Conformational Preferences and cisâ trans Isomerization of

J. Phys. Chem. B 2007, 111, 5377-5385

5377

Conformational Preferences and cis-trans Isomerization of Azaproline Residue Young Kee Kang* and Byung Jin Byun Department of Chemistry, Chungbuk National UniVersity, Cheongju, Chungbuk 361-763, South Korea ReceiVed: NoVember 25, 2006; In Final Form: March 2, 2007

The conformational study of N-acetyl-N′-methylamide of azaproline (Ac-azPro-NHMe, the azPro dipeptide) is carried out using ab initio HF and density functional methods with the self-consistent reaction field method to explore the effects of the replacement of the backbone CHR group by the nitrogen atom on the conformational preferences and prolyl cis-trans isomerization in the gas phase and in solution (chloroform and water). The incorporation of the NR atom into the prolyl ring results in the different puckering, backbone population, and barriers to prolyl cis-trans isomerization from those of Ac-Pro-NHMe (the Pro dipeptide). In particular, the azPro dipeptide has a dominant backbone conformation D (β2) with the cis peptide bond preceding the azPro residue in both the gas phase and solution. This may be ascribed to the favorable electrostatic interaction or intramolecular hydrogen bond between the prolyl nitrogen and the amide hydrogen following the azPro residue and to the absence of the unfavorable interactions between electron lone pairs of the acetyl carbonyl oxygen and the prolyl NR. This calculated higher population of the cis peptide bond is consistent with the results from X-ray and NMR experiments. As the solvent polarity increases, the conformations B and B* with the trans peptide bond become more populated and the cis population decreases more, which is opposite to the results for the Pro dipeptide. The conformation B lies between conformations D and A (R) and conformation B* is a mirror image of the conformation B on the φ-ψ map. The barriers to prolyl cis-trans isomerization for the azPro dipeptide increase with the increase of solvent polarity, and the cis-trans isomerization proceeds through only the clockwise rotation with ω′ ≈ +120° about the prolyl peptide bond for the azPro dipeptide in the gas phase and in solution, as seen for the Pro dipeptide. The pertinent distance d(N‚‚‚H-NNHMe) and the pyramidality of imide nitrogen can describe the role of this hydrogen bond in stabilizing the transition state structure and the lower rotational barriers for the azPro dipeptide than those for the Pro dipeptide in the gas phase and in solution.

Introduction Azapeptide is a peptide analogue in which the CHR group of the backbone has been replaced by a nitrogen atom. The incorporation of azapeptide residues into biologically active peptides has been attempted to improve their biological potencies.1-3 In particular, the azapeptide linkage has been known to prevent the proteolytic cleavage and been used to design inhibitors of several proteases. Azaproline (azPro) is a proline (Pro) analogue in which the CHR group of the backbone of the Pro residue has been replaced by a nitrogen atom (Figure 1). There are a limited number of works carried out to explore conformational and biological roles of the azPro residue to date. A series of the azPro dipeptides with various N-substituents were synthesized as potent inhibitors of two proline-specific serine proteases: dipeptidyl peptidase IV and prolyl oligopeptidase.4 In order to study how Nacetylgalactosamine transferase handles the MUC5AC mucin motif octapeptide TTSAPTTS and its azPro analogue in vitro, the O-glycosylation rates of these two substrates were measured.5 Analogues of thyrotropin-releasing hormone (TRH) containing the azPro residue were studied using computer simulations and NMR experiments to explore the receptor-bound conformation of TRH.6 Several FKBP12 ligands containing azPro derivatives were synthesized, and their neuroprotective effects were evaluated in vitro and in vivo.7 * To whom correspondence should be addressed. Telephone: +82-43-261-2285. Fax: +82-43-273-8328. E-mail: [email protected].

Figure 1. Definition of torsion angles and structural parameters for the azPro dipeptide.

