A Single Stereocenter Changes the Conformation of a Cyclic

Article pubs.acs.org/JPCB

Stereochemistry Rules: A Single Stereocenter Changes the Conformation of a Cyclic Tetrapeptide Fee Li,† Kenny Bravo-Rodriguez,‡ Miguel Fernandez-Oliva,† Juan M. Ramirez-Anguita,‡,⊥ Klaus Merz,§ Manuela Winter,¶ Christian W. Lehmann,‡ Wolfram Sander,*,† and Elsa Sanchez-Garcia*,‡ †

Lehrstuhl für Organische Chemie II, Ruhr-Universität Bochum, D-44801 Bochum, Germany Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany § Lehrstuhl für Anorganische Chemie I, Ruhr-Universität Bochum, D-44801 Bochum, Germany ¶ Lehrstuhl für Anorganische Chemie II, Ruhr-Universität Bochum, D-44801 Bochum, Germany ‡

S Supporting Information *

ABSTRACT: Two novel cyclo(Boc-Cys-Pro-Leu-Cys-OMe) peptides 1 and 2 containing the enantiomeric amino acids D-Leu and L-Leu, respectively, were synthesized to investigate the effect of chiral centers on peptide conformations. By combining a variety of experimental techniques (X-ray crystallography, 2D NMR spectroscopy, temperaturedependent 1H NMR and IR spectroscopy, and UV-CD spectroscopy) with replica exchange molecular dynamics (REMD) techniques and quantum mechanics/molecular dynamics (QM/MM) calculations, we establish that the stereochemistry of just one residue can noticeably influence the properties of the whole peptide and rationalize the origins of this effect, with potential implications for the rational design of peptides of chemical and biological relevance.

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INTRODUCTION Small peptides are useful models for the investigation of molecular interactions in more complex biological systems. Cyclic peptides are especially interesting since cyclization reduces the number of possible conformers while still maintaining the necessary conformational flexibility to mimic folding processes. Conformational studies of a number of cyclic peptides established that tetrapeptides containing proline and a cysteine−cysteine disulfide bridge ensure a β-turn and are thus more rigid than their linear analogues.1−7 The therapeutic potential of small cyclic peptides is well-known.8 Recently, the bioactivity of a conformationally constrained small cyclic peptide has been reviewed.9 Since peptides containing D-amino acids are protease resistant and less immunogenic than their stereoisomers with only L-amino acids,10,11 research in this field has gained relevance during the past years. D-Amino acids and D-peptides have shown potential for improving selectivity and maintaining activity of therapeutic agents,12 as well as for the diagnosis, disease monitoring, and inhibition of amyloid formation and cell toxicity in Alzheimer’s disease, among other applications.13−15 In different peptides with four to eight residues, the conformational change of L-amino acids to their D-forms was reported to result in antibacterial, neurological, and prospective anticancer activity, as well as more proteolytical stability.16−20 The relationship between one amino acid in the D-configuration and the biological activity was investigated, and the relevance of conformational changes in activity was emphasized.20 The use © 2013 American Chemical Society

of D-alanine derivatives to investigate the dynamics of peptidoglycan, an essential component of the bacterial cell wall, has been recently reported.21 Thus, it has become clear that a single D-substitution in various small cyclic peptides can be used to modulate biological response. However, since most studies focus on biological activity versus peptide configuration, the physical origin of this effect remains unclear. In this context, our paper provides a benchmark study of two diastereomeric model peptides cyclo(Boc-Cys-Pro-Leu-CysOMe) 1 and 2 containing D-(1) and L-(2) leucine, respectively, to investigate the effect of single chirality centers on peptide conformation (Figure 1). Crystallization of such peptides can

Figure 1. Chemical structure of cyclo(Boc-Pro-D-Leu-Cys-OMe) 1 (left-hand side) and cyclo(Boc-Pro-L-Leu-Cys-OMe) 2 (right-hand side). Received: July 1, 2013 Revised: August 13, 2013 Published: September 6, 2013 10785

