Synlett 2013; 24(18): 2454-2458
DOI: 10.1055/s-0033-1339853
letter
© Georg Thieme Verlag Stuttgart · New York

Efficient, Traceless Semi-Synthesis of α-Synuclein Labeled with a Fluoro­phore/Thioamide FRET Pair

Rebecca F. Wissner
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA   Fax: +1(215)5732112   Email: [email protected]
,
Anne M. Wagner
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA   Fax: +1(215)5732112   Email: [email protected]
,
John B. Warner
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA   Fax: +1(215)5732112   Email: [email protected]
,
E. James Petersson*
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA   Fax: +1(215)5732112   Email: [email protected]
› Author Affiliations
Further Information

Publication History

Received: 01 August 2013

Accepted after revision: 29 August 2013

Publication Date:
20 September 2013 (online)


Abstract

We have shown that thioamides can be incorporated into proteins through semi-synthesis and used as probes to monitor structural changes. To date, our methods have required the presence of a cysteine at the peptide ligation site, which may not be present in the native peptide sequence. Here, we present a strategy for the semi-synthesis of thioproteins using homocysteine as a ligation point with subsequent masking as methionine, making the ligation ‘traceless’.

Supporting Information

 
  • References and Notes

  • 1 Royer CA. Chem. Rev. 2006; 106: 1769
    • 2a Goldberg JM, Batjargal S, Petersson EJ. J. Am. Chem. Soc. 2010; 132: 14718
    • 2b Goldberg JM, Speight LC, Fegley MW, Petersson EJ. J. Am. Chem. Soc. 2012; 134: 6088
    • 2c Goldberg JM, Wissner RF, Klein AM, Petersson EJ. Chem. Commun. 2012; 48: 1550
    • 3a Miwa JH, Pallivathucal L, Gowda S, Lee KE. Org. Lett. 2002; 4: 4655
    • 3b Miwa JH, Patel AK, Vivatrat N, Popek SM, Meyer AM. Org. Lett. 2001; 3: 3373
    • 3c Reiner A, Wildemann D, Fischer G, Kiefhaber T. J. Am. Chem. Soc. 2008; 130: 8079
    • 3d Choudhary A, Raines RT. ChemBioChem 2012; 12: 1801
  • 4 Dawson PE, Muir TW, Clark-Lewis I, Kent SB. H. Science 1994; 266: 776
    • 5a Batjargal S, Wang YJ, Goldberg JM, Wissner RF, Petersson EJ. J. Am. Chem. Soc. 2012; 134: 9172
    • 5b Muir TW. Annu. Rev. Biochem. 2003; 72: 249
  • 6 Galvin JE, Lee VM. Y, Trojanowski JQ. Arch. Neurol. 2001; 58: 186
    • 7a Uversky VN, Li J, Fink AL. J. Biol. Chem. 2001; 276: 10737
    • 7b Auluck PK, Caraveo G, Lindquist S. Annu. Rev. Cell Dev. Biol. 2010; 26: 211
    • 7c Wood SJ, Wypych J, Steavenson S, Louis JC, Citron M, Biere AL. J. Biol. Chem. 1999; 274: 19509
  • 8 Drescher M, Huber M, Subramaniam V. ChemBioChem 2012; 13: 761
  • 9 Wissner RF, Batjargal S, Fadzen CM, Petersson EJ. J. Am. Chem. Soc. 2013; 135: 6529
