Synlett 2017; 28(14): 1789-1794
DOI: 10.1055/s-0036-1589027
letter
© Georg Thieme Verlag Stuttgart · New York

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
a  Department 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
b  Department of Chemistry, University of Pennsylvania, 231 S. 34th Street, Philadelphia, PA 19104, USA
,
Taylor M. Barrett
b  Department of Chemistry, University of Pennsylvania, 231 S. 34th Street, Philadelphia, PA 19104, USA
,
Eileen M. Hoang
b  Department of Chemistry, University of Pennsylvania, 231 S. 34th Street, Philadelphia, PA 19104, USA
c  Swarthmore College, 500 College Ave, Swarthmore, PA 19081, USA
,
E. James Petersson*
a  Department of Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104, USA   Email: ejpetersson@sas.upenn.edu
b  Department of Chemistry, University of Pennsylvania, 231 S. 34th Street, Philadelphia, PA 19104, USA
› Author Affiliations
This work was supported by funding from the National Science Foundation (NSF CHE-1150351 to E.J.P.). Instruments supported by the NSF and National Institutes of Health include: HRMS (NIH RR-023444) and MALDI-TOF MS (NSF MRI-0820996). C.R.W. thanks the NIH for funding through the Structural Biology and Molecular Biophysics Training Program (T32 GM008275). T.M.B. thanks the NIH for funding through the Chemistry-Biology Interface Training Program (T32 GM071399).
Further Information

Publication History

Received: 12 March 2017

Accepted after revision: 10 April 2017

Publication Date:
19 May 2017 (eFirst)

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.

Supporting Information

 
  • References and Notes

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  • 15 Thioamino acid precursors for SPPS were synthesized as Fmoc-thionitrobenzotriazolide monomers. The synthesis follows a standard route shown in Scheme 1. The synthesis of Fmoc-PheS-Nbt serves as an example. See the Supporting Information for structures of compounds S1S3. (9H-fluoren-9-yl)methyl(R)-{1-[(2-amino-5-nitrophenyl)amino]-1-oxo-3-phenylpropan-2-yl}carbamate (S1) Fmoc-Phe-OH (1.29 mmol, 500 mg) was dissolved in THF (15 mL) and stirred under argon. The solution was then cooled to –10 °C. NMM (2.58 mmol, 0.284 mL) was slowly added. Next, isobutyl chloroformate (1.42 mmol, 0.186 mL) was carefully added dropwise. The reaction was then stirred at –10 °C for 15 min, after which 4-nitro-o-phenylenediamine (1.42 mmol, 218 mg) was added. The reaction was stirred at –10 °C for 2 h and continued stirring at r.t. overnight under argon. The next day the solvent was removed in vacuo. The yellow solid was dissolved in DMF (20 mL), and the product was precipitated by addition of sat. KCl solution (100 mL). This precipitate was filtered and washed with cold water and Et2O. The product was dried under vacuum overnight. Compound S1 (1.13 mmol, 589 mg) was obtained in a crude yield of 87.4% and used without further purification
  • 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(AcS)-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(AcS)-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.