Design, construction and properties of peptide N-terminal cap templates devised to initiate α-helices. Part 3.† Caps derived from N-[(2S )-2-chloropropionyl]-(2S )-Pro-(2R)-Ala-(2S,4S )-4-thioPro-OMe

Abstract

The construction of a 12-membered macrocyclic template capable of entraining attached peptides in helical conformations from acyclic N-[(2S )-2-chloropropionyl]-(2S )-Pro-(2S )-Pro-(2S,4S )-4-thioPro-OMe precursors has been severely hampered by the problem of simultaneously aligning carboxamide dipoles in the transition state for cyclisation. Previously we provided a detailed conformational analysis of the system and tested two methods for forcing the acyclic precursor into the macrocyclic conformation required for helix initiation. First, the destabilisation of unwanted conformations in the transition state for cyclisation, and second, the stabilisation of the favoured transition state structure through the introduction of a hydrogen-bonding interaction. Both strategies were unsuccessful. A third strategy based upon removing the requirement for all of the carbonyl dipoles to align in the transition state at the same time was also tested and the results are presented here. The relaxation of the highly restrained C^α–N bond torsion for Pro³ in the acyclic precursor, through its substitution for a (2R)-alanine residue, effectively decouples the motion of the second carboxamide group from the C^α–N bond torsion and allows the second carboxamide group to rotate. This rotation allows a helical conformation to develop in the transition state to the macrocycle without the need to align all of the carboxamide dipoles and results in successful cyclisation to give template structures of the all trans (ttt ) form. Derivatives of the template were prepared by extending the C-terminus and these were characterised by NMR spectroscopy and restrained simulated annealing. In deuterochloroform solution at low temperature, separate sets of NMR signals were observed for two rapidly interconverting helical conformational isomers of the thioether macrocycle based on (2R)-N-propionyl-(2S )-Pro-(2R)-Ala-(2S )-Pro which possessed an appended trialkylammonium ion. The free energy of activation for the transition (ΔG_c^‡) was 48 kJ mol^–1. A similar time-averaged conformation was also observed in aqueous solution. At –80 °C in dichloromethane the rate of conformational exchange was slowed sufficiently to obtain resonance assignments and NOE data separately for each isomer. In the minor isomer (40%), the four carbonyl oxygen hydrogen-bond acceptors of the template are aligned in an α-helical conformation and in the major conformer the Pro² carbonyl dipole was anti-aligned with the other three dipoles. Thus, the conformers differ in the orientation of one carbonyl group. Molecular modelling calculations showed that the minor isomer was stabilised by coulombic interactions between the trialkylammonium salt and the carbonyl group dipole moments.

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