dx.doi.org/10.1021/jp406497r | J. Phys. Chem. B 2013, 117, 10785−10791

The Journal of Physical Chemistry B

Article

simulations of 1 and 2 in acetonitrile (four sets of simulations since in each case we started from both β-II and β-I structures) were also performed using Gromacs 4.629−31 and the OPLSA force field.32 The temperature range for these simulations was 290−400 K, and the temperature distribution was obtained as described by Patriksson and van der Spoel.33 Despite the fact that the solvent used in the simulation was not water, a good exchange ratio between replicas was obtained. The number of replicas used was 23. Initial equilibration of the replicas was achieved by performing 10 ns MDs simulation at constant pressure and 290 K and subsequent 10 ns MD simulations at constant volume and the corresponding temperature for each replica. The electrostatic interactions were treated using the PME method with a cutoff distance of 1.2 nm34 while for van der Waals interactions a twin range cutoff with a distance of 1.2 nm was employed.35 The exchange of replicas was attempted every 1 ps and the time step was 2 fs. Bonds containing hydrogen atoms were kept fixed with the LINCS algorithm.36 The cluster analysis of the results was performed with the Gromos method with a cutoff of 0.15 nm using the backbone atoms of the peptide for the least-squares fit algorithm.37 All REMD simulations ran for 60 ns. In the case of L-Leu with a βII structure as starting point additional 60 ns were calculated for a total simulation time of 120 ns. Selected snapshots from the MD simulations were optimized at the QM/MM level of theory using the program ChemShell v3.438 with Turbomole 5.1039 for the QM region and DL_POLY40 for the MM part. The QM region which includes all peptide atoms was described using the B3LYP density functional with empirical dispersive energy correction (B3LYPD2) and the SVP basis set from the Turbomole basis set library.41−43 An electrostatic embedding scheme was applied.44 The optimization was performed with the HDLC optimizer.45 All atoms within the active region (peptide and a solvation sphere of 136 solvent molecules) were allowed to move in each optimization step. D. Temperature-Dependent FTIR/ATR Spectra. The peptide solutions were transferred with a syringe into a thermostabilized flow through top-plate assembly with a ZnSe 45° crystal (6 reflections, 550 μL sample volume, temperature range −10 to 90 °C, Specac, UK), which was connected to a ATR optics unit (Specac Gateway ATR system). The ATR optics unit was placed in the sample compartment of the FTIR spectrometer (a Bruker IFS66v/S, Bruker Optics, Ettlingen). The thermostabilized top plate was connected to a thermostat (Haake Fison CH) to achieve temperature control. An electronic temperature sensor (Voltcraft 300 K Digital thermometer, Conrad Electronics, Germany) was used to check the temperature near the ZnSe crystal. All measurements were carried out with 4 cm−1 resolution in the spectral range between 400 and 3000 cm−1.46,47 The assignment of the carbonyl oscillators in the amide I region (∼1700−1600 cm−1) was done by comparison, based on the results obtained by pump−probe UV-IR and 2D-IR experiments of cyclo(Boc-ProCys-Aib-Cys-OMe) as reference molecule.7 E. UV-CD Spectra. The CD spectra were recorded on a Jasco J-815 spectropolarimeter by using 0.1 cm path length cells at temperatures of 10 and 60 °C. The peptide concentrations were of ∼0.1 mg/mL. CD data are expressed as molar ellipticity (deg cm2 dmol−1).

be difficult, and there are not many studies describing small peptides in the solid state and in solution with different solvents by state of the art computations and experiments. By combining organic synthesis, X-ray crystallography, and advanced spectroscopy with computational techniques, we aim to provide insight into the effect of stereocenters on the structure and dynamics of peptides.

