γ-Peptide

Article pubs.acs.org/JPCA

Role of Ring-Constrained γ‑Amino Acid Residues in α/γ-Peptide Folding: Single-Conformation UV and IR Spectroscopy Ryoji Kusaka,†,‡ Di Zhang,† Patrick S. Walsh,† Joseph R. Gord,† Brian F. Fisher,§ Samuel H. Gellman,§ and Timothy S. Zwier*,† †

Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907-2084, United States Department of Chemistry, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan § Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States ‡

S Supporting Information *

ABSTRACT: The capped α/γ-peptide foldamers Ac-γACHC-Ala-NH-benzyl (γα) and AcAla-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 γresidue 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 singleconformation 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/γpeptides 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. well-known tendency of short α-peptides to be unstructured in solution. Heterogeneous oligomers such as α/β-, α/γ-, and β/γpeptides enhance the conformational versatility among synthetic foldamers.4 Conventional helices formed in αpeptides are characterized by a pattern of repetitive H-bonded rings containing 10 atoms (C10, 310-helix, CO(i) ← HN(i + 3)) or 13 atoms (C13, α-helix, 413-helix, CO(i) ← HN(i + 4)). However, Gellman and co-workers have synthesized heterogeneous α/γ- and β/γ-peptides that form helix structures made of repetitive C12 and C13 CO(i) ← HN(i + 3) H-bonds in αγ and βγ subunits, respectively, both in solution and in crystalline form, in which a cyclohexyl ring constrains the γ-residue at the Cβ−Cγ bond.8,13 As the study of these new foldamers has matured, the need for predictive capabilities has fueled increasing interest in the

1. INTRODUCTION The secondary and tertiary structures of proteins are dictated by a delicate balance of effects, with intramolecular H-bonds that fold the polypeptide backbone playing a leading role in the preferred structures. In recent years, there have been intense efforts to test and extend our understanding of protein folding by creating new types of peptide oligomers (foldamers) that are designed to adopt specific secondary structures and mimic functional properties of proteins.1,2 Synthetic β- and γ-amino acid residues, which contain two- and three-carbon segments between their amide groups, respectively, have played an essential role in efforts to design synthetic polypeptides with discrete folding preferences.3−7 By virtue of their three-carbon segments, γ residues offer a richness in design possibilities that has recently come to the fore as a result of new procedures developed for their synthesis.8−12 One effective design strategy is to incorporate a ring that limits backbone torsional mobility, which can enhance the propensity for a specific secondary structure.13−16 This strategy has produced well-defined helices even for relatively short oligomers, which contrasts with the © 2013 American Chemical Society

Received: August 18, 2013 Revised: September 26, 2013 Published: September 26, 2013 10847

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the γ residues. The pure constrained γ-peptide results are particularly relevant to the present study and will serve as a natural point of comparison in what follows. Molecules γα and αγ can be considered as representing local motifs within the larger γαγα tetramer and αγαγαγ hexamer previously studied by Gellman and co-workers, both of which form a C12 CO(i) ← HN(i + 3) helix in CDCl3 solution and in the solid state.8 The γα and αγ peptides have the minimum length necessary to form a single turn of the C12 CO(i) ← HN(i + 3) helix, making them excellent models that test whether the C12 H-bond is intrinsically favorable. Furthermore, the study of γα and αγ provides detailed conformational insights on the ring-constrained γ-residue. As we shall see, the C12 H-bond forms within γα and αγ in the gas phase, but other H-bonded conformations are observed as well, including several that contain nearest-neighbor C5, C7, and C9 H-bonds, sometimes in combination. Even in these cases, however, the C9 ring displays backbone dihedral angles similar to those present in C12 ring. We surmise on this basis that the ringconstrained γ residue plays a major role in promoting 12-helix formation.

development of accurate force fields and ab initio MO theory that incorporate β and γ residues.17−22 Such development rests on a foundation of experimental tests. In the condensed phase, conformational and secondary structural analysis of peptides via crystallography and NMR spectroscopy has provided considerable insight regarding structural preferences. Here, the preferences are the net result of both intramolecular and intermolecular interactions, including solvation forces. Complementary information can be obtained by conformation-specific UV and IR spectroscopy in the gas phase, where a supersonic expansion can be used to isolate conformational isomers in their zero-point levels for spectroscopic interrogation in the absence of solvent. IR and UV spectra of individual conformations can be obtained using doubleresonance laser-based methods. The IR spectra are particularly important since they report directly on the intramolecular Hbonds present in each conformation via the amide NH stretch, CO stretch (amide I), and NH bend (amide II) fundamentals, which can be compared directly with quantum chemical calculations. Using these conformation specific techniques, our group has studied the conformational preferences of several model synthetic foldamers, including β-, γ-, and α/β-peptides.23−31 In every case, such studies have revealed the local, inherent conformational propensities, including the discovery of unanticipated structures such as those stabilized by amide stacking.26 Here, we present a study of two isomeric model α/γpeptides, Ac-γACHC-Ala-NH-benzyl (γα) and Ac-Ala-γACHC-NHbenzyl (αγ) (Figure 1). These molecules are acylated at the C-

2. EXPERIMENTAL AND COMPUTATIONAL METHODS γα and αγ were synthesized using procedures reported previously.8 Details regarding the synthesis can be found in the Supporting Information. The experimental methods for the laser spectroscopic measurements have been described in detail elsewhere.33 Briefly, the solid sample was wrapped in glass wool and placed in a glass container in an effort to reduce thermal decomposition, then inserted into a stainless steel sample holder. The sample holder was located immediately behind a pulsed valve (Parker General Valve, Series 9) with a 500 μm diameter nozzle orifice and heated to ∼200 °C to obtain enough vapor pressure of the sample. The sample vapor was expanded into vacuum with neon carrier gas (3 bar). The expansion was skimmed prior to entering the ionization region of a time-of-flight (TOF) mass spectrometer. Low total gas flow rates (∼5 bar·cm3/min) were utilized to minimize interference of the gas pulse with the conical skimmer. One-color resonant two-photon ionization (R2PI) spectroscopy was used to record the electronic spectra of the jet-cooled γα and αγ molecules in the S0−S1 region of the benzyl chromophore, monitoring the parent mass channel (m/z 387). The UV photons used for R2PI were generated by the third harmonic of a Nd:YAG (355 nm) pumped tunable dye laser at pulse energies of ∼0.2 mJ/pulse at a 20 Hz repetition rate. Conformation-specific IR spectra were recorded using resonant ion-dip infrared (RIDIR) spectroscopy in the amide NH stretch spectral region (3200−3500 cm−1) and amide I/II (1450−1750 cm−1) region. Scans in the 3500−3650 cm−1 region (not shown) were also carried out to ensure that no OH stretch absorptions due to H2O were present, confirming that all spectra were due to the monomer. These spectra were recorded by monitoring a unique vibronic transition in the R2PI spectrum, while scanning a seeded, Nd:YAG (1064 nm) pumped IR parametric converter (LaserVision), operating at 10 Hz. The IR beam was spatially overlapped with the UV beam, temporally preceding it by 200 ns. When the IR frequency was resonant with a vibrational transition of the conformer of interest, the ground state population of that conformer was depleted, appearing as a loss in the ion signal from the monitored vibronic transition. Given that the IR laser is operated at half the repetition rate of the UV laser, the

Figure 1. Chemical structures of the γα and αγ peptides investigated. Possible H-bonding patterns are represented by arrows, with solid lines indicating H-bonds observed in the present study. The Cn designation indicates the number of atoms involved in a H-bonding ring. These H-bonds can be classified into three groups based on the residue(s) involved: (1) α-residue, C5 and C7 H-bonds (red); (2) γresidue, C7 and C9 (blue); (3) α- and γ-residues, C10 and C12 (green). See text for more specifics of the nomenclature definitions.

terminus and incorporate an ultraviolet chromophore at the Nterminus, which is benzyl-capped. This placement of the aromatic ring removes it from the backbone and is synthetically convenient. Both molecules contain a γ-amino residue constrained by a cis-cyclohexyl ring at the Cβ−Cγ bond, with an ethyl group at the Cα position. The absolute configurations of all four chiral centers in the two peptides are S. In what follows, we will refer to the former molecule simply as γα and the latter as αγ to denote the order of the α and γ residues within these isomers. The present study is part of a larger investigation that includes studies of pure constrained γ-peptides32 and constrained β/γ-peptides with the same capping and constraints in 10848