  • 10 Ferreon AC. M, Moosa MM, Gambin Y, Deniz AA. Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 17826
    • 11a Pivato M, De Franceschi G, Tosatto L, Frare E, Kumar D, Aioanei D, Brucale M, Tessari I, Bisaglia M, Samori B, de Laureto PP, Bubacco L. PLoS One 2012; 7: e50027
    • 11b Zhou W, Freed CR. J. Biol. Chem. 2004; 279: 10128
  • 12 Hendrickson TL, Imperiali B. Biochemistry 1995; 34: 9444
    • 13a Tam JP, Yu QT. Biopolymers 1998; 46: 319
    • 13b Saporito A, Marasco D, Chambery A, Botti P, Monti SM, Pedone C, Ruvo M. Biopolymers 2006; 83: 508
    • 13c Aussedat B, Fasching B, Johnston E, Sane N, Nagorny P, Danishefsky SJ. J. Am. Chem. Soc. 2012; 134: 3532
    • 13d Pachamuthu K, Schmidt RR. Synlett 2003; 659
  • 14 Tanaka T, Wagner AM, Warner JB, Wang YJ, Petersson EJ. Angew. Chem. Int. Ed. 2013; 52: 6210
    • 15a Suto K, Shimizu Y, Watanabe K, Ueda T, Fukai S, Nureki O, Tomita K. EMBO J. 2006; 25: 5942
    • 15b Watanabe K, Toh Y, Suto K, Shimizu Y, Oka N, Wada T, Tomita K. Nature (London) 2007; 449: 867
  • 16 Link AJ, Vink MK. S, Agard NJ, Prescher JA, Bertozzi CR, Tirrell DA. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10180
  • 17 Shalaby MA, Grote CW, Rapoport H. J. Org. Chem. 1996; 61: 9045
  • 18 Nagalingam AC, Radford SE, Warriner SL. Synlett 2007; 2517
  • 19 Taskent-Sezgin H, Chung J, Patsalo V, Miyake-Stoner SJ, Miller AM, Brewer SH, Mehl RA, Green DF, Raleigh DP, Carrico I. Biochemistry 2009; 48: 9040
  • 20 Synthesis of α-N-Fmoc-l-aspartate-2-amino-5-nitroanilide (1): Fmoc-Asp(Ot-Bu)-OH (2.67 g, 6.50 mmol) was dissolved in THF (35 mL) under argon flow and the solution was cooled to –10 °C in a NaCl/ice (1:3) bath. N-Methylmorpholine (NMM, 1.43 mL, 13 mmol) and isobutyl chloroformate (IBCF, 0.85 mL, 6.5 mmol) were added dropwise with stirring. After 15 min, 4-nitro-o-phenylenediamine (1.0 g, 6.5 mmol) was added and the reaction was allowed to proceed with stirring under argon flow at –10 °C for 2 h. The reaction was then allowed to proceed for an additional 6 h with stirring at r.t. The reaction mixture was dried by rotary evaporation, resuspended in DMF (20 mL), and then poured into sat. KCl solution (200 mL). The precipitated product was filtered and washed with cold H2O. The precipitate was then dissolved in minimal EtOAc and purified over a silica gel column in hexanes–EtOAc (3:2) to afford 1 as a yellow solid in 88.9% yield; Rf 0.5 (hexanes–EtOAc, 1:1). 1H NMR (500 MHz, CDCl3): δ = 8.1 (s, 1H), 8.0 (s, 1H), 7.93 (dd, J = 2.5, 9.0 Hz, 1 H), 7.74 (dd, J = 3.6, 7.5 Hz, 2 H), 7.57 (dd, J = 3.4, 7.4 Hz, 2 H), 7.36–7.41 (m, 2 H), 7.26–7.30 (m, 2 H), 6.62 (d, J = 9.0 Hz, 1 H), 5.98 (d, J = 8.2 Hz, 1 H), 4.73 (s, H) 4.63 (br s, 1 H), 4.45–4.53 (m, 2 H), 4.19 (t, J = 6.3 Hz, 1 H), 2.97 (dd, J = 4.6, 17.2 Hz, 1 H), 2.72–2.80 (m, 1 H), 1.46 (s, 9 H). 13C NMR (125 MHz, CDCl3): δ = 172.5, 170.8, 157.3 , 149.0, 144.4, 142.3, 139.6, 128.8, 128.2, 125.9, 125.4, 124.5, 121.7, 121.1, 115.9, 83.6, 68.3, 52.7, 48.2, 38.3, 29.1. HRMS (ESI): m/z [M + H]+ calcd for C29H31N4O7: 547.219; found: 547.218.
