Brian F. Fisher

Heterogeneous H-Bonding in a Foldamer Helix

Structural characterization of new α/γ-peptide foldamers containing the cyclically constrained γ-amino acid I is described. Crystallographic and 2D NMR analysis shows that γ residue I promotes the formation of a 12/10-helical secondary structure in α/γ-peptides. This helix contains two different types of internal H-bond, and the data show that the 12-atom C═O(i) → H-N(i+3) H-bond is more favorable than the 10-atom C═O(i) → H-N(i-1) H-bond. Several foldamer helices featuring topologically distinct H-bonds have been discovered, but our findings are the first to show that such H-bonds may differ in their favorability.

Role of ring-constrained γ-amino acid residues in α/γ-peptide folding: single-conformation UV and IR spectroscopy.

The capped α/γ-peptide foldamers Ac-γACHC-Ala-NH-benzyl (γα) and Ac-Ala-γACHC-NH-benzyl (αγ) were studied in the gas phase under jet-cooled conditions using single-conformation spectroscopy. These molecules serve as models for local segments of larger heterogeneous 1:1 α/γ-peptides that have recently been synthesized and shown to form a 12-helix composed of repeating C12 H-bonded rings both in crystalline form and in solution [Guo, L.; et al. J. Am. Chem. Soc. 2009, 131, 16018]. The γα and αγ peptide subunits are structurally constrained at the Cβ-Cγ bond of the γ-residue with a cis-cyclohexyl ring and by an ethyl group at the Cα position. These triamides are the minimum length necessary for the formation of the C12 H-bond. Resonant two-photon ionization (R2PI) provides ultraviolet spectra that have contributions from all conformational isomers, while IR-UV hole-burning (IR-UV HB) and resonant ion-dip infrared (RIDIR) spectroscopies are used to record single-conformation UV and IR spectra, respectively. Four and six conformers are identified in the R2PI spectra of the γα and αγ peptides, respectively. RIDIR spectra in the NH stretch, amide I (C═O stretch), and amide II (NH bend) regions are compared with the predictions of density functional theory (DFT) calculations at the M05-2X/6-31+G* level, leading to definite assignments for the H-bonding architectures of the conformers. While the C12 H-bond is present in both γα and αγ, C9 rings are more prevalent, with seven of ten conformers incorporating a C9 H-bond involving in the γ-residue. Nevertheless, comparison of the assigned structures of gas-phase γα and αγ with the crystal structures for γα and larger α/γ-peptides reveals that the constrained γ-peptide backbone formed by the C9 ring is structurally similar to that formed by the larger C12 ring present in the 12-helix. These results confirm that the ACHC/ethyl constrained γ-residue is structurally preorganized to play a significant role in promoting C12 H-bond formation in larger α/γ-peptides.

Cyclic Constraints on Conformational Flexibility in γ-Peptides: Conformation Specific IR and UV Spectroscopy

Single-conformation spectroscopy has been used to study two cyclically constrained and capped γ-peptides: Ac-γACHC-NHBn (hereafter γACHC, Figure 1a), and Ac-γACHC-γACHC-NHBn (γγACHC, Figure 1b), under jet-cooled conditions in the gas phase. The γ-peptide backbone in both molecules contains a cyclohexane ring incorporated across each Cβ-Cγ bond and an ethyl group at each Cα. This substitution pattern was designed to stabilize a (g+, g+) torsion angle sequence across the Cα-Cβ-Cγ segment of each γ-amino acid residue. Resonant two-photon ionization (R2PI), infrared-ultraviolet hole-burning (IR-UV HB), and resonant ion-dip infrared (RIDIR) spectroscopy have been used to probe the single-conformation spectroscopy of these molecules. In both γACHC and γγACHC, all population is funneled into a single conformation. With RIDIR spectra in the NH stretch (3200-3500 cm(-1)) and amide I/II regions (1400-1800 cm(-1)), in conjunction with theoretical predictions, assignments have been made for the conformations observed in the molecular beam. γACHC forms a single nearest-neighbor C9 hydrogen-bonded ring whereas γγACHC takes up a next-nearest-neighbor C14 hydrogen-bonded structure. The gas-phase C14 conformation represents the beginning of a 2.614-helix, suggesting that the constraints imposed on the γ-peptide backbone by the ACHC and ethyl groups already impose this preference in the gas-phase di-γ-peptide, in which only a single C14 H-bond is possible, constituting one full turn of the helix. A similar conformational preference was previously documented in crystal structures and NMR analysis of longer γ-peptide oligomers containing the γACHC subunit [Guo, L., et al. Angew. Chem. Int. Ed. 2011, 50, 5843-5846]. In the gas phase, the γACHC-H2O complex was also observed and spectroscopically interrogated in the molecular beam. Here, the monosolvated γACHC retains the C9 hydrogen bond observed in the bare molecule, with the water acting as a bridge between the C-terminal carbonyl and the π-cloud of the UV chromophore. This is in contrast to the unconstrained γ-peptide-H2O complex, which incorporates H2O into both C9 and amide-stacked conformations.

