Synthesis of thanatin and derivatives 1 to 7

Synthesis of thanatin and derivatives 1-7

Peptides were synthesized by solid phase peptide synthesis (SPPS) based on a mixed solid/solution phase procedure using the Fmoc/tBu strategy.

Fmoc-Tyr(tBu)-OH or Fmoc-Nle-OH or Fmoc-D-Tyr(tBu)-OH was loaded onto 2-chlorotritylchloride resin in dichloromethane in presence of N,N-diisopropylethylamine (DIEA).

After capping (MeOH), a typical loading of 0.4–0.8 mmol/g was achieved. The peptide was assembled by standard automated Fmoc SPPS on 0.025 mmol scale (based on pre-loaded resin loading). Fmoc groups were deprotected with 20 % piperidine in dimethylformamide (DMF).

Amino acids were introduced by double coupling of 2x 15 min (2x 30 min after synthesis cycle 9) with amino acid (AA) / 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) / N-methylmorpholine (NMM) 8/7.6/24 molar equivalents (eq) respectively in N-methyl-2-pyrrolidone (NMP). For the unusual unnatural AA derivatives, e.g., Fmoc-Lys(azido-PEG4)-OH, the coupling was performed only once for 2 hours with AA/HATU/NMM 4/3.8/12 eq. Each coupling step was followed by a capping step with acetic anhydride (Ac2O) / NMM 12/12 eq. The synthesis cycle Fmoc deprotection / coupling / capping was repeated with the appropriate amino acid until the end of the elongation.

To introduce the N-terminal guanidine group in 5, N,N-bis-Boc-1-guanylpyrazole (10 eq) dissolved in a mixture of 0.5 mL of dimethylsulfoxide (DMSO) / DMF 1/1 v/v was added to the peptide resin after removal of the Fmoc of the last coupled amino acid (V6’, see Table 1). The resin suspension was shaken overnight at room temperature and washed with DMF.

After final Fmoc removal of the last coupled amino acid (V6’) or N-terminus functionalization (guanidine), the resin was washed with dichloromethane. The peptide was simultaneously cleaved from the resin and globally deprotected (removal of side chain protecting groups) by treatment with 6 mL of trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / thioanisole / anisole / water 82.5/5/5/5/2.5 v/v/v/v/v. After 2.5 hours, the resin was filtered, and the crude linear peptide was precipitated in 35 mL of cold diethyl ether (Et2O). The solid was isolated by centrifugation and washed 3 times with 20 mL of Et2O. After removal of residual Et2O by evaporation, the crude cyclic product was dissolved in 1.8 mL of water and 1.8 mL of ammonium acetate buffer (1 M, pH 6) containing 4 % of DMSO (v/v). The disulfide bridge formation was monitored by LC/MS and Ellman’s colorimetric test. Reaction time required to reach completion varied from 2 to 6 days depending on the peptide sequence. The crude cyclic product was purified by reversed phase HPLC (0.1% TFA in water/acetonitrile gradient, C18 column material). For analytical data, see Table S3.

Synthesis of fluorescently labeled compounds 6-FL The peptide precursor bearing the 6-azido-L-lysine derivative (Fig. S6) was synthesized and purified as described above. The purified peptide (10 mg, TFA salt) in water (0.7 mL) was 3submitted to a copper-catalysed azide-alkyne cycloaddition with 1 eq of the Alexa Fluor 647 alkyne (Fluoroprobes) in degassed methanol (MeOH) (20 mg/mL) and a mixture of 1 eq copper (II) sulfate pentahydrate (CuSO4*5H2O) (11 mg/mL) and 3 eq ascorbic acid (23 mg/mL) in water.

The reaction concentration was close to 10 mg/mL of peptide. After 1 h, partial precipitation of the Alexa Fluor 647 alkyne was observed. Additional degassed MeOH (0.5 mL) was added, and the reaction was stirred overnight. MeOH was removed by evaporation under reduced vacuum and the crude peptide was purified by reversed phase HPLC (0.1% TFA in water/acetonitrile gradient, C18 column material). For analytical data, see Tab. S3.