  • 21 Synthesis of α-N-Fmoc-l-thioaspartate-2-amino-5-nitroanilide (2): P2S5 (2.47 g, 5.56 mmol) and anhyd Na2CO3 (0.589 g, 5.56 mmol) were stirred in THF (30 mL) at r.t. under argon flow until a clear yellow solution was obtained. After cooling the solution to 0 °C on ice, 1 (3.04 g, 5.56 mmol) was added, and the reaction was carefully monitored by TLC. After approximately 1 h, the reaction was filtered through Celite® (Sigma-Aldrich) and dried by rotary evaporation. The crude reaction material was dissolved in EtOAc and purified over a silica gel column in hexanes–EtOAc (1:1) to afford 2 as a yellow foam (2.46 g, 78.6% yield); Rf 0.7 (hexanes–EtOAc, 1:1). 1H NMR (500 MHz, CDCl3): δ = 9.84 (br s, 1 H), 8.07 (br s, 1 H), 7.96 (d, J = 8.4 Hz, 1 H), 7.76 (d, J = 6.9 Hz, 2 H), 7.52 (dd, J = 7.3, 19.6 Hz, 2 H) 7.39–7.43 (m, 2 H), 7.28–7.32 (m, 2 H), 6.56 (d, J = 9.0 Hz, 1 H), 6.06 (d, J = 8.2 Hz, 1 H), 5.07 (br s, 1 H), 4.84 (br s, 2 H), 4.37 (br s, 2 H), 4.21–4.13 (m, 1 H), 3.18–3.05 (m, 2 H), 1.45 (s, 9 H). 13C NMR (125 MHz, CDCl3): δ = 204.9, 172.1, 157.2, 149.6, 144.3, 142.2, 139.0, 128.8, 128.1, 126.5, 125.9, 125.8, 122.8, 121.0, 115.6, 83.5, 68.4, 58.4, 47.8, 41.6, 28.9. HRMS (ESI): m/z [M + H]+ calcd for C29H31N4O6S: 563.196; found: 563.197.
  • 22 Synthesis of α-N-Fmoc-l-thioaspartatenitrobenzo-triazolide (3): Compound 2 (1.00 g, 1.78 mmol) was added to glacial acetic acid diluted with 5% H2O (25 mL). NaNO2 (0.16 g, 2.23 mmol) was added in small portions over 5 min with constant stirring at r.t. After 30 min, the reaction was quenched by the addition of ice water (500 mL). The resulting pale orange precipitate 3 was filtered, washed extensively with ice water, and allowed to dry under vacuum. The final product was characterized and used in peptide synthesis without any further purification; Rf 0.9 (hexanes–EtOAc, 1:1). 1H NMR (500 MHz, CDCl3): δ = 9.64 (s, 1 H), 8.46 (d, J = 8.6 Hz, 1 H), 8.33 (d, J = 8.7 Hz, 1 H), 7.77–7.78 (m, 2 H), 7.59–7.64 (m, 2 H) 7.38–7.44 (m, 2 H), 7.29–7.35 (m, 2 H), 6.45–6.52 (m, 1 H), 6.23 (d, J = 8.7 Hz, 1 H), 4.48–4.54 (m, 1 H), 4.34–4.41 (m, 1 H), 4.21–4.26 (m, 1 H), 3.07–3.15 (m, 1 H), 2.86–2.97 (m, 1 H), 1.42 (s, 9 H). 13C NMR (125 MHz, CDCl3): δ = 206.7, 169.3, 156.4, 150.6, 149.9, 144.6, 142.3, 132.8, 128.7, 128.0, 126.0, 123.2, 122.6, 121.0, 113.6, 83.4, 68.2, 59.0, 48.1, 41.9, 28.9. HRMS (ESI): m/z [M + Na]+ calcd for C29H27N5NaO6S: 596.158; found: 596.158.