Mimicking the First Turn of an α-Helix with an Unnatural Backbone: Conformation-Specific IR and UV Spectroscopy of Cyclically Constrained β/γ-Peptides

The folding preferences of two capped, constrained β/γ-dipeptide isomers, Ac-βACPC-γACHC-NHBn and Ac-γACHC-βACPC-NHBn, (designated βγ and γβ, respectively), have been investigated using single- and double-resonance ultraviolet and infrared spectroscopy in the gas phase. These capped β/γ-dipeptides have the same number of backbone atoms between their N- and C-termini as a capped α-tripeptide and thus serve as a minimal structural unit on which to test their ability to mimic the formation of the first turn of an α-helix. Resonant two-photon ionization and UV-UV hole-burning spectroscopy were performed in the S0-S1 region, revealing the presence of three unique conformations of βγ and a single conformation of γβ. Resonant ion-dip infrared spectra were obtained in the NH stretch region from 3300 to 3500 cm(-1) and in both the amide I and amide II regions from 1400 to 1800 cm(-1). These infrared spectra were compared to computational predictions from density functional theory calculations at the M05-2X/6-31+G(d) level, leading to assignments for the observed conformations. Two unique bifurcated C8/C13 H-bonded ring structures for βγ and a single bifurcated C9/C13 H-bonded ring structure for γβ were observed. In all cases, the H-bonding patterns faithfully mimic the first full turn of an α-helix, most notably by containing a 13-membered H-bonded cycle but also by orienting the interior amide group so that it is poised to engage in a second C13 H-bond as the β/γ-peptide lengthens in size. The structural characteristics of the β/γ-peptide version of the 13-helix turn are compared with the α-helix counterpart and with a reported crystal structure for a longer β/γ-peptide oligomer.

Impact of γ-Amino Acid Residue Preorganization on α/γ-Peptide Foldamer Helicity in Aqueous Solution.

α/γ-Peptide foldamers containing either γ(4)-amino acid residues or ring-constrained γ-amino acid residues have been reported to adopt 12-helical secondary structure in nonpolar solvents and in the solid state. These observations have engendered speculation that the seemingly flexible γ(4) residues have a high intrinsic helical propensity and that residue-based preorganization may not significantly stabilize the 12-helical conformation. However, the prior studies were conducted in environments that favor intramolecular H-bond formation. Here, we use 2D-NMR to compare the ability of γ(4) residues and cyclic γ residues to support 12-helix formation in more challenging environments, methanol and water. Both γ residue types support 12-helical folding in methanol, but only the cyclically constrained γ residues promote helicity in water. These results demonstrate the importance of residue-based preorganization strategies for achieving stable folding among short foldamers in aqueous solution.

Helix Propensities of Amino Acid Residues via Thioester Exchange

We describe the use of thioester exchange equilibria to measure the propensities of amino acid residues to participate in helical secondary structure at room temperature in the absence of denaturants. Thermally or chemically induced unfolding has previously been employed to measure α-helix propensities among proteinogenic α-amino acid residues, and quantitative comparison with precedents indicates that the thioester exchange system is reliable for residues that lack side chain charge. This system allows the measurement of α-helix propensities for d-α-amino acid residues and propensities of residues with nonproteinogenic backbones, such as those derived from a β-amino acid, to participate in an α-helix-like secondary structure.