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corresponds to CO(i) ← HN(i + 3) in terms of amino acids. In this article, we use the square bracket designation for H-bonds because it can individually specify each amide group. Since C7 H-bonds can be formed across either α or γ residues, the designations C7eq and C7ax are used for an α residue, and C7γ is used for a γ residue. The C7eq and C7ax designations differentiate whether the chiral side chain on the α residue takes up an axial or equatorial position relative to the nominal plane of the C7 ring.38 While nearest-neighbor C5, C7eq, C7ax, C7γ, and C9 H-bonds, in principle, can be formed in exactly the same way in both γα and αγ, it is not possible to form C10 NH[1] → OC[3] and C12 CO[1] ← HN[3] Hbonds in the same way for both γα and αγ because of the interchange of α and γ residues. For example, a C12 H-bond in γα involves the N-terminal cap CO and the C-terminal cap NH, while the C12 H-bond in αγ involves NH of γ and CO of α. Thus, we label large single ring H-bonds as C10αγ, C10γα, C12αγ, and C12γα. Solid arrows in Figure 1 indicate Hbonds we have observed in the present study; H-bonds indicated with dotted lines were not observed. IR bands are labeled based on the Cn[i] representation. In the NH stretch and NH bend regions, for instance, C9[2] refers to NH stretch and NH bend fundamentals, respectively, due to the NH[2] group forming a C9 H-bond. When CO groups act as acceptors in a bifurcated double ring, the CO stretch fundamental is labeled with both rings involved. For example, C12γα + C9[1] stands for a CO stretch fundamental due to CO[1] group on amide 1 doubly accepting C12γα and C9 H-bonds. In order to designate the H-bonding structures of individual conformations in γα and αγ, the total H-bonding pattern of the three NH groups in each molecule is used (NH[1]/NH[2]/ NH[3]). For instance, a C5/F/C9* label indicates that NH[1] and NH[3] form C5 and C9 H-bonds, respectively, and NH[2] is free of H-bonding interactions. An asterisk in the representation indicates that the NH[1] and NH[3] groups form a bifurcated H-bond to a CO group. In this labeling scheme, π stands for NH engaging in a H-bond with the π cloud of the benzyl group. A full structural designation would also indicate the dihedral angles in the γ residues and orientations of the benzyl group at the C-terminus and the ethyl group at Cα of the γ residue. Dihedral angles in γ residues are designated by ϕ for C(O)− N−Cγ−Cβ, θ for N−Cγ−Cβ−Cα, ζ for Cγ−Cβ−Cα−C( O), and ψ for Cβ−Cα−C(O)-N, and are not explicitly indicated in the labeling scheme. The phenyl ring orientation is given by a C(O)−N−CCH2−Cphenyl dihedral angle and the ethyl group orientation by the Cβ−Cα−CCH2−CCH3 dihedral angles, respectively. As is standard, gauche+ (g+) refers to a dihedral angle near +60°, gauche− (g−) to ∼−60°, and anti (a) to ∼180° range. Thus, the complete representation for a given structure becomes, for example, C5/F/C9*(g−)(a), (NH[1]/ NH[2]/NH[3](phenyl)(ethyl)). In the results of the calculations, there are several structures that can be designated by the same NH[1]/NH[2]/NH[3] Hbonding family but differ in phenyl and/or ethyl orientation. In some cases, the exact conformational assignments for phenyl and ethyl orientations cannot be made based on the singleconformation spectra because the IR spectra in the NH stretch and amide I/II regions are very similar for such conformers. In those cases, we choose to display results for the most stable structure, referring to it as the assigned structure in the subconformational family having the same H-bonding pattern.

difference in total ion signal between IR “on” and IR “off” was monitored directly using the active baseline subtraction mode of a gated integrator (Stanford Research Systems). For generation of IR light in the amide I/II regions, a AgGaSe2 crystal was placed downstream from the parametric converter output. IR laser powers were 3−5 mJ/pulse in the amide NH stretch region and 0.5−1.0 mJ/pulse in the amide I/II region. Conformation-specific UV spectra were recorded using IRUV hole-burning (IR-UV HB) spectroscopy. A unique IR transition observed in the RIDIR spectrum of a particular conformer was used to burn a hole in the UV-interrogated population. A configuration identical to that for RIDIRS was used, except that the wavelength of the UV laser was scanned while the IR laser was fixed. UV transitions of the conformer sharing the same ground state level as the IR excited population appear in the spectrum. For all IR-UV HB spectra, multiple IR transitions in the individual RIDIR spectra were checked to ensure that the IR band chosen for the hole-burn transition was unique to a particular conformer of interest. The IR bands used for recording IR-UV HB spectra shown here are labeled by an asterisk in RIDIR spectra in the NH stretch region. In order to identify the possible conformational minima associated with each molecule, a conformational search was carried out for each molecule using the Amber* force field34 within the MACROMODEL (Schrödinger) suite.35 Within a 50 kJ/mol energy window, about 300 structures were obtained for each molecule. About 100 of the lowest energy structures were further optimized using density functional theory (DFT) employing the M05-2X functional36 and the 6-31+G(d) basis set, available in the Gaussian09 program package.37 Several of the low-energy conformations were also optimized at the ωB97X-D/6-31++G** level of theory. In the cases tested, the order of the relative energies of the conformers was not changed significantly compared with ordering from the M05-2X calculations. Harmonic frequencies were scaled by 0.940 for the NH stretch and 0.955 for the amide I/II region. In the conformational assignment process, the vibrational frequencies for all DFT optimized structures within 30 kJ/mol of the global minimum were checked against the experimental IR spectra. As we shall see, the combined results from the NH stretch and amide I/II regions provide firm assignments for the H-bonding structures formed in the peptides.

3. STRUCTURAL NOMENCLATURE Three sets of NH and CO groups in γα and αγ peptides are numbered from the N- to the C-terminus. For instance in Figure 1, the NH and CO groups at the N-terminus are NH[1] and CO[1] in both γα and αγ. H-bonds formed in the peptides are represented by Cn, where n stands for the total number of atoms forming the H-bonded ring. In principle, as the arrows in Figure 1 show, six types of Cn H-bonds can be formed in γα and αγ. The six Cn H-bonds are classified into three types based on the residue(s) spanning donor NH and acceptor C O groups. Across an α residue, nearest-neighbor C5 NH[i] → OC[i + 1] and C7 CO[i] ← HN[i + 1] H-bonds can be formed (red in Figure 1). Across a γ residue, nearest-neighbor C7 NH[i] → OC[i + 1] and C9 CO[i] ← HN[i + 1] H-bonds can form (blue). When the H-bond connects terminal amide groups that span both α and γ residues, C10 NH[i] → OC[i + 2] and C12 CO[i] ← HN[i + 2] H-bonds are formed (green). Note that our numbering scheme is based on the amide groups involved rather than the amino acid sequence. Thus, our nomenclature for a C12 H-bond is CO[i] ← HN[i + 2], which 10849

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4.1.2. RIDIR Spectra in the NH Stretch and Amide I/II Regions. Figure 3a shows the RIDIR spectra of γα(A−D) in the NH stretch region (3200−3500 cm−1) , while Figure 3b shows the corresponding spectra in the amide I (CO stretch, 1650−1750 cm−1) and amide II (NH bend, 1450−1600 cm−1) regions. No bands were observed in the 3500−3650 cm−1 region for any of conformers A−D. The RIDIR spectra of A and B are similar in both the NH stretch and amide I/II regions, with only small shifts between them. The NH stretch spectra contain NH stretches at 3362(A) and 3360(B) cm−1 due to moderately strong H-bonds, weakly H-bonded NH stretches at 3428(A) and 3450(B) cm−1, and free NH stretches at 3472(A) and 3473(B) cm−1. RIDIR spectra of A and B in the amide I/II region show three CO stretches and three NH bends with similar frequency and intensity patterns. As expected based on the similarities in the R2PI spectra, the RIDIR spectra of C and D are also similar in their NH stretch and amide I/II IR spectra but are different than those of A and B. In the NH stretch region, there is one free NH stretch at 3477(C) and 3473(D) cm−1 and a pair of bands in D shifted down below 3300 cm−1 (3265 and 3296 cm−1), indicative of the formation of two strong NH···OC H-bonds. The spectrum of C shows a single, broad absorption centered at 3300 cm−1. The unusual breadth of this transition and the presence of only one other resolved transition in the spectrum makes it likely that the 3300 cm−1 band encompasses a pair of unresolved absorptions that are more closely spaced than in conformer C. It should be noted that conformers C and D show two transitions that are shifted to lower frequency than any seen in conformers A and B. This indicates the presence of two particularly strong H-bonds in conformers C and D. The amide II region reflects these strong H-bonds in its pair of bands above 1550 cm−1, which are clearly resolved in D (1554 and 1570 cm−1), but form a single broadened transition in C (centered at 1554 cm−1). We have recently compiled results on the amide I/II spectra for an array of gas phase α-, β-, γ-, and α/β-peptides31 and have shown that NH groups involved in C9 H-bonds of γ-peptides have higher frequency amide II fundamentals than all other Cn rings with n = 5−11, with values near 1560 cm−1, suggesting the presence of a C9 ring especially in conformer D of γα. 4.1.3. Calculated IR Spectra and Assigned Structures for γα. The calculated IR spectra of the assigned structures for A− D in the NH stretch and amide I/II regions are shown in the stick diagrams immediately below the RIDIR spectra in Figure 3. The assigned structures for A and D are shown in Figure 4 as representative members of the A/B and C/D pairs. The left side of Figure 4 shows the F/C9/C12γα*(g−)(a) structure assigned to conformer A in which the ethyl group is in the anti position, while the F/C9/C12γα*(g−)(g−) structure assigned for B (not shown) has the ethyl group in the gauche position. In both structures A and B, two H-bonds (C12γα CO[1] ← HN[3] and C9 CO[1] ← HN[2]) are formed with CO[1] as the acceptor group, leading to a bifurcated double-ring structure. The structure on the right side of Figure 4 is that assigned to conformer D. Both assigned structures C and D are represented as F/C9/C7eq(g+)(a), differing only slightly in the N−C− CphenylCphenyl dihedral (phenyl plane) angle. The C and D conformers form C9 CO[1] ← HN[2] and C7eq CO[2] ← HN[3] H-bonds, resulting in a sequential double ring Hbonding structure composed of C9 and C7eq nearest-neighbor H-bonds.

It is noteworthy that isomers with anti-ethyl orientation are always more stable than those with g+ or g− orientation, according to the calculations, which is consistent with the observation of exclusively anti-ethyl conformations in the crystal structures of the α/γ-peptide tetramer and hexamer.8 The cyclohexyl ring can take on either chair or boat conformations in principle, but in practice only the chair conformation is observed. Calculations suggest that the boat conformation is less stable than the chair by ∼20 kJ/mol. Chair conformations are observed exclusively in the crystal structures.