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METHODS A. Single-Crystal Structure Determination. The cyclic tetrapeptides 1 and 2 were prepared using a sequential synthesis strategy (Supporting Information, pages 2−6), and were crystallized from a hexane/ethyl acetate mixture. The single-crystal X-ray diffraction measurement of 1 was carried out on a Bruker Smart 1000 CCD diffractometer using graphite-monochromated Mo Kα radiation (λ = 0.710 73). The structure of 1 was solved by direct methods, and all nonhydrogen atoms were refined anisotropically on F2 (program SHELXTL-97, G. M. Sheldrick, University of Göttingen, Göttingen, Germany). All H atoms were visible in difference maps. The amino H atom H1 was refined isotropically, while the amino H atom H3d and those H atoms attached to C atoms were positioned geometrically with C−H = 0.98−0.99 Å and refined as riding atoms. The absolute configuration was assigned from the known configuration of the starting material and confirmed by a Flack x parameter of −0.10(12). Single crystal data for 2 were measured using a Bruker Proteum diffractometer housed in front of a FR591 rotating Cu anode (λ = 1.541 83 Å) equipped with focusing Montel optics. As in 1, for the structure solution and refinement of 2 all amide protons were kept in geometrically optimized positions. The Flack parameter was established as −0.003(12). B. NMR Studies. 2D NMR spectroscopy (HSQC, HMBC, TOCSY) experiments were conducted in DMSO-d6 and CD3CN for both peptides. NOE (nuclear Overhauser effects) contacts were also measured in all cases. The NMR samples of 1 and 2 were prepared in DMSO-d6 or CD3CN. The concentrations of 1 and 2 were adjusted to 4.9 mM. The 1D 1 H NMR spectra were recorded at 303 K with a time domain of 32k data points and a spectral width of 12 019.23 Hz. The sweep width of the 2D homonuclear spectra for 1 in DMSO-d6 was 4084.97 Hz in the direct and in the indirect 1H dimension. The free-induction decay was acquired for 200.00 ms with a dwell time set to 122.40 μs. For all peptides in CD3CN, spectral width of 3019.32 Hz was chosen. The free-induction decay was acquired for 191.28 ms with a dwell time set to 165.60 μs. All spectra were zero-filled prior to Fourier transformation and sine apodization functions were applied in both dimensions. Manipulation and assignment of the NMR spectra was carried out using MestReNova and SPARKY 3. The NMR structure calculation was carried out with Xplor-NIH 2.33 using distance constraints extracted from the corresponding NOESY spectra.22,23 A set of 100 structures was generated by standard slow cooling protocols for simulated annealing, as implemented in the Xplor-NIH program and the 10 structures with lower energies were selected.24 VMD-Xplor was used for structure manipulation and visualization.25 C. Computational Details. Initially, 200 ns molecular dynamics (MD) simulations of 1 and 2 in acetonitrile (CH3CN) were performed at 303 and 353 K. The NAMD 2.7 program and the CHARMM22 force field were used.26,27 The parameters reported by Nikitin et al.28 were employed for CH3CN. Replica exchange molecular dynamics (REMD) 10786



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Figure 2. Intermolecular NH···O hydrogen bonds of 1 (left) and 2 (right). Carbon bound hydrogen atoms are omitted for clarity. Symmetry codes for 1 are x − 1, y, z and x + 1, y, z and for 2 x + 1/2, y − 3/2, z − 1.

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RESULTS AND DISCUSSION We found that peptide 1 crystallizes in the space group P21 with Z′ = 1, whereas 2 crystallizes in the orthorhombic space group P212121 with Z′ = 2 (Figure S1 and Table S1 in the Supporting Information (SI)). The presence of two molecular conformations 2a and 2b in the asymmetric unit (Z′) of 2 can be rationalized since, according to Desiraju, Z′ > 1 structures occur when the crystals are “frozen” in some high-energy kinetic forms.48 The growing number of molecular conformations for Z′ > 1 could also be considered as “snapshots” of the crystallization reaction.49 Both conformers 2a and 2b exhibit similar torsional angles of the peptide backbone (Table S2, SI). 1, 2a and 2b feature a trans peptidyl−prolyl bond and a β-turn II structure that could be identified by comparing the relevant dihedral angles of the peptide backbone ΦPro2, ΨPro2, ΦLeu3, and ΨLeu3 to the ideal angles from the literature (Table S2, SI).50 However, the dihedral angles of 1 are slightly closer to the values of an ideal β-turn II structure than those of 2. The Cαi− Cαi+3 distances are also shorter than the 7 Å criteria defining a β-turn51 (1 (5.36 Å), 2a (5.34 Å), and 2b (5.40 Å)). The disulfide bridges of 1 and 2 adopt a left-handed conformation. The COCys1···HNCys4 distances in 1 (2.20 Å), 2a (2.35 Å), and 2b (2.27 Å) as well as the NCys4−HCys4−OCys1 angles (148° in 1, 151° in 2a, and 158° in 2b) are in agreement with an intramolecular hydrogen bond being present in each of the cyclic peptides. In addition, the crystal structures of 1 and 2 are stabilized by intermolecular hydrogen bonds. Tetrapeptide 1 affords hydrogen bonds between those molecules translated along the αvector by one unit cell. One such intermolecular hydrogen bond is formed by COPro2···HND‑Leu, the second by the amide proton of Cys1 and the carbonyl oxygen of the Boc-protecting group (COBoc···HNCys1). The two crystallographically independent molecules of the L-Leu-peptide 2 form dimers in the solid state, which are stabilized by the same set of hydrogen bonds as found in 1. A noncrystallographic translation symmetry operation along [1 0 1] is employed for this interaction between 2a and 2b. These dimers are further interconnected by two additional hydrogen bonds. NCys1H··· OCL‑Leu is formed between two molecules of 2a, while COL‑Leu···HNL‑Leu is formed between the corresponding amino acids of 2a and 2b leading to a zigzag chain of dimers, where the dimers are almost orthogonal to each other (Figure 2). NMR spectroscopy (1H, 13C, TOCSY, HSQC, HMBC, and NOESY) provided information about the structure of both

