Thieme Chemistry Journals Awardees – Where Are They Now?Improved Fmoc Deprotection Methods for the Synthesis of 
Thioamide-Containing Peptides and Proteins

D. Miklos Szantai-Kis; Christopher R. Walters; Taylor M. Barrett; Eileen M. Hoang; E. James Petersson

doi:10.1055/s-0036-1589027

Synlett, Table of Contents

Synlett 2017; 28(14): 1789-1794
DOI: 10.1055/s-0036-1589027

letter

Thieme Chemistry Journals Awardees – Where Are They Now?
Improved Fmoc Deprotection Methods for the Synthesis of Thioamide-Containing Peptides and Proteins

D. Miklos Szantai-Kis

^aDepartment of Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104, USA Email: ejpetersson@sas.upenn.edu

,

Christopher R. Walters

^bDepartment of Chemistry, University of Pennsylvania, 231 S. 34^th Street, Philadelphia, PA 19104, USA

,

Taylor M. Barrett

^bDepartment of Chemistry, University of Pennsylvania, 231 S. 34^th Street, Philadelphia, PA 19104, USA

,

Eileen M. Hoang

^bDepartment of Chemistry, University of Pennsylvania, 231 S. 34^th Street, Philadelphia, PA 19104, USA

^cSwarthmore College, 500 College Ave, Swarthmore, PA 19081, USA

,

E. James Petersson*

^aDepartment of Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104, USA Email: ejpetersson@sas.upenn.edu

^bDepartment of Chemistry, University of Pennsylvania, 231 S. 34^th Street, Philadelphia, PA 19104, USA

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Abstract

All articles of this category

^‡ These authors contributed equally to this work

Abstract

Site-selective incorporation of thioamides into peptides and proteins provides a useful tool for a wide range of applications. Current incorporation methods suffer from low yields as well as epimerization. Here, we describe how the use of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) rather than piperidine in fluorenylmethyloxycarbonyl (Fmoc) deprotection reduces epimerization and increases yields of thioamide-containing peptides. Furthermore, we demonstrate that the use of DBU avoids byproduct formation when synthesizing peptides containing side-chain thioamides.