4. RESULTS AND ANALYSIS 4.1. Ac-γACHC-Ala-NH-benzyl (γα). 4.1.1. R2PI and IR-UV HB Spectra. Figure 2 shows the R2PI spectrum of the γα

Figure 2. R2PI (top trace) and IR-UV HB spectra (lower traces) of γα. Asterisks denote transitions of αγ arising from residual sample in the sample holder. IR-UV HB spectra of conformers B and D are not shown due to interference from incomplete subtraction of signals from the other conformers. See text for further discussion.

peptide, in which four conformers (A−D) are identified by RIDIR and IR-UV HB spectroscopy. The IR-UV HB spectra of A and C are shown below the R2PI spectrum. The S0−S1 origin band positions of conformers A and B appear close to one another in the R2PI spectrum, suggesting that these two conformers share similar structures. In the same way, conformers C and D also form a closely spaced pair of transitions, implying structural similarity between C and D. As we shall see later, their RIDIR spectra in the NH stretch and the amide I/II regions also reflect these structural pairings. The IR-UV HB spectra of B and D were not measured because of interference from strong bands in the vicinity, leading to incomplete subtractions that mask the transitions due to B and D. An R2PI scan taken to the red of the region shown in Figure 2 (37 030−37 300 cm−1) revealed no additional transitions due to other conformers. The relatively strong bands just above the origins of C and D are identified as overlapped vibronic transitions of A, C, and D because their RIDIR spectra showed contributions from all three conformers. The S0−S1 origin positions (in cm−1) of all conformers of γα and αγ are included in the Supporting Information (Table S1). 10850

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Figure 3. RIDIR spectra in the (a) NH stretch and (b) amide I/II regions of γα (A−D). Stick spectra underneath were simulated based on calculations carried out at the DFT M05-2X/6-31+G* level of theory. The assigned structures of A and D are shown in Figure 4. Asterisks in the NH stretch region show the transitions used for recording IR-UV HB spectra.

acceptor. This shortens the C7eq H-bond by ∼0.1 Å relative to its length in isolation and lowers the NH stretch frequency by about 50−60 cm−1. These structures are the α/γ-peptide equivalent of the C8/C7eq structures that are pervasive in α/βpeptides, sharing much in common with them.25,27 These two archetypal H-bonded sequential double ring structures are also reflected in the spectra in the amide I and amide II regions. The interior CO[2] group is involved in a C7eq H-bond but is also affected by the C9 H-bond formed to NH[2]. This leads to a large downshift in frequency for the C7eq CO stretch fundamental to 1668/1664 cm−1 in C and D, respectively. The strengthening of the interior amide group H-bonds in the sequential double ring also leads to an unusually large shift up in frequency for the C9[2] amide II fundamental (NH bend) to above 1550 cm−1. Second, even though our formal nomenclature lists the structures of A and B as C9/C12 bifurcated double rings, a closer look at the structures confirms that the C12γα CO[1] ← HN[3] H-bond dominates the bifurcated structure and weakens/lengthens the C9 CO[1] ← HN[2] H-bond significantly relative to an isolated C9 ring.32 Indeed, while an isolated C9 ring has its NH stretch fundamental 3301 cm−1,32 the corresponding C9 rings in the C9/C12 bifurcated double ring structures appear at 3428 and 3450 cm−1, some 130−150 cm−1 higher in frequency. This leads to a spectrum in which the C12 NH stretch frequency is well below that of the C9 ring. The weak nature of the C9 H-bond is also reflected in the lack of a corresponding shift to higher frequency in its amide II fundamentals of conformers A/B relative to C/D. 4.1.4. X-ray Crystal Structure for γα. An X-ray-quality crystal of γα was grown by slow evaporation of an isopropanol solution. Intramolecular H-bonding is observed of the type CO[1] ← HN[3] (C12), a hydrogen-bonding pattern that is assigned to conformers A and B in the gas phase. In the crystals, intermolecular H-bonding is observed for NH[2] and CO[2] with distinct cocrystallized isopropanol molecules. In the gasphase structures for conformers A and B, however, NH[2] participates in H-bonding with CO[1], while CO[2] is free of H-bonding interactions. The crystalline arrangement of γα also

Figure 4. Calculated structures assigned for A and D of γα at the DFT M05-2X/6-31+G* level of theory. Most hydrogen atoms are omitted for clarity.

The comparison presented in Figure 3 shows the close match between experiment and theory in both NH stretch and amide I/II regions, with only subtle differences between the members of the A/B and C/D pairs. On the basis of these fits, the groups involved in a given IR transition are revealed by the calculations, which in turn shed further light on structural differences between the conformers. We consider such structural differences in greater detail in the Discussion section but mention the most important comparisons here since they inform the spectral interpretation. The full set of IR band assignments and H-bond geometries for γα and αγ are listed in the Supporting Information (Table S1). First, in conformers C and D, the pair of NH stretch fundamentals near 3300 cm−1 is due to the C9 and C7eq Hbonds in the sequential double ring structures, both of which are short, well-oriented H-bonds. In addition, these H-bonds are strengthened by each other’s presence since they are linked by the interior amide group [2], which acts both as donor and 10851

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features intermolecular H-bonding between NH[1] of one γα and CO[3] of a neighboring γα, a stabilization not present in the isolated molecule structures of conformers A and B. The Xray crystal structure for γα is available in the Supporting Information (Figure S1). 4.2. Ac -Ala-γACHC -NH-benzyl (αγ). 4.2.1. R2PI and IR-UV HB Spectra. Figure 5 shows the R2PI spectrum of αγ, in which

transitions. Because of the small intensity of transitions due to conformers E and F in the R2PI spectrum, their IR-UV HB spectra were not possible to measure. They were identified by not being among the transitions that burned out in the four IRUV HB spectra and were confirmed as unique conformers by having unique IR spectra. The small shift in the band origins of conformers A (37307.1 cm−1) and B (37308.5 cm−1), and their similar vibronic patterns indicate formation of similar structures. The IR-UV HB spectrum of conformer C is unique both in its large blue shift (37 716 cm−1) and in its long progression in an ∼10 cm−1 mode, making its electronic origin very weak. We surmise that the environment surrounding the phenyl ring in conformer C is unusual compared to those of the others. The higher electronic transition energy of C might arise from interaction between the chromophore and an amide CO group, analogous to the blue shift observed in βαL(A) and βαD(A′).25 4.2.2. RIDIR Spectra of αγ(A) and αγ(B). Figure 6 shows RIDIR spectra of αγ(A) and αγ(B) in the (a) NH stretch and (b) amide I/II regions. The NH stretch and amide I/II spectra of Ac-γACHC-NHBn (hereafter referred to simply as ‘γ’) are also included in Figure 6 as a logical point of comparison with the γresidue in αγ. As the chemical structures on the left side of Figure 7 shows, ‘γ’ has the same constrained γ-residue and benzyl cap as the C-terminal end of αγ. A more detailed analysis of this spectrum will be taken up elsewhere as a part of our study on pure constrained γ-peptides.32 We briefly review here the spectrum of γ in order to set the present work in context. In the NH stretch region, the spectrum of γ shows two NH stretch fundamentals, a sharp transition at 3476 cm−1 due to a free NH, and an intense, broadened band centered at 3301 cm−1 due to a strongly Hbonded NH group that is part of a C9 ring. The effects of the strong C9 H-bond are less dramatic in the amide I region, where the free and H-bonded CO stretch fundamentals appear at 1704 and 1690 cm−1. The amide II region (NH bend) is more sensitive and follows the opposite trend, with free and C9 H-bonded NH groups at 1523 and 1558 cm−1, respectively.32 The spectra for αγ(A) and αγ(B) in the NH stretch region are almost identical, with experimentally resolvable differences

Figure 5. R2PI (top trace) and IR-UV HB spectra (lower traces) of αγ. IR-UV HB spectra of minor conformers E and F are not shown due to interference from incomplete subtraction of signals from the other conformers. See text for further discussion.

six conformers (A−F) are identified by RIDIR and IR-UV HB spectroscopy. The IR-UV HB spectra of four conformers (A− D) are shown below the R2PI spectrum. The origin band positions of the conformers are spread over a surprisingly large range of almost 500 cm−1 (37 300−37 800 cm−1), indicating the existence of some conformers in which the phenyl rings are in distinctly different environments. Careful searches in the 37 040−37 300 cm−1 lower frequency region showed no additional

Figure 6. RIDIR spectra in the regions of (a) NH stretch and (b) amide I/II of γ, αγ(A), and αγ(B). The stick spectra below are calculated at the DFT M05-2X/6-31+G* level. Assigned structures A and B are shown in Figure 7. Asterisks in the NH stretch region indicate the bands that are used for recording IR-UV HB spectra. 10852