peptides in solution (deuterated DMSO and CD3CN). NMR structure calculations were carried out to determine the structures of both peptides in CD3CN. An ensemble of 100 structures was generated to match the sets of interproton distance constraints extracted from each of the corresponding NOESY spectra. The 10 structures with the lower energies were selected for each peptide. The corresponding structural statistics suggest a well-ordered solution structure for 1 and 2 in CD3CN (Table S3, SI). As expected, a greater mobility was found for the Boc and OMe protective groups, as well as for the region around the disulfide bridge, due to the sparseness of experimental constraints in these regions of the molecules. Furthermore, the distance between CαCys1 and CαCys4 is below 7 Å in the calculated structures of both peptides, supporting the hypothesis of a β-turn structure in solution. The NOESY spectra showed in all cases a cross peak correlating HNCys1 and HδPro2 protons which suggests the occurrence of a trans peptidyl-prolyl bond in both solvents (Figures S4 and S5, SI). The presence of two strong cross peaks correlating HαPro2 with HND‑Leu and HND‑Leu with HNCys4, along with a weak cross peak correlating HαPro2 with HNCys4 in the NOESY spectra of 1 in both solvents, suggests that 1 adopts a type-II β-turn structure in both solvents. In contrast to 1, the NMR spectra of 2 reveal the presence of several conformations in DMSO-d6, reflecting its increased conformational flexibility. The additional 1H-resonances of the amide protons (Figure S2, SI) as well as the additional set of signals in the 2D NMR spectra suggest the presence of at least three conformations. This can be related to the isomerization along the urethane group of Boc, which has been suggested to take place in cyclo(Boc-Cys-Pro-L-Phe-Cys-OMe).2 The NOESY spectra of 2 show an additional NOE cross peak between HδPro2 and HNL‑Leu3, along with two cross peaks correlating HαX3 with HNL‑Leu3 and HNL‑Leu3 with HNCys4 in DMSO-d6 and CD3CN. These cross peaks indicate the presence of a second β-turn structure (type I) of 2 in both solvents. Correspondingly, the REMD and MD simulations of 1 suggest that in acetonitrile at 302 K this peptide exclusively adopts the β-II turn structure. Simulations starting with β-I structures of 1 result in a fast conversion to the β-II motif. On the contrary, for 2 both conformers coexist (Table S4, SI), and 120 ns REMD simulations of 2 result in 84% of β-II-like structures at 302 K. Thus, the substitution of an L-amino acid by its D-enantiomer leads to a greater conformational rigidity of the whole peptide in both solvents. 10787



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conversion in 1. For 2, both groups of conformers (β-II and βI) are found in the simulations and the optimized structures have very similar QM energies (Figure 3). The different behavior of 1 and 2 can be rationalized in terms of intramolecular Cys−Cys and intermolecular peptide−solvent interactions. For 2 the strong Cys−Cys interaction is conserved in both β-turns (COCys1···HNCys4 distances of 1.89−1.97 Å for β-II snapshots and of 1.89−2.02 Å for β-I structures) (Figure 3). In addition, two molecules of solvent form strong hydrogen bonds with the HN group of Cys1 (which is not part of the cyclic core of the peptide, and thus not discussed here), and with the HN group of Leu (NHL‑Leu···NCH3CN distances of 1.75−1.80 Å for β-II snapshots of 2 and 1.76−1.87 Å for β-I structures of 2). In 1, the Cys−Cys interaction can only take place in structures with the β-II motif (COCys1···HNCys4 distances of 1.93−2.10 Å for β-II snapshots and 4.45−5.0 Å for β-I structures). This Cys−Cys hydrogen bond is then replaced in some β-I snapshots by Pro−Cys interactions which introduce additional strain in the molecule. Moreover, the peptide−solvent hydrogen bonding is less pronounced (and even missing in some snapshots) for β-I geometries (Figure 3). The FTIR spectra of the amide I region of 1 and 2 also provided evidence for the presence of a β-turn stabilized by intramolecular hydrogen bonding between COCys1 and HNCys4 or HND‑Leu. The IR spectrum of 2 is significantly different from that of 1 (Figure S10, SI). In 2, overlapping bands in the amide I region prevent the correct assignment of the carbonyl vibrations of Pro2 and L-Leu3. The carbonyl vibration of Cys1 in 2 is blue-shifted by 3 cm−1 compared to that in 1, in agreement with the NMR and QM/MM results which indicate a shorter COCys1···HNCys4 hydrogen bond in 2. The temperature dependence of the IR spectra (Figure 4) indicates that the Cys1 carbonyl band (1641 cm−1) of 2 is involved in intramolecular hydrogen bonding.

