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General procedure: Examples 4, 5, 6, 74, 77 and 78 are shown in Table 1. Procedure B, as described above, was used for the preparation of the peptides. The peptides were synthesized starting with the amino acid Fmoc-Thr(tBu)-OH, which was grafted to the resin (Fmoc-Thr(tBu)-2-chlorotrityl resin). The linear peptides were synthesized on the solid support according to method A, as described above, using Fmoc protection for the alpha-amino group and allyl protection for the a-carboxyl group for the addition of the amino acid residue at P13. Assembly of the peptides was in the following sequence: Subsequently, allyl deprotection at P13, macrolactam cycle formation (module A) by an amide bond between the liberated alpha-carboxyl group of the amino acid residue at P13 and the alpha-amino group of the amino acid residue at P14, ivDde deprotection at Q1, cleavage of the peptide from the resin, macrolactam cycle formation (module B) by an amide bond between the alpha-carboxyl group of Thr at Q7 and the gamma-amino group of Dab at Q1, and full deprotection were performed as indicated in procedure B above. Finally, the peptides were purified, as described above, and characterized by HPLC-MS. For analytical data, see Ex. 4, 5, 6, 74, 77 and 78 in Table 2. 1.1.1.1 Coupling to the resin via a carboxyl group In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1% crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1, v/v) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1, v/v/v) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Thr(tBu)-2-chlorotrityl resin, Fmoc-DThr(tBu)-2-chlorotrityl resin, Fmoc-Val-2-chlorotrityl resin, Fmoc-DVal-2- chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-DArg(Pbf)-2-chlorotrityl resin. 1.1.2 Methods for synthesis on solid support of the fully protected peptide fragment and of fully protected peptide fragments for fragment coupling The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. Unless otherwise indicated, in each vessel were placed 0.05 mMol of the resin, obtained from procedure 1.1.1.1 as described above, and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out as described in the methods A - L, as described herein below: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20% piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 a) 3.6 eq appropriately protected amino acid and 3.6 eq HOAt in DMF or NMP + 3.6 eq DIC in DMF 1 x 40 min 6 3.6 eq appropriately protected amino acid and 3.6 eq HOAt in DMF or NMP + 3.6 eq HATU + 7.2 eq DIPEA in NMP l x 40 min 7 DMF, wash 5 x 1 min 8 20% piperidine/DMF 1 x 5 min and 1 x 15 min or 2 x 2 min b) 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min a) In the coupling cycle following coupling of an N-alkyl amino acid residue and for coupling of the first protected amino acid residue to Sieber amide resin, step 5 was omitted and step 6 was performed twice instead, b) Reduced times were used for Fmoc deprotection of an amino acid residue having a carboxyl group protected as allyl ester, and for the Fmoc deprotection step of the following coupling cycle. 1.1.2.1 Method A The reaction cycles, as described herein above, were applied for the assembly of the fully protected peptide fragment, using appropriately protected Fmoc amino acid building blocks. Steps 5 to 9 are repeated to add each amino acid residue. 1.1.3.1 Procedure B: Preparation of a peptide having macrolactam cycles in module A and module B The linear peptide was assembled on solid support according to Method A, as described above, and subsequently the following steps were performed: Allyl deprotection (module A) Selective removal of the allyl protecting group from a carboxyl function was performed as described in the corresponding section in procedure A for removal of the alloc protecting group. Macrolactam cycle formation (module A) To the resin in CH2CI2, 2 eq FDPP in 0.5 mL DMF and 2 eq DIPEA in 0.5 mL CH2CI2 were added. After stirring the reaction mixture for approximately 16 h, the resin was filtered off, and fresh solutions of reagents were added to repeat the procedure. Subsequently, the resin... |
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