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weakly H-bonded or free CO groups. The amide II region shows just two resolved transitions, making it difficult to analyze or interpret without further knowledge of the structures. 4.2.3. Calculated IR Spectra and Assigned Structures for αγ(A) and αγ(B). The calculated IR spectra of the assigned structures for αγ(A) and αγ(B) are shown below the RIDIR spectra in Figure 6. For comparison, the analogous diagram for the assigned structure of γ is reproduced as well.32 The right side of Figure 7 compares the assigned C9 structure for γ from Walsh et al.32 (F/C9(g−)(a)) with the structure assigned to αγ(A or B). The two structures assigned to A and B differ only in the ethyl side chain position, which is anti (a) in one and gauche (g+) in the other. According to our labeling scheme, we label one as C5/F/C9*(g−)(a) (shown) and the other as C5/ F/C9*(g−)(g+) (not shown). The differences between the two are sufficiently small, that we cannot confidently assign which of these two is conformer A and which is B. Note that the corresponding (g−) conformer, C5/F/C9*(g−)(g−), is excluded as an assignable structure because its IR spectrum is distinctly different than the other two, with shifts that are substantially larger than the pair observed experimentally. This is consistent with its higher energy (20.51 kJ/mol). Perhaps more importantly, the assigned structures of γ and αγ are also very similar. Both form a C9 CO[2] ← HN[3] Hbond and share the same phenyl ring position (g−). In αγ, however, the N-terminal α-peptide subunit incorporates an additional C5 NH[1] → OC[2] H-bond that makes the structure a bifurcated double ring with both C5 and C9 Hbonds sharing the same CO[2] acceptor group. The calculated stick spectra in Figure 6 replicate well the IR spectral patterns and the way in which they shift in going from an isolated C9 in γ to a bifurcated C5/C9 structure in αγ. Several aspects of this comparison deserve mention. (1) The C9[3] NH stretch bands of αγ A and B shift up in frequency by about 70 cm−1 relative to the corresponding C9 NH stretch of γ. On the basis of the assigned structures in Figure 7, this shift of the C9[3] NH stretch band must be due to the fact that the C9 CO[2] ← HN[3] H-bond is weakened

Figure 7. Chemical and 3D optimized structures of γ and αγ(A or B), showing the close similarity between the two.

between the two of 1 cm−1 or less. Transitions due to strongly H-bonded NH stretch at 3372/71 cm−1 (A/B), weakly Hbonded NH stretch (3443/44 cm−1, A/B), and free NH stretch (3460/60 cm−1, A/B) are apparent. In addition, the high frequency tail of the H-bonded NH stretch displays a fourth transition, which appears in both conformers at 3384/87 cm−1 (A/B). There are two possible sources of these transitions. Most likely to us is a Fermi resonance between the H-bonded NH stretch and combinations of two CO stretches (1683 + 1706 = 3389 cm−1). Alternatively, the satellite bands could be due to a low frequency vibration (∼15 cm−1) built off the Hbonded NH stretch. Further discussion of these bands is included in Supporting Information. The frequency separation between the satellite bands provides a means for recording the IR-UV HB spectra. The spectra of αγ(A) and αγ(B) in the amide I region are also nearly identical, with CO stretch fundamentals at 1683, 1706, and 1711 cm−1. On the basis of the spectrum of γ, this frequency pattern is consistent with the presence of one strongly H-bonded CO group and two

Figure 8. RIDIR spectra in the (a) NH stretch and (b) amide I/II region of αγ (C−F). The stick spectra below are calculated at the DFT M05-2X/ 6-31+G* level. Assigned structures C−F are shown in Figure 9. Asterisks in the NH stretch region indicate the bands that are used for recording IRUV HB spectra. 10853

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Figure 9. (a) Assigned structures for αγ calculated at the DFT M05-2X/6-31+G* level of theory. Most hydrogen atoms are omitted for clarity. (b) Mirror-reflected αγαγαγ hexamer crystal structure (ref 8).

consequence of the two H-bonds formed to CO[2], which is changing the electrostatic environment of the NH on the same amide group. 4.2.4. RIDIR Spectra in the NH Stretch and Amide I/II Regions of αγ(C−F). Figure 8 shows RIDIR spectra of conformers C−F of αγ in the (a) NH stretch and (b) amide I/II regions of the infrared. When compared with the close similarities present between conformers A and B, the IR spectra of αγ(C−F) are each quite different from one another, indicating that they form different types of H-bonded structures. Here we focus primary attention on the qualitative appearance of the NH stretch spectra, leaving discussion of the amide I/II until the calculations are considered. The RIDIR spectrum of conformer C in the NH stretch region shows one band due to a strongly H-bonded NH at 3364 cm−1, and two free NH fundamentals at 3465 and 3481 cm−1. In this sense, the general appearance of the spectrum of αγ(C) (Figure 8C) is similar to that of A and B (Figure 6A,B). However, both of the free NH stretch fundamentals are well above 3450 cm−1. This structure is likely due to a singly Hbonded ring. The RIDIR spectrum of conformer D in the NH stretch region shows one band due to a strongly H-bonded NH at 3363 cm−1, and two bands due to weakly H-bonded NH groups at 3420 and 3447 cm−1. The spectrum of conformer E in the NH stretch region has two bands due to strong H-bonded NH at 3301 and 3330 cm−1 and a single free NH stretch transition at 3471 cm−1. The spectral features of E both in the NH stretch and amide I/II regions are very similar to those of γα(D), pointing to αγ(E) as a sequential double ring H-bonding structure. Finally, conformer F has one NH stretch band due to strongly H-bonded NH groups at 3336 cm−1, a weakly Hbonded NH stretch at 3412 cm−1, and a free NH at 3458 cm−1. 4.2.5. Calculated IR Spectra and Assigned Structures of αγ(C−F). The calculated spectra of the assigned structures are shown below each RIDIR spectrum in Figure 8. The assigned structures themselves are shown in Figure 9a and are summarized in Table 1. Interestingly, the four structures

by formation of a bifurcated H-bond in which the same CO group now also accepts a H-bond from the C5 ring. While the calculations show a qualitative shift to higher frequency in accord with experiment, the magnitude of the effect is underestimated, raising the possibility that the calculations do not fully account for some aspects of bifurcated ring formation. We will look more closely at the weakening induced by bifurcated ring formation, an anticooperative effect, in the Discussion section. (2) The free NH stretch band (F[2]) of αγ is 16 cm−1 lower in frequency than the free NH stretch of γ (3476 vs 3460 cm−1). This shift is striking because the free NH[2] is not directly involved in the bifurcated double ring but experiences them only indirectly through the two H-bonds to the CO[2] group, potentially revealing another spectral signature of anticooperativity in the bifurcated H-bond. (3) The C5[1] NH stretch band of αγ is located lower in frequency than F[2] NH stretch by 12 cm −1 . The corresponding experimental separation is 16 cm−1. The C5 NH stretch of αγ(A) and αγ(B) (3443 and 3444 cm−1) are close to those of αβ peptide conformers forming similar bifurcated C5/F/C8* H-bond (3448 cm−1 for αβ and 3454 and 3450 cm−1 for αβACPC)25,27 but are higher than the C5 NH fundamental in an α-peptide that forms a single C5 H-bond (3438 cm−1).39 (4) The C5 + C9[2] CO stretch band of αγ shifts down in frequency to 1683 cm−1 compared with the C9[2] CO stretch of γ (1690 cm−1). This shift is due to additional C5 NH[1] → OC[2] H-bond formation in αγ. Interestingly, this shift indicates that the two weak C9 and C5 H-bonds to the same CO[2] in αγ(A) and αγ(B) lead to a net larger downshift relative to the shift caused by the formation of the single strong C9 H-bond to the CO[2] in γ. (5) Finally, despite the fact that experiment does not clearly resolve the three amide II fundamentals, the calculations predict that the NH bend on the interior free amide NH[2] is shifted to approximately 1550 cm−1, almost as far as if it was involved in a strong H-bond. This again is a predicted 10854

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Table 1. Dihedral Angles in γ- and α-Residues Calculated at the DFT M05-2X/6-31+G* Level of Theory, for the Indicated Conformers of γα and αγa γ-residue label γα

αγ

γc αc αγαγαγd

A B C D crystal Ab Bb C D E F anti gauche+ crystal

α-residue

H-bond assignment

ϕ

θ

ζ

ψ

ϕ

ψ

F/C9/C12γα* F/C9/C12γα* F/C9/C7eq F/C9/C7eq F/F/C12γα C5/F/C9* C5/F/C9* F/C7eq/F π/C7ax/C12αγ* F/C7eq/C9 F/C7eq/C9b F/C9 F/C7eq F/C7eq F/F/C12γα/C12αγ/C12γα/C12αγ

−105 −98 −101 −103 −131 −103 −101 -151 -150 −107 −116 −106

62 64 67 64 64 66 67 58 56 66 70 66

71 68 71 78 54 76 74 51 46 75 -82 75

-126 -141 -119 −101 −131 −103 −103 −110 −108 −103 98 −103

−67 −67 −85 −82 −106 −161 −161 −85 77 −85 −85

−30 −25 57 61 2 160 159 74 −63 70 64

−83 −83 −76 −56 −67

84 55 −23 −41 −46

−134 −134 −162

58 57 55

51 56 51

−109 −110 −116

The values in the X-ray structures are shown for γα and the αγαγαγ hexamer crystals. bαγ(A) row = C5/F/C9*(g−)(a) and αγ(B) row = C5/F/ C9*(g−)(g+), differing in the ethyl group dihedrals. cα = Ac-Phe-NHMe; γ = Ac-γACHC-NHBn. dFrom ref 8. Dihedral angles are mirror-reflected values of the structure having R configuration at all chiral carbons. a