The temperature dependence of the amide protons chemical shifts was used to describe solvent accessibility of the amide protons and the formation of intramolecular hydrogen bonds (Figures S6−S9, SI). The relatively low values (around 2 × 10−3 ppm/K)52 of the temperature coefficients for HNCys4 in both peptides suggest that this proton is not exposed to the solvent, supporting the idea of an intramolecular hydrogen bond between HNCys4 and COCys1 (Table 1). On the Table 1. Temperature Coefficients for the Amide Protons (Δδ/ΔT × 10−3 ppm/K) peptide DMSO-d6 1 2a CD3CN 1 2a

Cys1

D-Leu

L-Leu

Cys4

9.7 7.9

5.4 −

− 3.5

1.8 1.6

3.7 3.5

2.4 −

− 1.5

1.8 2.0

a

Major conformation (Figures S2, S3, S7, and S9 in the Supporting Information).

contrary, the high values of the coefficients for HNCys1, particularly in deuterated DMSO (over 8 × 10−3 ppm/K), suggest that this is the amide proton that is most solventexposed, as expected. To understand these tendencies, we looked in detail at the molecular interactions in the peptide (Figure 3). QM(B3LYPD2/SVP)/CHARMM optimizations of several snapshots of 1 and 2 in acetonitrile show that for 1 structures with a β-II turn are in average 18 kcal/mol more stable than the β-I conformers (B3LYP-D2/SVP energies, 16 kcal/mol for the snapshots shown in Figure 3). This is in agreement with the MD simulations that show a rapid and nonreversible β-I → β-II

Figure 3. Representative QM/MM optimized structures of 1 (top) and 2 (bottom) with β-II and β-I turns; the interaction with one molecule of acetonitrile is also shown (distances are in Å). 10788



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Figure 4. Top: Temperature-dependent ATR-IR spectra of cyclo(Boc-Cys-Pro-D-Leu-Cys-OMe) (1) in CH3CN (left) and in CHCl3 (right). Bottom: Temperature-dependent ATR-IR spectra of cyclo(Boc-Cys-Pro-L-Leu-Cys-OMe) (2) in CH3CN (left) and in CHCl3 (right). In all cases the concentration was 40 mM and the resolution 4 cm−1.

and OMe. No significant blue shift can be observed for the IR band of COPro2/L‑Leu3. Similar experiments in CHCl3 reveal a relatively strong temperature dependence of the amide I region of 1, and indicate a β-turn structure at even higher temperatures (Figure 4). Although the spectra of 2 show partially overlapping bands, the band at 1645 cm−1, which indicates that the Cys1 carbonyl group is involved in a hydrogen bond, is conserved even at elevated temperatures. Circular dichroism (CD) experiments of 1 in CH3CN show a positive band at 203 nm due to the π → π* transition of the peptide amide bond and a negative band at ∼230 nm attributed to the peptide n → π* transition (Figure 5).4 Hence, 1 seems to adopt a rigid backbone conformation with a β-turn structure. This structure is in accordance with the high thermal stability (up to 60 °C) observed in the temperature-dependent CD spectra. The CD spectra of 2 show two maxima at 195 and 204 nm, as well as a shifted minimum at 240 nm compared to 1, as expected for a β-turn.4,5 On the other hand, the differences between the CD spectra of 1 and 2 suggest that 2 could adopt a different type of β-turn, in agreement with the NMR and REMD results. The CD spectra also indicate that the substitution of D-Leu by L-Leu strongly affects the conformational behavior of the cyclic tetrapeptides, resulting in the destabilization of the β-turn structure in 2 (Figure 5).

