Skip to main content
SpringerLink
Log in

Comparative syntheses of peptides and peptide thioesters derived from mouse and human prion proteins

  • Original Article
  • Published:
Amino Acids Aims and scope Submit manuscript

Abstract

Prions are suspected as causative agents of several neuropathogenic diseases, even though the mode of their action is still not clear. A combination of chemical and recombinant syntheses can provide suitable probes for explanation of prions role in pathogenesis of neurodegenerative diseases. However, the prions contain several difficult sequences for synthesis by Fmoc/tBu approach. For that reason, the peptide thioesters as the key building blocks for chemical syntheses of proteins by native chemical ligation were employed. A scan of the mouse prion domain 93–231 was carried out in order to discover availability of derived thioesters as the suitable building blocks for a total chemical synthesis of the prion protein based probes. The synthesis on 2-chlorotritylchloride resin was utilized and after a deprotection of the samples for analysis, the peptide segments were purified and characterized. If the problems were detected during the synthesis, the segment was re-synthesized either using the special pseudoproline dipeptides or by splitting its molecule to two or three smaller segments, which were prepared easier. The protected segments, prepared correctly without any deletion and in sufficient amounts, were coupled either with EtSH after DIC/DMAP activation or with p-Ac-NH-Ph-SH using PyBOP activation to yield corresponding thioesters. In some special cases, the other techniques of thioester formation, like sulfonamide-safety catch and/or trimethylaluminium approach were utilized.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

AA:

Amino acid analysis

ACN:

Acetonitrile

Asi:

Aspartic acid imide

Boc:

tert-Butoxycarbonyl

ClTrt:

2-Chlorotrityl

DIC:

N,N′-Diisopropylcarbodiimide

DCM:

Dichloromethane

DIEA:

N,N-Diisopropylethylamine

DMAP:

4-(Dimethylamino)pyridine

Dmb:

Dimethylbenzyl

DMF:

N,N-Dimethylformamide

EDT:

1,2-Ethanedithiol

ESI MS:

Electro-spray ionization mass spectrometry

Et:

Ethyl

Fmoc:

[(Fluoren-1-yl-methoxy]carbonyl

HBTU:

O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate

HFIP:

Hexafluoroisopropanol

HOBt:

1-Hydroxybenzotriazole

HPLC:

High performance liquid chromatography

MALDI TOF:

Matrix-assisted laser desorption ionization time of flight

Mpa:

Mercaptopropionic acid

NMP:

N-Methylpyrrolidone

PyBOP:

(Benzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium hexafluorophosphate

Sec:

Selenocysteine

SPPS:

Solid-phase peptide synthesis

tBu:

tert-Butyl

TFA:

Trifluoroacetic acid

THF:

Tetrahydrofuran

Thz:

Thiazolidine-4-carboxylic acid

TIS:

Tri-isopropylsilane

TMS:

Trimethylsilyl

References

  • Backes BJ, Ellman JA (1999) An alkanesulfonamide safety-catch linker for solid-phase synthesis. J Org Chem 64:2322–2330

    Article  CAS  Google Scholar 

  • Ball HL, King DS, Cohen FE, Prusiner SB, Baldwin MA (2001) Engineering the prion protein using chemical synthesis. J Peptide Res 58:357–374

    Article  CAS  Google Scholar 

  • Bang D, Kent SBH (2004) A one-pot total synthesis of crambin. Angew Chem Int Ed 43:2534–2538

    Article  CAS  Google Scholar 

  • Bons N, Mestre-Frances N, Belli P, Cathala F, Gajdusek DC, Brown P (1999) Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents. Proc Natl Acad Sci USA 96:4046–4051

    Article  PubMed  CAS  Google Scholar 

  • Brask J, Albericio F, Jensen KJ (2003) Fmoc solid-phase synthesis of peptide thioesters by masking as trithioortho esters. Org Lett 5:2951–2953

    Article  PubMed  CAS  Google Scholar 

  • Bueler H, Fisher M, Lang Y, Bluethmann H, Lipp HP, DeArmond SJ, Prusiner SB, Aguet M, Weissmann C (1992) Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356:577–582

    Article  PubMed  CAS  Google Scholar 

  • Cardona V, Eberle I, Barthelemy S, Beythien J, Doerner B, Schneeberger P, Keyte J, White P (2008) Application of Dmb-dipeptides in the Fmoc SPPS of difficult and aspartimide-prone sequences. Int J Pept Res Therapeutics 14:285–292

