SYNTHESIS AND NMR STUDIES OF A β-TURN MIMETIC MOLECULAR TORSION BALANCE

The attainment of precise measurements of the molecular forces that influence protein folding is important in order to further understand peptide dynamics and stability. A hybrid synthetic-natural peptide motif, combining an o,o,o’-trisubstituted biphenyl with an (ortho-tolyl)-amide, was synthesized in multiple formats and studied by NMR to probe the effects of amino acid substitutions on antiparallel beta-sheet configuration and stability. The potential of this “molecular torsion balance” as a beta-turn mimic was demonstrated by quantifying the rotational barriers about several axes. The free-energy rotational barrier of the aryl-aryl bond was found to be 35.7 kcal mol-1 at 418 K in hexanes. EXSY analysis was also used to measure barriers about the N-aryl (20.9 kcal mol-1 at 343 K in toluene-d8) and N-CO bonds (17.2 kcal mol-1 at 298 K in chloroform-d). The N-aryl barriers of a zwitterionic torsion balance containing a single alanine residue (19.7 kcal mol-1 in acetonitrile-d3 at 343 K, and 22.6 kcal mol-1 in deuterated buffer pD 6.9 at 373 K) showed that rotation about this bond is slower in water, most likely due to the propensity of water to form hydrogen bonds with the charged moieties participating in a salt bridge. Torsion balances were used to study intramolecular hydrogen bond preference by analyzing 1H NMR peak data. An amino acid chain (a single alanine or glycine residue) was found to preferably hydrogen bond with an amide versus an ester carbonyl (1.4:1.0 at 298 K in toluene-d8), and with a secondary amide versus a tertiary amide carbonyl (observation of tertiary amide proton coalescence via variable temperature NMR, from 303 K to 343 K, in toluene-d8). The overall findings suggest that this hybrid torsion balance is a valuable tool that can provide data on conformational dynamics and can examine hydrogen bond and salt bridge interactions of an antiparallel beta-sheet scaffold