A detailed analysis of the spectra indicates that the amide I bands of Pro2 and D-Leu3 at 1690 and 1670 cm−1 of 1 in CH3CN can be attributed to carbonyl groups that are not hydrogen bonded and are sterically constrained. The remaining two IR bands can be attributed to COOMe, usually appearing at ∼1752 cm−1 and to COBoc (∼1711 cm−1), which were assigned by deprotection of the Boc group.7 The carbonyl bands of Pro2 and D-Leu3 as well as Boc show only small intensity loss at higher temperatures and almost no frequency shifts which suggests that these carbonyl groups are most likely not involved in intramolecular hydrogen bonds (Figure 4). This is in agreement with the QM/MM calculations indicating that the β-I turn structures (which can be stabilized by an intramolecular hydrogen bond between the CO group of Pro2 and the NH group of Cys4, Figure 3) are much less favored in 1 with respect to the β-II turn motif. The Cys1 carbonyl band appearing at 1638 cm−1 in the spectra of 1 shows a higher temperature dependence in terms of intensity compared to the other carbonyl bands in the amide I region, but no shift in frequency with increasing temperature occurred, indicating the maintenance of a β-turn structure in CH3CN. The temperature dependence of the IR spectra (Figure 4) shows that, unlike 1, four IR bands of 2 are well resolved in the amide I region. At 1678 cm−1, only one IR band is observable for the carbonyl oscillators of Pro2 and L-Leu3 due to overlapping of bands. The carbonyl bands of Pro2/L-Leu3 and Cys1 are more strongly decreasing in intensity with temperature increase compared to the carbonyl bands of Boc 10789



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CONCLUSION We found that while 1 crystallizes with a single, well-defined structural motif, two crystal isoforms are present in 2. In solution, 2 shows a distinctive contribution of type I β-turn structures, in contrast to 1, which shows much less conformational flexibility. Thus, our work establishes that the substitution of the natural occurring L-Leu in peptide 2 by its D-enantiomer in peptide 1 results in a more rigid peptide structure in both the solid state and in solution. The fact that the stereochemistry of a single stereocenter controls the peptide’s properties is rationalized in terms of crystal packing in the solid state and intramolecular and peptide−solvent interactions in solution. In the solid state, the peptide−solvent interactions are replaced by peptide−peptide interactions resulting in very similar conformations. Our studies provide a basis for the understanding of the conformational flexibilities of L-peptides compared to their D-counterparts. This in turn affects other biological and chemical properties that make Dpeptides interesting as therapeutic agents, among other applications. ASSOCIATED CONTENT