already suggested that the higher electronic transition energy of C might arise from interaction between the phenyl ring and an amide CO group, analogous to that observed in βαL(A) and βαD(A′).25 Now we see that the proposed F/C7eq/F(a)(a) structure possesses a free CO[2] that approaches the phenyl ring from the side (RCO···H = 2.57 Å from the nearest aromatic H), much as it does in βαL(A) and βαD(A′) (RCO···H = 2.59 Å).25 The lowest-frequency mode in F/C7eq/F(a)(a) (16 cm−1 in S0) involves motion of the phenyl ring rocking against C O[2] and the Ala side chain, which is positioned directly above the phenyl ring (the closest distance between aromatic C and Ala H is 2.91 Å). It seems likely then that this mode is the one responsible for the long Franck−Condon progression observed. Indeed, the TDDFT optimized S1 state geometry for F/C7eq/ F(a)(a) has the CO[2] group bending up relative to the amide plane, consistent with this group’s involvement with the long progression observed. The bifurcated double ring structure for conformer D, π/ C7ax/C12αγ*(a)(a), incorporates a C7ax CO[1] ← HN[2] Hbond. Previous studies on α-peptides have identified two types of C7 rings, labeled C7eq and C7ax, with the C7eq rings calculated to be ∼10 kJ/mol more stable than C7ax.40−42 As a result, it is initially surprising that the π/C7ax/C12αγ*(a)(a) structure assigned to αγ(D) is predicted by the calculations as the global minimum. It is noteworthy that this structure is the only one of those observed that incorporates three H-bonds, with the C7ax ring complemented by both π and C12αγ Hbonds. However, the H-bond distances and IR vibrational frequencies indicate that the C7ax H-bond dominates the structure, hindering optimal C12 H-bonding formation. In fact, the H-bond distances associated with C7ax RCO[1]←HN[2] and C12αγ RCO[1]←HN[3] H-bonds are 1.79 and 3.14 Å, respectively. As a consequence, in the NH stretch region, the C12αγ[3] NH stretch band appears at 3447 cm−1, just slightly lower in frequency than a free NH stretch (>3450 cm−1). The π[1] NH stretch at 3420 cm−1 is also quite unique and is only matched

provide examples of four unique H-bonded family types. Conformer C is assigned to a single-ring C7eq structure labeled as F/C7eq/F(a)(a) involving a CO[1] ← HN[2] nearestneighbor H-bond. Conformer D is a bifurcated double ring structure, π/C7ax/C12αγ*(a)(a), with C7ax CO[1] ← HN[2] and C12αγ CO[1] ← HN[3] H-bonds. Conformers E and F are both sequential double ring C7/C9 structures, with E assigned to F/C7eq/C9(g−)(a) and F to F/C7eq/C9b(g+)(a). While both E and F have nominally C7eq CO[1] ← HN[2] and C9 CO[2] ← HN[3] H-bonds, the two differ quite significantly due to the unique type of C9 ring formed in F (labeled C9b in Figure 9a). The predicted IR spectrum for F/C7eq/F(a)(a) provides a satisfactory match with experiment for conformer C of αγ. It is one of only a few low-energy structures that possess a single Hbonded NH and two free NH stretch transitions. The strongly H-bonded NH stretch fundamental at 3364 cm−1 is accurately predicted by the C7eq H-bonded NH stretch, which is very close to C7eq NH stretch frequencies (3350 and 3362 cm−1) of the conformers of the α-peptide Ac-α-Phe-NHMe.39 While the presence of two free NH groups is unequivocal from the spectrum, the calculated separation between the two free NH stretch transitions F[3] and F[1] (1.7 cm−1) is much smaller than the experimental separation (16 cm−1). The reason for this discrepancy is unclear; however, it should be noted that the calculated splitting increases to 5.4 cm−1 when the ωB97X-D/ 6-31++G** level of theory was used, suggesting that subtle dispersive interactions may modulate this splitting. The amide I/II regions of C are also nicely reproduced by the F/C7eq/F H-bonding structure. The C7eq[1] CO stretch fundamental is not clearly observed in the experimental spectrum but is tentatively assigned to the very weak transition at 1679 cm−1. Final pieces of evidence in support of the assignment of F/ C7eq/F(a)(a) to αγ(C) come from its unique R2PI spectrum (Figure 5C), with an S0−S1 origin shifted further blue than all others and possessing a long Franck−Condon progression involving a low frequency vibration (∼10 cm−1). We have 10855

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by the π/C7ax/C12αγ*(a)(a) structure, strengthening the assignment of D to this structure. The fit in the amide I/II region is less satisfactory. We note that the match-up between experiment and calculation in the amide I/II region is often compromised in bifurcated double ring structures, suggesting that anticooperative effects may represent a particular challenge for theory. In the amide I region, the calculation predicts that F[2] and F[3] should be resolved; however, the single strong broadened band at 1712 cm−1 is likely an unresolved pair of bands due to F[2] and F[3]. We do not clearly observe the transition due to C7ax + C12[1] CO stretch, which is calculated to be quite weak. In the amide II region, there are four observed bands. From the calculated spectrum, the bands at 1537, 1507, and 1494 cm−1 are tentatively assigned to C7ax[2], π[1], and C12αγ[3] NH bend vibrations, respectively. Conformer E of αγ is assigned to the F/C7eq/C9(g−)(a) sequential double ring structure. The similarity between the spectrum of αγ(E) and that of γα(D) is striking. The latter is an F/C9/C7eq sequential double-ring structure with C9 and C7 positions swapped relative to those in αγ(E). The calculations identify the NH stretch fundamentals at 3301, 3330, and 3471 cm−1 with NH groups localized on the C9[3], C7eq[2], and F[1] NH groups, respectively. The C9[3] NH stretch band of E appears at the same frequency as the corresponding C9 fundamental in γ (3301 cm−1). While the calculated, scaled frequencies are not shifted to low enough frequency (3332 for γ, 3336 for αγ(E)), they predict C9[3] bands in close proximity to one another as observed. On this basis, we surmise that NH[3] in αγ(E) is independently H-bonded to CO[2], as it is in γ, and is distinct from that found in αγ(A) and αγ(B), which form bifurcated H-bonds. The experimental frequency of the C7eq[2] amide NH stretch fundamental (3330 cm−1) of αγ(E) is over 30 cm−1 lower than those of C (3364 cm−1) and D (3363 cm−1), indicating that the C7eq[2] H-bond in E is stronger than those of C and D due to cooperative strengthening involving the interior amide group.25,27 We will consider this further in the Discussion section. In the amide I region, the three CO stretch fundamentals are coupled strongly enough to one another that we choose not to label individual bands as arising from a single CO group. In the amide II region, the band at 1565 cm−1 is due to C7eq[2], consistent with the strong Hbond formed with NH[2], about 40 cm−1 higher in frequency than the free NH[1]. These shifts are reproduced well by the calculation. The band due to C9[3] NH bend is not observed in the RIDIR spectrum because of its weak intensity. Finally, the tentatively assigned structure for Conformer F, F/C7eq/C9b(g+)(a), is a C7/C9b sequential double ring structure that differs in the type of C9 ring formed. The C7eq H-bond CO[1] ← HN[2] appears (3336 cm−1) at a frequency similar to that of the transition in αγ(E) (3330 cm−1). However, the C9b NH[3] band appears more than 100 cm−1 higher in frequency than the other C9 H-bonded NH groups found in γ, αγ(A), αγ(B), and αγ(E). In the C9b ring, the Hbond distance RCO[2]←HN[3] is substantially longer (2.15 Ǻ ) than the corresponding C9 H-bond distance in γ (1.92 Ǻ ), αγ(A/B) (1.94 Ǻ ), and αγ(E) (1.93 Ǻ ), consistent with its shift to higher frequency.

Ala-γACHC-NH-benzyl (αγ), which incorporate a constrained γ residue, has identified four conformers of γα and six conformers of αγ. The observed structures form a diverse set that includes one C7eq single-ring structure, four C9/C7eq, and C7eq/C9 sequential double ring structures, two C5/C9 bifurcated double rings, two C9/C12 bifurcated double rings, and one C7ax/C12 bifurcated double ring. 5.1. Role of the Ring-Constrained γ-Residue. A primary motivation for the present study has been to understand the influence of the ring-constrained γ residue on the folding preferences of α/γ-peptides. Of the ten structures collectively identified for γα and αγ, seven of them, γα(A−D) and αγ(A, B, E), form a nearest-neighbor C9 H-bond, which was previously observed to be favored by the simpler molecule Ac-γACHCNHBn (single γ residue only).32 The remaining three conformers of γα and αγ either do not form the C9 H-bond (αγ(C), F/C7eq/F, and αγ(D), π/C7ax/C12αγ*) or take up a nonstandard C9 ring type (αγ(F), F/C7eq/C9b). The dihedral angle θ is dictated by the ACHC ring, which locks in a gauche(+) dihedral angle 56° < θ < 70° in all ten observed conformers (Table 1). At the same time, the ACHC ring and the ethyl side chain imposes a pronounced gauche(+) preference on the dihedral angle ζ, which in nine of the ten conformers falls in the range 46° < ζ < 78° (Table 1). In combination, these two standard g+ dihedral angles dispose the γ-peptide residue for C9 H-bond formation. Indeed, because conformers γα(A−D) and αγ(A, B, E) all possess a C9 H-bond between the two amide groups that span the γ residue, all four dihedral angles in the γ residue (ϕ, θ, ζ, ψ) are very similar in all of these conformers, as shown in Table 1, with the ranges −107° < ϕ < −98°, 62° < θ < 67°, 68° < ζ < 78°, and −141° < ψ < −101°. Importantly, the ϕ, θ, ζ, and ψ dihedral angles of αγ(C) (−151°, 58°, 51°, −110°) and αγ(D) (−150°, 56°, 46°, −108°), which do not form a C9 H-bond, are still remarkably similar to those of the conformers that form a C9 H-bond, differing primarily in the value of ϕ, which increases to ϕ = −150/−151° in the absence of the C9 H-bond. In these two structures (Figure 9), CO[2] is free, and NH[2] swings around toward CO[1] to form a C7 H-bond across the α residue. The similarities in the γ residue dihedral angles indicate that the ring-constrained γ residue in γα and αγ locks in the same overall peptide backbone architecture, even for αγ(C) and αγ(D), which do not form a C9 H-bond. One of the spectral consequences of this structural rigidity in the γ residue is directly seen in the RIDIR spectra in the NH stretch region of γα (Figure 3a). In all four conformers of γα, the free NH[1] fundamentals appear at virtually the same position (∼3475 cm−1). Thus, the strong structural preference in the constrained γ residue fixes NH[1] in a specific position away from the other amide groups, preventing formation of a H-bond with the NH[1] group. The rigidity of the γ residue has been assumed to favor sequential C12 H-bond formation (12-helix) that the larger α/ γ-peptides display in crystalline form and in solution. Figure 9b shows a mirror-image version of the crystal structure of αγαγαγ hexamer, which forms a 12-helix. We show the mirror image because all of the chiral centers in the hexamer are R, while the molecules studied here are all S.8 As highlighted by blue shading in Figures 4 and 9, the γ residue conformations in all γα and αγ conformers except αγ(F) are similar to those in the crystal structure. This similarity to the larger peptide is obvious also in the γ residue dihedral angles listed in Table 1.