Key words

peptides - sulfur - solid-phase synthesis - protecting groups - proteins

Full Text

References

References and Notes

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16 (9H-Fluoren-9-yl)methyl-(R)-(1-[(2-amino-5-nitrophenyl)amino]-3-phenyl-1-thioxopropan-2-yl)carbamate (S2) Na₂CO₃ (1.13 mmol, 120 mg) and P₄S₁₀ (1.13 mmol, 502 mg) were suspended in THF (20 mL) and was stirred under argon atmosphere at r.t. for 30 min. The reaction was cooled to 0 °C. Compound S1 (1.13 mmol, 589 mg) was dissolved in THF (10 mL) and added to the reaction. After 1.5 h, the reaction was complete and condensed to dark yellow oil by rotary evaporation. The crude product was run over a short silica plug column to remove insoluble P₄S₁₀ aggregates. The crude product was purified by flash using EtOAc–hexanes (1:1). The desired product S2 was obtained as an orange powder in 54.2% yield (0.612 mmol, 330 mg). ¹H NMR (500 MHz, CDCl₃): δ = 9.07 (s, 1 H), 7.90 (dd, J = 9.0, 2.6 Hz, 1 H), 7.73 (dd, J = 10.6, 7.5 Hz, 2 H), 7.58 (s, 1 H), 7.48 (d, J = 7.5 Hz, 1 H), 7.41 – 7.32 (m, 3 H), 7.30–7.17 (m, 9 H), 6.48 (d, J = 9.0 Hz, 1 H), 5.78 (s, 1 H), 4.86 (s, 1 H), 4.10 (dt, J = 16.7, 8.3 Hz, 5 H), 3.21 (d, J = 9.2 Hz, 0 H), 1.59 (s, 1 H), 1.36–1.04 (m, 1 H).
17 (9H-Fluoren-9-yl)methyl-(R)-(1-{6-nitro-1H-benzo[d][1,2,3]triazol-1-yl}-3-phenyl-1-thioxopropan-2-yl)carbamate (S3) Compound S2 (0.612 mmol, 330 mg) was dissolved in 95% AcOH_(aq) (10 mL) and cooled to 0 °C. After 5 min, NaNO₂ (0.765 mmol, 52.8 mg) was added slowly to the reaction. After 30 min, ice-cold Milli-Q water (100 mL) was added to the reaction. The resulting orange precipitate was filtered and washed with additional cold Milli-Q water. After drying the product was obtained as an orange powder in 84.0% yield (0.514 mmol, 256 mg) and was used directly in SPPS without further purification. ¹H NMR (500 MHz, CDCl₃, major rotamer only): δ = 9.61 (s, 1 H), 8.44 (d, J = 8.9 Hz, 1 H), 8.29 (d, J = 8.9 Hz, 1 H), 7.74 (d, J = 7.6 Hz, 2 H), 7.52 (t, J = 6.9 Hz, 2 H), 7.38 (t, J = 7.6 Hz, 2 H), 7.28 (q, J = 8.0 Hz, 2 H), 7.23–7.13 (m, 5 H), 6.56 (d, J = 7.4 Hz, 1 H), 5.66 (d, J = 9.3 Hz, 1 H), 4.46–4.35 (m, 1 H), 4.33 (t, J = 9.0 Hz, 1 H), 3.40 (dd, J = 14.0, 5.4 Hz, 1 H), 3.10 (dd, J = 13.8, 8.0 Hz, 1 H). ESI⁺-HRMS: m/z calcd for C₃₀H₂₃N₅O₄S⁺: 550.1549; found [M + H]⁺: 550.1550.
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28 N ²-(tert-Butoxycarbonyl)-N ⁶-ethanethioyl-l-lysine (Boc-Lys(Ac^S)-OH, 14) Boc-Lys-OH (13, 369 mg, 1.50 mmol, 1.0 equiv) was suspended in EtOH (4.4 mL) and 10% (w/v) Na₂CO₃ solution (4.0 mL) added. Ethyl dithioacetate (189 μL, 1.65 mmol, 1.1 equiv) added and stirred overnight. The solvent was removed in vacuo and the solid redissolved in H₂O (10 mL). The reaction mixture was acidified with 3 M HCl until the solution became milky white (ca. pH 2). The aqueous phase was extracted three times with CHCl₃ (10 mL). The combined organic phase was dried over Na₂SO₄, and the solvent was removed in vacuo. The product was obtained as yellow foam in high yield (423 mg, 1.39 mmol, 92.4%). ¹H NMR (500 MHz, CDCl₃): δ = 10.88 (s, 1 H), 8.37 (s, 1 H), 5.35 (d, J = 7.7 Hz, 1 H), 4.07 (d, J = 75.4 Hz, 1 H), 3.52 (s, 2 H), 2.50–2.34 (m, 3 H), 1.77 (s, 1 H), 1.69–1.49 (m, 3 H), 1.42–1.23 (m, 11 H). ESI⁺-HRMS: m/z calcd for C1₃H₂₅N₂O₄S⁺: 305.1535; found [M + H]⁺: 305.1556.
29 N ²-(tert-Butoxycarbonyl)-N ⁶-[1-(piperidin-1-yl)ethylidene]-l-lysine (15) Boc-Lys(Ac^S)-OH (14, 133 mg, 0.438 mmol) was dissolved in 50% (v/v) piperidine in DMF (2 mL). After stirring for 5 h, the reaction mixture was diluted with 0.1% TFA in H₂O and purified by reverse phase HPLC. Fractions containing product 15 or unreacted starting material 14 were collected separately and lyophilized. After lyophilization starting material 14 was dissolved in 50% (v/v) piperidine in DMF and, after 5 h the reaction was purified as before. This procedure was repeated one more time until enough product was collected for NMR analysis (1.90 mg, 4.05 μmol, 0.9%). ¹H NMR (500 MHz, DMSO-d ₆): δ = 12.58 (s, 1 H), 8.68 (s, 1 H), 7.05 (d, J = 7.8 Hz, 1 H), 3.84 (td, J = 8.8, 4.6 Hz, 1 H), 3.57 (s, 4 H), 3.32 (s, 2 H), 2.28 (s, 3 H), 1.67–1.54 (m, 8 H), 1.49 (dt, J = 13.3, 7.0 Hz, 2 H), 1.37 (s, 9 H), 1.36–1.28 (m, 2 H). ¹³C NMR (126 MHz, DMSO): δ = 174.28, 162.44, 155.65, 78.02, 53.36 (+), 49.45 (–), 46.48 (–), 43.89 (–), 30.38 (–), 28.86 (–), 28.25 (+), 25.64 (–), 24.74 (–), 23.06 (–), 22.65 (–), 14.35 (+). ESI⁺-HRMS: m/z calcd for C₂₃H₂₇N₂O₄S⁺: 356.2549; found [M + H]⁺: 356.2558. Additional 2D NMR correlations are given in the Supporting Information.

Supplementary Material

Supporting Information

Thieme Chemistry Journals Awardees – Where Are They Now?Improved Fmoc Deprotection Methods for the Synthesis of Thioamide-Containing Peptides and Proteins

Thieme Chemistry Journals Awardees – Where Are They Now?
Improved Fmoc Deprotection Methods for the Synthesis of Thioamide-Containing Peptides and Proteins