    Article  CAS  Google Scholar 

  • Castilla J, Saá P, Hetz C, Soto C (2005) In vitro generation of infectious scrapie prions. Cell 121:195–206

    Article  PubMed  CAS  Google Scholar 

  • Caughey B (2000) Transmissible spongiform encephalopathies, amyloidoses and yeast prions: Common threads? Nat Med 6:751–754

    Article  PubMed  CAS  Google Scholar 

  • Cohen FE, Prusiner SB (1998) In vitro generation of infectious scrapie prions. Annu Rev Biochem 67:793–819

    Article  PubMed  CAS  Google Scholar 

  • Come JH, Fraser PE, Lansbury PT (1993) A kinetic model for amyloid formation in the prion diseases: importance of seeding. Proc Natl Acad Sci USA 90:5959–5963

    Google Scholar 

  • Dawson PE, Kent SBH (2000) Synthesis of native proteins by chemical ligation. Ann Rev Biochem 69:923–960

    Google Scholar 

  • Dawson PE, Muir TW, Clark-Lewis I, Kent SBH (1994) Synthesis of proteins by native chemical ligation. Science 266:776–779

    Article  PubMed  CAS  Google Scholar 

  • Fields GB, Noble RL (1990) Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int J Pept Protein Res 35:161–214

    Article  PubMed  CAS  Google Scholar 

  • Forloni G, Angeretti N, Chiesa R, Monzani E, Salmona M, Bugiani O, Tagliavini F (1993) Neurotoxicity of a prion protein fragment. Nature 362:543–546

    Article  PubMed  CAS  Google Scholar 

  • GPCRDB: Swiss-Prot entry (2011) http://www.receptors.org/Prion/seq/all/PRIO_HUMAN.SW.html

  • Hagenmaier H (1970) The influence of the chain length on the coupling reaction in solid phase peptide synthesis. Tetrahedron Lett 11:283–286

    Article  Google Scholar 

  • Huang Z, Prusiner SB, Cohen FE (1996) Scrapie prions: a three-dimensional model of an infectious fragment. Fold Des 1:13–19

    Article  PubMed  CAS  Google Scholar 

  • Jackson GS, Hosszu LLP, Power A, Hill AF, Kenney J, Saibil H, Craven CJ, Waltho JP, Clarke AR, Collinge J (1999) Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations. Science 283:1935–1937

    Article  PubMed  CAS  Google Scholar 

  • Jarrett JT, Lansbury PT (1993) Seeding “one-dimensional crystallization” of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell 73:1055–1058

    Google Scholar 

  • Jobling M, Barrow C, White A, Masters C, Collins S, Cappai R (1999) The synthesis and spectroscopic analysis of the neurotoxic prion peptide 106–126: Comparative use of manual Boc and Fmoc chemistry. Lett Pept Sci 6:129–134

    CAS  Google Scholar 

  • Kaiser E, Colescott RL, Bossinger CD, Cook PI (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem 34:595–598

    Article  PubMed  CAS  Google Scholar 

  • Kaneko K, Ball HL, Wille H, Zhang H, Groth D, Torchia M, Tremblay P, Safar J, Prusiner SB, DeArmond SJ, Baldwin MA, Cohen FE (2000) A synthetic peptide initiates Gerstmann-Sträussler-Scheinker (GSS) disease in transgenic mice. J Mol Biol 295:997–1007

    Google Scholar 

  • Krchňák V, Vágner J, Šafář P, Lebl M (1988a) Non-invasive continuous monitoring of solid phase peptide synthesis by acid-base indicator. Coll Czech Chem Comm 53:2542–2548

    Article  Google Scholar 

  • Krchňák V, Vágner J, Lebl M (1988b) Noninvasive continuous monitoring of solid-phase peptide synthesis by acid-base indicator. Int J Pept Protein Res 32:415–416

    Article  PubMed  Google Scholar 

  • Legname G, Baskakov IV, Nguyen HO, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB (2004) Synthetic mammalian prions. Science 305:673–676

    Article  PubMed  CAS  Google Scholar 

  • Milton RC, Milton SCF, Kent SBH (1992) Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show demonstration of reciprocal chiral substrate specificity. Science 256:1445–1448

    Google Scholar 

  • Pan K-M, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Huang Z, Fletterick RJ, Cohen FE, Prusiner SB (1993) Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA 90:10962–10966

