Anthranilic acid-containing cyclic tetrapeptides: at the crossroads of conformational rigidity and synthetic accessibility

Abstract

Each amino acid in a peptide contributes three atom units to main-chains, hence natural cyclic peptides can be 9, 12, 15, …. ie 3n membered-rings, where n is the number of amino acids. Cyclic peptides that are 9 or 12-membered ring compounds tend to be hard to prepare because of strain, while their one amino acid homologs (15-membered cyclic pentapeptides) are not conformationally homogeneous unless constrained by strategically placed proline or D-amino acid residues. We hypothesized that replacing one genetically encoded amino acid in a cyclic tetrapeptide with a rigid β-amino acid would render peptidomimetic designs that rest at a useful crossroads between synthetic accessibility and conformational rigidity. Thus this research explored non-proline containing 13-membered ring peptides 1 featuring one anthranilic acid (Anth) residue. Twelve cyclic peptides of this type were prepared, and in doing so the viability of both solution- and solid-phase methods was demonstrated. The library produced contained a complete set of four diastereoisomers of the sequence 1aaf (ie cyclo-AlaAlaPheAnth). Without exception, these four diastereoisomers each adopted one predominant conformation in solution; basically these conformations feature amide N-H vectors puckering above and below the equatorial plane, and approximately oriented their N-H atoms towards the polar axis. Moreover, the shapes of these conformers varied in a logical and predictable way (NOE, temperature coefficient, D/H exchange, circular dichroism). Comparisons were made of the side-chain orientations presented by compounds 1aaa in solution with ideal secondary structures and protein-protein interaction interfaces. Various 1aaa stereoisomers in solution present side-chains in similar orientations to regular and inverse γ-turns, and to the most common β-turns (types I and II). Consistent with this, compounds 1aaa have a tendency to mimic various turns and bends at protein-protein interfaces. Finally, proteolytic- and hydrolytic stabilities of the compounds at different pHs indicate they are robust relative to related linear peptides, and rates of permeability through an artificial membrane indicate their structures are conducive to cell permeability.

Crystal and NMR Structures of a Peptidomimetic β‐Turn That Provides Facile Synthesis of 13‐Membered Cyclic Tetrapeptides

“…Importantly, NMR studies revealed the solution structure of these CTPs to be conformationally homogeneous and the structure of 4 to be in agreement with its crystal structure. This all‐ trans conformation appears to be independant of N ‐methylation, at least in the position prior to 2‐Abz, and the stereoconfiguration of the additional residues, in accordance with the recent solution structure calcuations of Xin and Burgess The predictable and versatile nature of these 2‐Abz CTPs provides significant merit as scaffolds for rational design. Interestingly, the 2‐Abz containing cyclic pentapeptide, cycloaspeptide A, demonstrated four trans ‐amides and a single cis ‐amide bond preceeding one of the two N ‐methylated residues .…”

“…Interestingly, 3 and 4 bear different orientations of the peptide bond at d ‐Phe (Figure ), a consequence of the steric effect from N ‐methylation. The bond orientation observed for 3 is in accordance with solution conformations calculated for similar non‐ N ‐methylated CTPs by Xin and Burgess . The space‐fill models of 3 and 4 (Figure S7) demonstrate the lack of available space for N ‐methylation in the geometry of 3 resulting in the observed bond flipping in 4 .…”

“…The NMR ensemble is very well defined with an average backbone RMSD of only 0.03 Å. The intra‐residue H‐bond of 2‐Abz that is present in both its linear precursor 1 and also compound 4 is now absent, similar to structures calculated by Xin and Burgess . Figure S9 demonstrates the major bond rotations that occur upon cyclization of compound 1 to form compound 3 .…”

“…Recent work of Xin and Burgess reported 2‐Abz containing CTPs, however, their cyclizations yielded only 31–55 % from linear precursors bearing 2‐Abz as the C‐terminal residue. Our improved ring dissection strategy produces linear precursors 1 and 2 with pre‐organised structures, whereby 2‐Abz is favourably placed in the third position.…”

“…While linear peptides mimicing β‐turn geometries provide further applicability for the design of longer sequences as bioactive β‐hairpins, CTPs are also known to mimic a variety of turn conformations and benefit from reduced conformational freedom . Despite providing an attractive approach to probing molecular recognition events or rational drug design, CTPs are often limited by poor synthetic accessibility due to poor efficiency during cyclisation ,. The design of synthetic frameworks with inherently high propensity for cyclisation is therefore of importance to advancing medicinal chemistry.…”