The synthesis of cyclic decapeptides based on the loloatin family of bioactive cyclic peptides is described. Two procedures were used. In the first procedure, developed at Bayer AG Central Research, the resin-linked linear decapeptides were assembled manually in the C- to N-direction by iterative coupling reaction employing TentaGel S RAM resin, with coupling reactions performed in DMF with excess of Fmoc amino acid derivatives, and excess of the coupling reagent DCC/HOBt. Fmoc deprotection was effected with 20% piperidine in DMF, Pd(PPh₃)₄ was used for allyl deprotection, and cyclization was effected with TBTU and HOBt in N-methylpyrrolidone solution with DIEA. Residues were purified by reverse-phase HPLC, and products identified by FAB-MS and NMR spectroscopy. In this way were synt...[ Read more ]

The synthesis of cyclic decapeptides based on the loloatin family of bioactive cyclic peptides is described. Two procedures were used. In the first procedure, developed at Bayer AG Central Research, the resin-linked linear decapeptides were assembled manually in the C- to N-direction by iterative coupling reaction employing TentaGel S RAM resin, with coupling reactions performed in DMF with excess of Fmoc amino acid derivatives, and excess of the coupling reagent DCC/HOBt. Fmoc deprotection was effected with 20% piperidine in DMF, Pd(PPh₃)₄ was used for allyl deprotection, and cyclization was effected with TBTU and HOBt in N-methylpyrrolidone solution with DIEA. Residues were purified by reverse-phase HPLC, and products identified by FAB-MS and NMR spectroscopy. In this way were synthesized all-L loloatin A [cyclic (L-valyl-L-ornithyl-L-leucyl-L-tyrosyl-L-prolyl-L-phenyl-alanyl-L-phenylalanyl-L-asparaginyl-L-aspartyl-L-tyrosyl) (10%); an unidentified product corresponding partly to the structure of all-L loloatin B [cyclic (L-valyl-L-ornithyl-L-leucyl-L-tyrosyl-L-prolyl-L-phenyl- alanyl-L-phenylalanyl-L-asparaginyl-L-aspartyl-L-tryptophanyl)] (12%), and all-L loloatin C [cyclic (L-valyl-L-ornithyl-L-leucyl-L-tyrosyl-L-prolyl-L-tryptophanyl-L- phenylalanyl-L-asparaginyl-L-aspartyl-L-tryptophanyl) (19%), whose structure was confirmed by FAB-MS, and NMR data.

Because of low yields, and difficulties in purification, the resin and peptide coupling was substantially modified for the preparation of natural loloatins A-C, as follows. Polystyrene AM-RAM resin was used, and coupling reactions performed in 25% DMF in 1,2-dichloroethane with 3-fold excess of Fmoc amino acid derivatives, and 3-fold excess of PyBOP/DIEA coupling reagent. The reaction progress was monitored with bromophenol blue. Fmoc- and allyl deprotection was carried out as described above. An excess of HATU/HOAt/DIEA was used for cyclizing the peptide. The residues were purified by reverse-phase HPLC, and FAB-MS and NMR spectra were acquired to confirm the structures of target compounds. In this way were synthesized loloatin A [cyclic (L-valyl-L-ornithyl-L-leucyl-D-tyrosyl-L-prolyl-L-phenylalanyl-D-phenylalanyl-L-asparaginyl-L-aspartyl-L-tyrosyl)] (30.9%), loloatin B [cyclic (L-valyl-L-ornithyl-L-leucyl-D-tyrosyl-L-prolyl-D-phenylalanyl-L-phenylalanyl-L-asparaginyl-L-aspartyl-L-tryptophanyl)] (34.6%), and loloatin C [cyclic (L-valyl-L-ornithyl-L-leucyl-D-tyrosyl-L-prolyl-L-tryptophanyl-D-phenylalanyl-L-asparaginyl-L-asparaginyl-L-tryptophanyl) (36.6%), where all the structures were consistently confirmed by FAB-MS, HRMS and ¹H-NMR and 2D NMR data. In addition, the new procedure was applied to synthesis of all-L loloatin B [cyclic (L-valyl-L-ornithyl-L-leucyl-L-tyrosyl-L-prolyl-L-phenylalanyl-L-phenylalanyl-L-asparaginyl-L-aspartyl-L-tryptophanyl)] (35.4%), of which the structure was also confirmed.

By use of NOESY spectra, it was established preliminarily the 3D structures of loloatins. There are no turn or sheet structures in loloatin A. Whilst for loloation B, there is a γ-turn between Asp⁹, Tyr¹⁰, Val¹, with a hydrogen bond between Asp-CO and Val-NH, and a hydrogen bond between Val-CO and D-Phe-NH, suggesting the presence of a β-sheet among D-Phe⁷, Asn⁸, Asp⁹, Tyr¹, Val¹. As for loloatin C, there is a type I β-turn between Leu³, Tyr⁴, Pro⁵, and a hydrogen bond between Pro-CO and Leu-NH, implying the presence of a 10-membered ring within the cyclic peptide; a intramolucular hydrogen bond also is found between Asn⁸-NH and Val¹-CO.

The CNS-X-Plor program was applied to establish the solution conformations of loloatin C in three different solutions. In DMSO solution, the molecule was shown to possess a hydrophobic aromatic "wall" consisting of Trp⁶and Phe⁷, and a type I β-turn structure involving Val¹, Tip¹⁰, Asp⁹ and Asn⁸ with a hydrophobic “head” at Val¹, Trp¹⁰ and a hydrophilic “tail” at Asp⁹ and Asn⁸; another type I β-turn between Leu³, Tyr⁴, Pro⁵, and Trp⁶ was also located. However, in 70/30 TFE-d3/HZO, loloatin C is shown to possess a dumbbell structure. The 3D structure of loloatin C in 30/70 TFE-d₃/H₂O is shown to possess a dumbbell structure. The 3D structure of loloation C in 30/70 TFE-d₃/H₂O is similar to that in DMSO.

A simple although expeditious 'Sliding Window’ device has been applied to locate the pharmocophore of loloatin C. The device was used to identify three cyclic hexapeptide substructures of loloatin C, namely the 'Southern', the 'Central', and the 'Northern' cyclic hexapeptides. These were then synthesized. Whilst solid phase methods were successful for synthesis of the first two, a solution phase method has to be used for the 'Northern' peptide. The key of the solution phase synthesis was the development of an in situ cyclization method to conduct the final cyclization of the linear peptide. The solution phase methods were then used to synthesize the 'Central' and 'Southern' sequence cyclic peptides as well. The yields for the cyclization were very good, ranging between 70 to 85%.

Structures of the cyclic hexapeptides were confirmed by a combination of FAB-MS and NMR data. Antibiotic tests show that the ‘Northern’ cyclic hexapeptide possesses the highest activity amongst the peptide mimetics. Therefore the basic pharmacophore of loloatin C is suggested to be -Trp-D-Tyr-. Synthesis of other peptide mimics based on reverse turn mimetics and conformational restriction were not successful.