5. DISCUSSION The present study of the single-conformation spectroscopy of α/γ-peptide foldamers Ac-γACHC-Ala-NH-benzyl (γα) and Ac10856

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accommodate diverse H-bond arrangements, which depend to some degree on the order of α and γ residues. In γα, the NH[3] group associated with the α residue can form C12γα (γα(A,B)) and C7eq (γα(C,D)) H-bonds, while in αγ, NH[1] engages in C5 (αγ(A,B)), C7eq (αγ(C,E)), C7ax (αγ(D)), and C12αγ, (αγ(D)) H-bonds. The additional conformational choices offered to αγ might arise partly from the direction of the H-bonds that can form. For example, as can be seen in Figure 1, the bifurcated C5 NH[1] → OC[2] and C9 CO[2] ← HN[3] H-bonds are specific for αγ. In order for γα to form C5 and C9 H-bonds, a different kind of bifurcated H-bonding arrangement is required, involving a single donor NH[2] group and two acceptors, CO[1] and CO[3], an arrangement that is energetically unfavorable. Figure 10a,d displays energy level diagrams for all the structures calculated to be within 20 kJ/mol of the global

Collectively, these structures are consistent with the expectation that the rigid γ residue promotes a g+/g+ conformational preference for the Cα−Cβ/Cβ−Cγ backbone bond sequence, and the structures show that this γ residue conformation can be accommodated in diverse local H-bonding patterns. The inherent propensity for C12 H-bond formation is evident also in the crystal structure of γα, which features a C12 ring as its only intramolecular H-bond (Figure S1, Supporting Information). Both intramolecular and intermolecular H-bonds are observed within this crystal, making it difficult to deduce whether the overall stabilization of the structure is the result of its intramolecular H-bond. To this point, we have argued that the rigidity built into the γ-peptide is the combined consequence of the cyclohexyl ring incorporated at Cβ−Cγ and the ethyl group at Cα. However, recent studies of unconstrained γ-peptides with a γ4-amino acid residue (single substitution on Cγ, Figure 1) also show a strong preference for helix formation and in fact have dihedral angles better suited to mimic the α-helix than the (θ, ζ) = (g+, g+) conformation preferred by the cyclohexyl/ethyl combination.43 Furthermore, Bandyopadhyay et al.44 and Basuroy et al.45 have recently reported that α/γ-peptides with γ4-substitution show C12 H-bond formation in crystalline form. Thus, it is probable that substitution at Cγ is most crucial to C12 H-bond formation in α/γ peptides. It would be interesting in future work to study the conformational preferences of α/γ4-peptides in the gas phase. Previous studies of 1:1 α/γ peptides containing the gabapentin γ residue suggest a preference for C12 formation in these systems.46,47 The γ3-geminal disubstitution of the gabapentin residue restricts the θ and ζ dihedral angles to gauche values (±60°), as required for C12 H-bond formation.46,47 The predisposition of the Cα−Cβ and Cβ− Cγ to gauche conformations, in turn, produces a turn in the γpeptide backbone, as is necessary for formation of the 12-helix; this predisposition can apparently be achieved either through the constraints employed in our study or through spiro cyclohexyl substitution at the γ3 position as in gabapentin.46,47 It remains to be seen whether one type of constrained residue displays a larger 12-helical propensity than the other. One of the reasons why γα and αγ do not always form the C12 H-bond is the flexibility of the α residue. In contrast to the consistent conformation of the γ residue, the α residue in γα and αγ displays diverse torsion angles among the gas-phase structures. Apart from γα(A,B), these α residue torsion angles are very different from those of the crystalline form of the hexamer (Table 1). Thus, the α-residue is flexible enough that γα and αγ can form not only the C12 H-bond but also a variety of other H-bonds including the C7eq (γα(C, D)) and C5 (αγ(A, B)) H-bonded rings. Another contributor to the structural diversity observed for γα and αγ in the gas phase is a tendency to maximize the number of H-bonds. If γα or αγ were to take up the H-bonding pattern observed for the hexamer or γα in the crystal, the gas phase dimer would form a F/F/C12 structure, with a single intramolecular H-bond and several unpaired H-bond donors and acceptors. However, in the crystalline state, backbone amides in the α/γ-peptides can form intermolecular H-bonds that contribute to its overall stabilization. 5.2. Differences between γα and αγ. In this section, we focus on the way in which the residue order (γα vs αγ) affects structural preferences in the gas phase. As discussed above, the α residue in γα and αγ is flexible and therefore free to

Figure 10. Energy level diagrams for all conformational minima of (a) γα and (d) αγ within 20 kJ/mol of the global minimum, calculated at the DFT M05-2X/6-31+G* level of theory. (b,e) Calculated energies of optimized structures in which the −CH2CH3 group was replaced by −CH3 in the γ2-position. (c,f) Calculated energies of optimized structures that additionally replace −C6H5 by −H. Conformers that contain a C12 H-bond are shown in red, C9 in blue, and all others in black.

minimum in γα and αγ, respectively. The energy levels are classified by color based on H-bonding structures that the peptides form, with C12 H-bonds in red, C9 H-bonds in blue, and all others in black. Several important deductions can be made based on the results in Figure 10. First, one can clearly see that all structures within 8 kJ/mol of the global minima are C9 or C12 structures, confirming the strong preference for these structures in both γα and αγ. Second, there are almost three times more conformational minima of αγ (51) within 20 kJ/mol of the global minimum than there are in γα (19). We take this as some indication that αγ is more flexible than γα. The reduced number of low-energy conformations in γα is in part a natural consequence of the unusual stability of the four lowest-energy conformations. Indeed, the global minimum structure of γα is 6.75 kJ/mol more stable than the αγ global 10857

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Table 2. Experimental IR Frequencies of NH Stretch, NH Bend, CO Stretch, and Calculated Structural Parameters of γα and αγ Conformers Forming Sequential Double Ring Structuresa

Single-ring structures for αγ(C) and the γ and α monomer peptides are included for comparison. The parameters due to the C9 H-bond ring are highlighted by red for clarity. bRef 39. cRef 31. dα = Ac-Phe-NHMe; γ = Ac-γACHC-NHBn.

a

minimum, so all four conformations γα(A−D) are lower in energy than any of the conformations of αγ. Part of the structural complexity present in these peptides arises from the presence of conformations that share the same H-bonding architecture but differ in the ethyl group or phenyl ring configurations. To probe the extent to which these seemingly more trivial conformational differences affect the energy level diagram, we carried out geometry optimizations on modified versions of αγ and γα in which (i) the −CH2CH3 group at Cα was replaced by −CH3, or (ii) the phenyl ring (−C6H5) was replaced by H and −CH2CH3 group at Cα was replaced by −CH3. The resultant energy level diagrams for the ethyl to methyl substitution are shown in Figure 10b,e, while those from removal of the phenyl ring and the ethyl to methyl substitution are in Figure 10c,f. Replacing ethyl with methyl (Figure 10b,e) reduces the number of lower energy isomers by almost a factor of 2 in both cases, dropping from 19 to 10 in γα and 51 to 27 in αγ. However, the number of low energy conformations is still almost three times larger in αγ (27) than in γα (10). Furthermore, the global minimum structure for γα is now more stable than the lowest energy αγ conformer by a slightly greater amount (7.8 kJ/mol) relative to the comparison with ethyl groups intact. Somewhat surprisingly, replacement of the phenyl ring with H in γα (Figure 10b,c) increases the number of structures within 20 kJ/mol of the global minimum, as steric constraints imposed by interactions of the phenyl ring with the peptide backbone in γα are removed. As a result, there are now nearly the same number of structures in γα (16) as αγ (15) (Figure 10c,f), and the energy difference between their global minima has dropped to only 0.72 kJ/mol. These results point strongly to the phenyl substituent as the source of the larger number of low-energy conformers available to αγ relative to γα. The energy level diagrams shown in Figure 10a predict that the four observed conformers of γα are also the four most stable conformers. The relative intensities of the four conformers in the R2PI spectrum (Figure 2) are in keeping

with their calculated relative energies, with conformer A most intense, B least intense, and C and D with comparable intermediate intensity. However, for αγ, there is only partial correlation between experimental intensity and relative energy: αγ(D) and αγ(E) are the two lowest-energy structures, but A− C and F are considerably higher, with several missing conformations in between. The assignment of one member of the A/B conformer pair to the C5/F/C9*(g−)(g+) conformer, with relative energy 18 kJ/mol above the global minimum, is especially puzzling. The two structures associated with conformers A/B differ only in the ethyl group orientation but have energies that differ by ∼10 kJ/mol. Several possible reasons for this can be postulated. First, the conformational populations might be dictated by ΔG rather than ΔE. Second, high-energy conformers can retain population during cooling if the barrier(s) to cooling are sufficiently high to trap population behind these barriers in the collisional cooling process.38,53 This may be the case for ethyl group, with only the A/B pair split out from one another in the R2PI spectrum by an amount sufficient to separate the two. Third, conformation-dependent excited state lifetimes could modulate the apparent intensities of the conformers, as has been documented recently for phenylalanine conformers.54,55 In the Supporting Information, we consider each of these possible reasons in somewhat greater detail. 5.3. Spectroscopic and Energetic Consequences of Sequential and Bifurcated Double H-Bonded Ring Formation. One of the intriguing aspects of the present results is the degree to which bifurcated double H-bonded ring structures compete with sequential double rings. Of the ten observed conformers, half of them are bifurcated double rings: γα(A) and γα(B) are C9/C12, αγ(A) and αγ(B) are C5/C9, and αγ(D) is a C7ax/C12 structure. Since bifurcated H-bonds are typically associated with a weakening and lengthening of both H-bonds involved,25,27 it is worth considering why they are formed in such abundance. We also seek to establish and distinguish the spectroscopic consequences of sequential and bifurcated double ring formation. 10858