S Supporting Information *

Synthesis strategy, experimental and computational details. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Bredenbeck, J.; Helbing, J.; Sieg, A.; Schrader, T.; Zinth, W.; Renner, C.; Behrendt, R.; Moroder, L.; Wachtveitl, J.; Hamm, P. Picosecond Conformational Transition and Equilibration of a Cyclic Peptide. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6452−6457. (2) Kolano, C.; Helbing, J.; Bucher, G.; Sander, W.; Hamm, P. Intramolecular Disulfide Bridges as a Phototrigger to Monitor the Dynamics of Small Cyclic Peptides. J. Phys. Chem. B 2007, 111, 11297−11302. (3) Mantsch, H. H.; Perczel, A.; Hollosi, M.; Fasman, G. D. Characterization of B-turns in Cyclic Hexapeptides in Solution by Fourier Transform IR Spectroscopy. Biopolymers 1993, 33, 201−207. (4) Woody, R. W. Peptides, Polypeptides & Proteins; John Wiley & Sons: New York, 1974; pp 338−350. (5) Borics, A.; Murphy, R. F.; Lovas, S. Optical Spectroscopic Elucidation of B-turns in Disulfide Bridged Cyclic Tetrapeptides. Biopolymers 2007, 85, 1−11. (6) Denkewalter, R. G.; Schwam, H.; Strachan, R. G.; Beesley, T. E.; Veber, D. F.; Schoenewaldt, E. F.; Barkemeyer, H.; Paleveda, W. J.; Jacob, T. A.; Hirschmann, R. The Controlled Synthesis of Peptides in Aqueous Medium. I. The Use of α-Amino Acid N-Carboxyanhydrides. J. Am. Chem. Soc. 1966, 88, 3163−3164. (7) Kolano, C.; Helbing, J.; Kozinski, M.; Sander, W.; Hamm, P. Watching Hydrogen-bond Dynamics in a Beta-turn by Transient Twodimensional Infrared Spectroscopy. Nature 2006, 444, 469−472. (8) Miranda, E.; Nordgren, I. K.; Male, A. L.; Lawrence, C. E.; Hoakwie, F.; Cuda, F.; Court, W.; Fox, K. R.; Townsend, P. A. Packham, et al. A Cyclic Peptide Inhibitor of HIF-1 Heterodimerization That Inhibits Hypoxia Signaling in Cancer Cells. J. Am. Chem. Soc. 2013, 135, 10418−10425. (9) Bock, J. E.; Gavenonis, J.; Kritzer, J. A. Getting in Shape: Controlling Peptide Bioactivity and Bioavailability Using Conformational Constraints. ACS Chem. Biol. 2012, 8, 488−499. (10) Wiesehan, K.; Willbold, D. Mirror-image Phage Display: Aiming at the Mirror. ChemBioChem 2003, 4, 811−815. (11) Schumacher, T. N. M.; Mayr, L. M.; Minor, D. L.; Milhollen, M. A.; Burgess, M. W.; Kim, P. S. Identification of d-Peptide Ligands Through Mirror-Image Phage Display. Science 1996, 271, 1854−1857. (12) Li, J.; Kuang, Y.; Gao, Y.; Du, X.; Shi, J.; Xu, B. d-Amino Acids Boost the Selectivity and Confer Supramolecular Hydrogels of a Nonsteroidal Anti-Inflammatory Drug (NSAID). J. Am. Chem. Soc. 2012, 135, 542−545. (13) Tjernberg, L. O.; Lilliehöök, C.; Callaway, D. J. E.; Näslund, J.; Hahne, S.; Thyberg, J.; Terenius, L.; Nordstedt, C. Controlling Amyloid β-Peptide Fibril Formation with Protease-stable Ligands. J. Biol. Chem. 1997, 272, 12601−12605. (14) van Groen, T.; Wiesehan, K.; Funke, S. A.; Kadish, I.; NagelSteger, L.; Willbold, D. Reduction of Alzheimer’s Disease Amyloid Plaque Load in Transgenic Mice by D3, a D-Enantiomeric Peptide Identified by Mirror Image Phage Display. ChemMedChem 2008, 3, 1848−1852. (15) van Groen, T.; Kadish, I.; Wiesehan, K.; Funke, S. A.; Willbold, D. In Vitro and in Vivo Staining Characteristics of Small, Fluorescent, Aβ42-Binding D-Enantiomeric Peptides in Transgenic AD Mouse Models. ChemMedChem 2009, 4, 276−282. (16) Fernandez-Lopez, S.; Kim, H.-S.; Choi, E. C.; Delgado, M.; Granja, J. R.; Khasanov, A.; Kraehenbuehl, K.; Long, G.; Weinberger, D. A.; Wilcoxen, K. M.; et al. Antibacterial Agents Based on the Cyclic D,l-α-peptide Architecture. Nature 2001, 412, 452−455. (17) Kreil, G.; Barra, D.; Simmaco, M.; Erspamer, V.; Falconieri Erspamer, G.; Negri, L.; Severini, C.; Corsi, R.; Melchiorri, P. Deltorphin. A Novel Amphibian Skin Peptide with High Selectivity and Affinity for δ Opioid Receptors. Eur. J. Pharmacol. 1989, 162, 123−128. (18) Bai, L.; Romanova, E. V; Sweedler, J. V. Distinguishing Endogenous d-Amino Acid-Containing Neuropeptides in Individual Neurons Using Tandem Mass Spectrometry. Anal. Chem. 2011, 83, 2794−2800.

Figure 5. Temperature-dependent UV-CD spectra of cyclo(Boc-CysPro-X-Cys-OMe), X = D-Leu (1) or L-Leu (2) in CH3CN (peptide concentration ∼0.1 mg/mL, 0.1 cm path length cell).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Present Address ⊥

Research Programme on Biomedical Informatics (GRIB), Universitat Pompeu Fabra, Barcelona, Spain. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS E.S-G. acknowledges a Liebig stipend and K.B-R. a predoctoral stipend, both from the Fonds der Chemischen Industrie, Germany. This work was supported by the Cluster of Excellence RESOLV (EXC 1069) funded by the Deutsche Forschungsgemeinschaft. 10790



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A Single Stereocenter Changes the Conformation of a Cyclic

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