    Google Scholar 

  • Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95:13363–13383

  • Sarin VK, Kent SBH, Tam JP, Merrifield RB (1981) Quantitative monitoring of solid phase peptide synthesis by the ninhydrin reaction. Anal Biochem 117:147–157

    Article  PubMed  CAS  Google Scholar 

  • Šebestik J, Matějka P, Hlaváček J, Stibor I (2004) Solid-phase synthesis of head and tail bis-acridinylated peptides. Tetrahedron Lett 45:1203–1205

    Article  Google Scholar 

  • Šebestik J, Hlaváček J, Stibor I (2006) Rational design and synthesis of a double-stranded DNA-binder library. Biopolymers 84:400–407

    Article  PubMed  Google Scholar 

  • Sewing A, Hilvert D (2001) Fmoc compatible solid-phase peptide synthesis of long C-terminal thioesters. Angew Chem Int Ed. 40:3395–3396

    Article  Google Scholar 

  • Spielhaupter C, Schaetzl HM (2001) PrPC directly interacts with proteins involved in signaling pathways. J Biol Chem 276:44604–44612

    Article  PubMed  CAS  Google Scholar 

  • Supattapone S, Bosque P, Muramoto T, Wille H, Aagaard C, Peretz D, Nguyen H-OB, Heinrich C, Torchia M, Safar J, Cohen FE, DeArmond SJ, Prusiner SB, Scott M (1999) Prion protein of 106 residues creates an artificial transmission barrier for prion replication in transgenic mice. Cell 96:869–878

    Google Scholar 

  • Vojkovský T (1995) Detection of secondary amines on solid phase. Pept Res 8:236–237

    PubMed  Google Scholar 

  • Von Eggelkraut-Gottanka R, Klose A, Beck-Sickinger AG, Beyermann M (2003) Peptide thioester formation using standard Fmoc-chemistry. Tetrahedron Lett 44:3551–3554

    Article  Google Scholar 

  • Wan Q, Danishefsky SJ (2007) Free-radical-based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew Chem Int Ed 46:9248–9252

    Article  CAS  Google Scholar 

  • Weissman C (1999) Molecular genetics of transmissible spongiform encephalopathies. J Biol Chem 274:3–6

    Google Scholar 

  • White P, Keyte JW, Bailey K, Bloomberg G (2004) Expediting the Fmoc solid phase synthesis of long peptides through the application of dimethyloxazolidine dipeptides. J Pept Sci 10:18–26

    Article  PubMed  CAS  Google Scholar 

  • Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch A, Poser S, Pocchiari M, Hofman A, Smith PG (1996) A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347:921–925

    Google Scholar 

  • Wollscheid B, Bausch-Fluck D, Henderson C, O’Brien R, Bibel M, Schiess R, Aebersold R, Watts JD (2009) Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins. Nat Biotechnol 27:378–386

    Article  PubMed  CAS  Google Scholar 

  • Zahariev S, Guarnaccia C, Zanuttin F, Pintar A, Esposito G, Maravic G, Krust B, Hovanessian AG, Pongor S (2005) Efficient synthesis and comparative studies of the arginine and Nω, Nω-dimethylarginine forms of the human nucleolin glycine/arginine rich domain. J Pept Sci 11:17–28

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grant of Czech Science Foundation (GA CR) No. 203/07/1517 and Research Project Z40550506. We greatly thanks to Miroslava Blechová and Alexandrina Prychodska for assembly of peptide sequences on automated peptide synthesizer; and to Lenka Monincová for some ESI-MS measurements.

Author information

Authors and Affiliations

  1. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10, Prague 6, Czech Republic

    Jaroslav Šebestík, Zbigniew Zawada, Martin Šafařík & Jan Hlaváček

Authors
  1. Jaroslav Šebestík
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zbigniew Zawada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin Šafařík
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jan Hlaváček
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jan Hlaváček.

Additional information

The nomenclature and symbols of the amino acids follow the Recommendations of the IUPAC/IUB Joint Commission on Biochemical Nomenclature. Eur J Biochem (1984) 138:9–37.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Šebestík, J., Zawada, Z., Šafařík, M. et al. Comparative syntheses of peptides and peptide thioesters derived from mouse and human prion proteins. Amino Acids 43, 1297–1309 (2012). https://doi.org/10.1007/s00726-011-1203-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-011-1203-9

Keywords

Search

Navigation