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Table 3. Experimental IR Frequencies of NH Stretch, NH Bend, CO Stretch, and Calculated Structural Parameters for γα and αγ Conformers Forming Bifurcated Double-Ring Structuresa experimental frequency (cm−1) γα

label

H-bonding structure

NH stretch

NH bend

CO stretch

RN−H

RCO

RCO ← HN

CO ← H angle

O ← HN angle

A

C9 CO[1] ← HN[2] C12γα CO[1] ← HN[3] C9 CO[1] ← HN[2] C12γα CO[1] ← HN[3] C5 NH[1] → OC[2] C9 CO[2] ← HN[3] C5 NH[1] → OC[2] C9 CO[2] ← HN[3] C7ax CO[1] ← HN[2] C12αγ CO[1] ← HN[3] C9 CO ← HN C5 NH → OC

3428 3362 3450 3360 3443 3372 3444 3371 3363 3447 3301 3438b

1509 1557 1503 1557 1522 1551 1522 1551 1537 1494 1558 1499c

1682

1.011 1.014 1.010 1.014 1.010 1.015 1.010 1.015 1.015 1.009 1.016 1.010

1.237

2.180 2.021 2.398 1.955 2.201 1.946 2.206 1.941 1.978 3.140 1.926 2.210

121 136 130 141 86 124 86 125 106 57 126 85

130 172 117 168 105 165 105 166 145 155 165 105

B αγ

Ad Bd D

γe αe

calculated structural parameters (Å or deg)c

1689 1683 1683

1690 1714c

1.235 1.235 1.235 1.233 1.232 1.229

a Corresponding data on γ and α monomer peptides are included for comparison. bRef 39. cRef 31. dαγ(A) row = C5/F/C9*(g−)(a) and αγ(B) row = C5/F/C9*(g−)(g+), differing in the ethyl group dihedrals. eα = Ac-Phe-NHMe; γ = Ac-γACHC-NHBn.

we compare C9 NH stretch and CO stretch fundamentals in γα and αγ with those in γ(Ac-γACHC-NHBn)32 and C7eq fundamentals with those in the C7(g+) conformer of α(AcPhe-NHMe).39 Consider first the effect of the C7eq ring on the C9 rings. Two cases are involved, depending on the order of C7 and C9 rings. In γα(C) and γα(D), the NH group involved in the C9 ring is a part of the interior amide group NH[2]. We see that the C9 NH[2] stretch fundamental in γα(D) (3265 cm−1) is lowered by −36 cm −1 relative to γ (3301 cm −1 ), a strengthening ascribable to the presence of the C7eq C O[2] ← NH[3] H-bond. The C9 NH[2] stretch fundamental in γα(C) is not shifted appreciably relative to the isolated C9 ring nor is the external C9 H-bond (CO[2] ← NH[3]) in αγ(E). Alternatively, consider the effect of the C9 H-bond on the NH and CO stretch fundamentals of the C7eq ring. Again, two cases are involved depending on the ordering of the C7 and C9 rings in the structure. When the C7eq ring is formed with CO[2] ← NH[3], as in γα(C,D), the NH[3] C7eq fundamentals are lowered in the presence of the C9 CO[1] ← NH[2] ring by −36 and −54 cm−1, relative to that of the C7(g+) α-peptide, respectively (Table 2). The CO[2] frequency also responds, lowering by −20 and −24 cm−1, relative to those of the C7(g+) conformer of the α-peptide. However, in αγ(E,F), it is the central amide NH[2] that acts as donor in forming a C7eq ring (CO[1] ← NH[2]). In this case, a smaller shift (−20, −14 cm−1) is observed for C7eq stretches. This frequency lowering is also reflected in a small but noticeable decrease in the H-bond length (−0.03 Å) and in slight increases in the bond lengths of the N−H bonds involved. In summary, it is the γα C9/C7eq structure that shows greatest responsiveness to the presence of two back-toback H-bonds, with the NH and CO groups involved in the C7eq ring undergoing shifts of about −50 and −25 cm−1, respectively, when combined sequentially with a C9 ring. However, in the bifurcated H-bond structures, a single CO group accepts H-bonds from two NH groups. γα(A,B) and αγ(D) contain CO[1] ← HN[2] and CO[1] ← HN[3] Hbonds (either C9/C12 or C7ax/C12), while αγ(A, B) incorporates NH[1] → OC[2] and CO[2] ← HN[3] Hbonds in forming C5/C9 bifurcated double rings. Here, the strength of one H-bond to the CO group may be weakened

Sequential double rings have an interior amide group [2] in which both NH and CO groups are involved in H-bonds. If the polarization induced in the amide group by one H-bond strengthens the second H-bond, and vice versa, the H-bonds are said to be cooperatively strengthened. In larger polypeptides, cooperative effects are involved in stabilizing secondary structures such as helices or beta sheets.48,49 If the CO[1] ← NH[2] H-bond was strengthened by the presence of the additional CO[2] ← HN[3], we anticipate it to show up in decreased frequencies of the NH[2] and CO[1] stretches relative to its single-ring value. Alternatively, if the CO[2] ← NH[3] H-bond is strengthened by the presence of a CO[1] ← NH[2] H-bond, we should see this appear in shifts to lower frequency both in the NH[3] stretch and the CO[2] stretch fundamentals. We evaluate here the extent to which these predictions are borne out by the present data. C9/C7eq sequential double rings are formed in γα(C,D) and their C7eq/C9 counterparts in αγ(E,F). The structural and spectroscopic consequences of sequential double ring formation are summarized in Table 2. The experimental H-bonded NH stretch, NH bend, CO stretch frequencies, and calculated H-bond parameters of γα and αγ are listed in Tables 2 and 3, with those of single-residue reference compounds, diamides derived from the ring-constrained γ-amino acid (″γ″), or from phenylalanine (Ac-PheNHMe, (″α″)).31,32,39 A complete set of the experimental transition frequencies, including free NH stretch and amide I/II frequencies, is included in the Supporting Information (Table S1). The C7/C9 and C9/C7 structures share a structural resemblance with the C8/C7eq and C7eq/C8 sequential double rings that are formed pervasively in short α/βpeptides.25,27 Using the infrared data as a probe of cooperative effects, a comparison was made of the vibrational frequencies between members of the same structural type (e.g., γα(C) with γα(D)) showing shifts that reflect not only cooperative effects but also the structural differences between the two conformers. In order to get a clearer sense of the effects of cooperativity apart from these structural changes, we make primary comparison with the frequencies of isolated single rings of the same (nominal) ring size as is present in the sequential double ring, paying attention to the degree to which the structures of the rings in the two circumstances are similar. Using the dihedral angles as a guide, 10859

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strained α/γ-peptides, this leads to formation of the 12-helix,8 with the NH group involved in the C9 ring reorienting with little change of dihedral angles to form the next member of the C12 sequence.

by the presence of a second one to the same group, a circumstance sometimes referred to as anti-cooperativity or negative cooperativity because the two H-bonds hinder one another instead of working synergistically with one another.50−52 The primary diagnostic for anticooperativity in the present work is the effect of the presence of one H-bond to a given CO group on the frequency of the NH stretch of the other NH group H-bonded to the same group. As with the cooperative effect, it is difficult to distinguish how much of this shift to higher frequency is due to an indirect anticooperative effect mediated by the CO group, and how much of a direct repulsion between the two groups is involved, leading to structural changes that produce longer H-bonds that are weaker. This is a topic deserving further theoretical study. C9 rings are present in both C9/C12 and C5/C9 bifurcated double ring structures. However, a close look at the C9/C12 structures suggests that they are only nominally bifurcated double rings, with primary stabilization provided by the C12 Hbond. This is evident in the fact that the O ← HN H-bond angles of the C9 H-bonds in γα(A,B) are very different from those found in the isolated C9 ring in γ (Table 3), indicating that C9 H-bond formation is inhibited by the more dominant C12γα H-bond to the same CO group. Indeed, in γα(A,B), the O ← HN H-bond approach angles in the C12γα rings are almost 180°, with the NH group approaching the CO endon, thereby blocking optimal approach of the second NH group in forming the C9 ring. This steric hindrance is responsible, at least in part, for the NH stretching frequencies of the C9 Hbond in γα(A,B) appearing at frequencies 130−150 cm−1 higher (3428, 3450 cm−1) than in an isolated C9 ring (3301 cm−1). The effects on the amide II fundamentals are no less dramatic, with the C9 NH bend in C9/C12 conformers shifted ∼50 cm−1 lower in frequency than the C9 fundamental in γ. The C5/C9 bifurcated double ring structures of αγ(A) and αγ(B) are especially well-suited to display the effects of anticooperativity because there is no steric hindrance between the two NH groups in bifurcated double rings that contain a C5 ring. As evidence for this lack of steric hindrance, the CO ← H and O ← HN H-bond angles in the two C5/C9 structures are nearly identical to those found in the γ and α monopeptides (Table 3). Nevertheless, the frequencies of the C9 and C5 NH stretch fundamentals in αγ(A) and αγ(B) shift up in frequency by ∼70 and ∼5 cm−1 relative to the frequencies of isolated C9 and C5 rings, respectively, signaling a distinct weakening of the C9 H-bond induced by the presence of the C5 ring. The effects of bifurcated ring formation are also apparent, albeit weakly, in the amide I/II regions. Interestingly, the C9 NH bend shifts down in frequency by −6 cm−1, while C5 NH bend shifts up by a similar amount. In the amide I region, the C9 CO stretch is lowered by about 7−8 cm−1 relative to that in γ, showing the cumulative effect of the presence of two bifurcated H-bonds formed to the same CO group. Thus, the spectroscopic signatures of sequential and bifurcated double ring α/γ-peptides point to a cooperative strengthening present in the former and an anticooperative weakening in the latter. In the C9/C12 structures, we have argued that the weakened C9 H-bond is a result of the end-on formation of the C12 ring, which displaces the C9 ring from its optimal approach. The fact that the γα(A) bifurcated C9/C12γα structure (Figure 4A) is the most stable of all conformers of γα and αγ points to the underlying stability of the C12 ring imparted by the cyclohexyl/ethyl constrained γ residue. In longer con-

