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

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’.

Key words

proteins - peptides - thiols - chromophores - bioorganic chemistry

• References and Notes

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• 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; R_f 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); R_f 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; R_f 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.