Synthesis of TNFα peptide derivatives

The peptide Ac-EALPKK(NS)XGG-NH₂ (X = T, Y(NO₂) or Mcm) was synthesized by standard manual solid-phase-peptide synthesis using Fmoc-protected amino acid derivatives. Rink amide MBHA resin was treated with N,N-dimethylformamide (DMF) at room temperature (RT) for 10 min. The Fmoc-protecting group was removed with 20% piperidine in DMF (2 × 10 min). After washing with DMF (5 × 5 min) the resin was incubated with 4 eq of amino acid derivative, 4 eq HBTU and 8 eq of N,N-diisopropylethylamine (DIPEA) in DMF at RT (60 min). The N-terminus was modified with 4 eq acetic anhydride and 8 eq DIPEA in DCM (60 min). Nosyl-group was cleaved using 5 eq 1,8-Diazabicyclo[5.4.0]undec-7-en (DBU) and 5 eq thiophenol in DMF (2 × 90 min). Afterwards the resin was washed with DMF. Free lysine side chain was modified on-resin with HBTU (4 eq.), DIPEA (8 eq.) in DCM/DMF mixure (1:1) and myristic acid (1a, 2a), 6-(Fmoc-amino)-caproic acid and N-Boc-anthranilic acid (4, 5) or 8-(Fmoc-amino)-octanoic acid and N-Boc-anthranilic acid (4a). For 3 free lysine residue was acylated with 11-azidoundecanoic acid⁶⁹ according to the method used for peptides 1a and 1b. The resin was treated with a solution of triphenylphosphine (5 eq) in tetrahydrofuran (THF)/H₂O (95:5) for several days (small portions of resin were taken for the test cleavage and MS-analysis). After washing N-Boc-anthranilic acid was coupled by the standard method (see peptides 1a and 2b). 6 and 7 were prepared like peptide 3 with (4-N,N-dimethylamino-1,8-naphthalimid)-acetic acid⁶⁴ (6)or 2-amino-5-nitrobenzoic acid (7) instead of N-Boc-anthranilic acid. For 8 the resin bound peptide was incubated with the solution of carboxymethyl dithiomyristoate (3 eq) and DIPEA (3 eq) in DMF for 3 h and cleaved as described in general procedure. For 9 an an ice-cooling prepared solution of 5 eq of triphenylphosphine, 5 eq of diethyl azodicarboxylate (DIAD) and 10 eq of dried MeOH in dried DCM was added to the resin-bound fully protected peptide with the ε-Nosyl-protected lysine (Mitsunoby reaction). After one hour of incubation the resin was washed 5 times with DMF. Nosyl-group was removed as described above and resin was treated with a solution of carboxymethyl dithiomyristoate (3 eq) and DIPEA (3 eq) in DMF for 3 h.

Peptides 10 and 11 are based on a CPS1-peptide (Bz-GVLKEYGV-NH₂). To a DMF solution of the CPS1 peptide, ethyl dithioacetate (1.2 eq) and triethylamine (5 eq) (10) or methyl 3-[(methylthio)thiocarbonyl]propanoate (1.1 eq) and triethylamine (5 eq) (11) were added. Reaction mixture was stirred for 3–5 h. For 11 1 M NaOH (6 eq) was added and stirring continued for another 2 h.

The cyclic peptide inhibitor S2iL5 was synthesized by standard Fmoc-based solid phase peptide synthesis as described by Yamagata et al.⁶⁰.

All 3-nitrotyrosine containing peptides (2a, 2b, 3, 4, 4a, 5 and 6) require an additional piperidine treatment step (20% piperidine in DMF, 2 × 10 min) to remove acyl-group from 3-nitrotyrosine before cleavage.

The resin was washed with DCM (5 × 4 min), methanol (3 × 4 min) and DCM again and treated with TFA/H₂O (98:2) (2 × 60 min). Combined TFA solutions were evaporated in vacuum and re-dissolved in ACN/H₂O solution (50:50). HPLC purification and subsequent lyophilization yielded pure peptides.