6. CONCLUSIONS Ac-γACHC-Ala-NH-benzyl (γα) and Ac-Ala-γACHC-NH-benzyl (αγ) peptide foldamers have been studied by recording the conformation-specific UV and IR spectra of the isolated molecules cooled in a supersonic expansion. This is the first study of the conformational preferences of α/γ-peptides in isolated form. One of our primary goals was to characterize the role of the ring-constrained γ residue in the folding preferences of these model α/γ-peptides. In solution and in the solid state, constrained 1:1 α/γ-peptides are known to preferentially form C12 rings, leading in larger peptides to a 12-helix. We sought to determine if this same preference would manifest itself in our model peptides, or whether it would be superseded by other factors unique to the gas phase environment. A total of ten conformers were observed and characterized, four for αγ and six for γα. A variety of structural types are present, falling into one of three classifications: sequential double rings (CO[1] ← HN[2]-CO[2]←HN[3]), bifurcated double rings (NH[2] → CO[1] ← HN[3] or NH[1] → OC[2] ← HN[3]), and single-ring nearest-neighbor (CO[1] ← NH[2]) structures. The C12 H-bond is present in three of the ten conformers, but C9 rings are more pervasive, contributing to seven of the ten observed. By comparing all the assigned structures of γα and αγ and the larger peptide crystal structure, it is clear that the four dihedral angles of the γ residue in γα and αγ are quite similar, whether a C9 or C12 ring is being formed. This is because the two central dihedrals θ and ζ, are dictated by the cyclohexyl ring and ethyl group, respectively, and are locked into g+ conformations. The peripheral dihedral angles can then adjust to form either C9 or C12 H-bonded rings with a relatively minor reorientation of the amide groups. Thus, the preference for 12-helix in the larger α/ γ-peptides is present already in these short capped triamide models. This preference is borne out also by the formation of a single C12 intramolecular H-bond in the crystal structure of γα. The variety of H-bonding structures observed for γα and αγ is due in part to the flexibility of the α residue and in part to the preference for forming multiple H-bonds in the gas phase, where intermolecular stabilization does not exist.

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ASSOCIATED CONTENT

S Supporting Information *

Complete set of experimental S0−S1 and IR transition frequencies. Discussion on the satellite IR bands of αγ(A) and αγ(B) in the NH stretch region, and relative energies of conformers. X-ray crystal structure of γα. Details of synthesis of γα and αγ. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(T.S.Z.) E-mail: [email protected]. Notes

The authors declare no competing financial interest. 10860

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(19) Baldauf, C.; Gunther, R.; Hofmann, H. J. Helix Formation in alpha,gamma- and beta,gamma-Hybrid Peptides: Theoretical Insights into Mimicry of alpha- and beta-Peptides. J. Org. Chem. 2006, 71, 1200−1208. (20) Baldauf, C.; Gunther, R.; Hofmann, H. J. Theoretical Prediction of the Basic Helix Types in alpha,beta-Hybrid Peptides. Biopolymers 2006, 84, 408−413. (21) Zhu, X.; Koenig, P.; Gellman, S. H.; Yethiraj, A.; Cui, Q. Establishing Effective Simulation Protocols for beta- and alpha/betaPeptides. II. Molecular Mechanical (MM) Model for a Cyclic betaResidue. J. Phys. Chem. B 2008, 112, 5439−5448. (22) Baldauf, C.; Hofmann, H. J. Ab Initio MO Theory. An Important Tool in Foldamer Research: Prediction of Helices in Oligomers of omega-Amino Acids. Helv. Chim. Acta 2012, 95, 2348− 2383. (23) Baquero, E. E.; James, W. H.; Choi, S. H.; Gellman, S. H.; Zwier, T. S. Single-Conformation Ultraviolet and Infrared Spectroscopy of Model Synthetic Foldamers: beta-Peptides Ac-beta(3)-hPhe-NHMe and Ac-beta(3)-hTyr-NHMe. J. Am. Chem. Soc. 2008, 130, 4784− 4794. (24) Baquero, E. E.; James, W. H.; Choi, S. H.; Gellman, S. H.; Zwier, T. S. Single-Conformation Ultraviolet and Infrared Spectroscopy of Model Synthetic Foldamers: beta-Peptides Ac-beta(3)-hPhe-beta(3)hAla-NHMe and Ac-beta(3)-hAla-beta(3)-hPhe-NHMe. J. Am. Chem. Soc. 2008, 130, 4795−4807. (25) James, W. H.; Baquero, E. E.; Shubert, V. A.; Choi, S. H.; Gellman, S. H.; Zwier, T. S. Single-Conformation and Diastereomer Specific Ultraviolet and Infrared Spectroscopy of Model Synthetic Foldamers: alpha/beta-Peptides. J. Am. Chem. Soc. 2009, 131, 6574− 6590. (26) James, W. H.; Muller, C. W.; Buchanan, E. G.; Nix, M. G. D.; Guo, L.; Roskop, L.; Gordon, M. S.; Slipchenko, L. V.; Gellman, S. H.; Zwier, T. S. Intramolecular Amide Stacking and Its Competition with Hydrogen Bonding in a Small Foldamer. J. Am. Chem. Soc. 2009, 131, 14243−14245. (27) James, W. H.; Baquero, E. E.; Choi, S. H.; Gellman, S. H.; Zwier, T. S. Laser Spectroscopy of Conformationally Constrained alpha/betaPeptides: Ac-ACPC-Phe-NHMe and Ac-Phe-ACPC-NHMe. J. Phys. Chem. A 2010, 114, 1581−1591. (28) Buchanan, E. G.; James, W. H.; Gutberlet, A.; Dean, J. C.; Guo, L.; Gellman, S. H.; Zwier, T. S. Single-Conformation Spectroscopy and Population Analysis of Model gamma-Peptides: New Tests of Amide Stacking. Faraday Discuss. 2011, 150, 209−226. (29) James, W. H.; Buchanan, E. G.; Guo, L.; Geman, S. H.; Zwier, T. S. Competition between Amide Stacking and Intramolecular H Bonds in gamma-Peptide Derivatives: Controlling Nearest-Neighbor Preferences. J. Phys. Chem. A 2011, 115, 11960−11970. (30) James, W. H.; Buchanan, E. G.; Muller, C. W.; Dean, J. C.; Kosenkov, D.; Slipchenko, L. V.; Guo, L.; Reidenbach, A. G.; Gellman, S. H.; Zwier, T. S. Evolution of Amide Stacking in Larger gammaPeptides: Triamide H-Bonded Cycles. J. Phys. Chem. A 2011, 115, 13783−13798. (31) Buchanan, E. G.; James, W. H.; Choi, S. H.; Guo, L.; Gellman, S. H.; Muller, C. W.; Zwier, T. S. Single-Conformation Infrared Spectra of Model Peptides in the Amide I and Amide II regions: ExperimentBased Determination of Local Mode Frequencies and Inter-Mode Coupling. J. Chem. Phys. 2012, 137, 094301. (32) Walsh, P. S.; Kusaka, R.; Buchanan, E. G.; James, W. H.; Fisher, B. F., Gellman, S. H.; Zwier, T. S. Cyclic Constraints on Conformational Flexibility in gamma-Peptides: Conformation Specific IR and UV Spectroscopy. J. Phys. Chem. A 2013, submitted. (33) Shubert, V. A.; Baquero, E. E.; Clarkson, J. R.; James, W. H.; Turk, J. A.; Hare, A. A.; Worrel, K.; Lipton, M. A.; Schofield, D. P.; Jordan, K. D.; et al. Entropy-Driven Population Distributions in a Prototypical Molecule with Two Flexible Side Chains: O-(2acetamidoethyl)-N-acetyltyramine. J. Chem. Phys. 2007, 127, 234315. (34) Weiner, P. K.; Kollman, P. A. AmberL Assisted Model-Building with Energy Refinement. A General Program for Modeling Molecules and Their Interactions. J. Comput. Chem. 1981, 2, 287−303.

ACKNOWLEDGMENTS We gratefully acknowledge support for this work from the National Science Foundation (NSF CHE 1213289 and CHE 1307365). R.K. was supported by JSPS through the program “Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation.”

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