.

Figure 2. UPLC Chromatogram of CTEDQY(pS)LVED-NH2

Figure 3. UPLC Chromatogram of CPSPA(pT)DPSLY-NH2

Figure 4. UPLC Chromatogram of CSDGG(pY)MDMSK-NH2

Conclusion

Microwave-enhanced SPPS on the Liberty Blue automated microwave peptide synthesizer rapidly synthesizes high quality, high purity phosphopeptides. These include CTEDQY(pS)LVEDNH2, CPSPA(pT)DPSLY-NH2, and CSDGG(pY)MDMSK-NH2. Room temperature deprotection following the insertion of phosphoserine minimizes dephosphorylation in the synthesis of CTEDQY(pS)LVED-NH2. Employment of Fmoc-Asp(OMpe)- OH minimizes the occurrence of aspartimide formation in susceptible sequences, especially CSDGG(pY)MDMSK-NH2. Microwave-enhanced SPPS can improve direct and subsequent couplings of phosphorylated residues while minimizing undesired side reactions.

References

(1) Prabakaran, S.; Lippens, G.; Steen, H.; Gunawardena, J. Wiley Interdiscip. Rev. Syst. Biol. Med. 2012, 4 (6), 565–583.

(2) Chen, J.; Shinde, S.; Koch, M.-H.; Eisenacher, M.; Galozzi, S.; Lerari, T.; Barkovits, K.; Subedi, P.; Krüger, R.; Kuhlmann, K.; Sellergren, B.; Helling, S.; Marcus, K. Sci. Rep. 2015, 5 (1), 11438.

(3) Dephoure, N.; Gould, K. L.; Gygi, S. P.; Kellogg, D. R. Mol. Biol. Cell 2013, 24 (5), 535–542.

(4) White, P. D. In Fmoc Solid Phase Peptide Synthesis: A Practical Approach; Chan, W. C., White, P. D., Eds.; Oxford University Press: New York, 2000; pp 187–189.

(5) Perich, J. W.; Ede, N. J.; Eagle, S.; Bray, A. M. Lett. Pept. Sci. 1999, 6 (2/3), 91–97.

(6) CEM Application Note (AP0124) - “CarboMAX - Enhanced Peptide Coupling at Elevated Temperature.”

This information has been sourced, reviewed and adapted from materials provided by CEM Corporation - Life Science.

For more information on this source, please visit CEM Corporation - Life Science.

Last updated: Mar 29, 2018 at 8:25 AM

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