AzPro-containing peptides have been known to adopt preferentially the type VI β-turn with a cis peptide bond preceding the azPro residue in crystal8-10 and in organic solution.11 In X-ray structures of azPro-containing peptides,10 the azPro NRCO bond is shorter by about 0.10 Å than the Pro CR-CO bond, and the amide bond preceding the azPro residue is longer by about 0.03 Å than that of the Pro residue. In particular, the NR atom is out of the plane defined by the three atoms bonded to it and can have the same R or S chirality depending on the adjacent residues. The nonplanarity of the NR atom drives the azPro pyrazolidine ring to have a different puckering from that of the Pro pyrrolidine ring. The instability of the trans conformer for the azPro residue than its cis conformer was attributed to the unfavorable overlapping of the residual lone pair of the NR atom with that of the preceding carbonyl oxygen atom. The backbone torsion angle φ of about (110° for the X-ray structure

10.1021/jp067826t CCC: $37.00 © 2007 American Chemical Society Published on Web 04/18/2007

5378 J. Phys. Chem. B, Vol. 111, No. 19, 2007 with the cis azPro peptide bond was suggested to be due to the steric hindrances between the adjacent carbonyls. From 1H NMR and IR experiments for azPro-containing peptides in organic solvents,11 the azPro residue was proven to favor the type VI β-turn and the population of cis conformers was estimated to be about 75%. Considerable computations have been carried out on the peptides containing the Pro residue and its derivatives (or analogues), especially on N-acetyl-N′-methylamide of proline (Ac-Pro-NHMe, the Pro dipeptide) to understand the conformational preferences and cis-trans isomerization of the Pro residue12-26 as well as the puckering transition of the prolyl ring.27-29 However, there are a limited number of works that report on the conformational preferences for the cis conformer of azPro-containing peptides to form the type VI β-turn.6,30,31 The conformational preferences of azPro-containing peptides and azPro-substituted TRH derivatives were studied using Monte Carlo/stochastic dynamics (MC/SD) simulations in water and NMR experiments in polar solvents.6 Only representative trans and cis conformers of Ac-azPro-NHMe (the azPro dipeptide) were considered to explain quantum-mechanically the preference to adopt the cis conformer in the gas phase and water.30,31 Although the populations of cis and trans conformers for the azPro dipeptide were analyzed using the Boltzmann distribution of the conformational electronic energies along the backbone torsion angle ψ,31 all feasible conformations, obtained on the basis of conformational free energies in both the gas phase and solution, should be taken into account to explain reasonably the observed conformational preferences. In particular, the changes in rotational barriers for the prolyl cis-trans isomerization and in the prolyl puckering by the replacement of the CR atom with the nitrogen atom have never been experimentally or computationally studied. We report here the results on the azPro dipeptide calculated using ab initio Hartree-Fock (HF) and density functional theory (DFT) levels of theory with the self-consistent reaction field (SCRF) method to determine the effects of the replacement of the CHR group by the nitrogen atom on the conformational preferences and prolyl cis-trans isomerization in the gas phase and in solution. In particular, the conformational preferences were analyzed utilizing all feasible conformations found from the survey of the backbone potential energy surface (PES) depending on the azaprolyl puckering and from the search of local minima and transition states for the azPro dipeptide in the gas phase and in solution. Computational Methods Chemical structures and torsional parameters for the azPro dipeptide are defined in Figure 1. All ab initio HF and density functional calculations were carried out using the Gaussian 9832 and Gaussian 0333 packages. Here, each backbone conformation of the dipeptide is represented by a capital letter depending on its values of φ and ψ for the backbone.34 Conformations C, A, F, and D are equivalent to the γ-turn (C7eq), R-helical (RR), polyproline-like (β or PII), and β2 structures in other works, respectively. Conformation B lies between conformations D and A on the φ-ψ map. Conformations B* and F* are mirror images of conformations B and F, respectively. The trans and cis conformations for the Ac-X (X ) azPro and Pro) imide bond are denoted by “t” and “c”, respectively. The azPro ring can have distorted down- and up-puckerings, which are represented by “d” and “u”, respectively. The azaprolyl puckering is described by the endocyclic torsion angle χ1 for the N-NRCβ-Cγ sequence (i.e., positive and negative χ1 for the down-

Kang and Byun and up-puckered structures, respectively), as defined for the Pro residue.21,25,26 In addition, the azPro ring can adopt envelope conformations “e” and “e′”, in which the Cγ and Cδ atoms lie above the plane defined by the other four atoms of the ring, respectively (Figure 1). The seven local minima of Ac-Pro-NHMe with trans and cis imide bonds21,26 and the two local minima tFd and tFu of AcPro-NMe223 optimized at the HF/6-31+G(d) level were edited by replacing the CRH group by the NR using the Chem3D program35 to generate starting conformations for optimization of the azPro dipeptide. After minimizations at the HF/6-31+G(d) level, six initial conformations tCd, tCu, cAd, cFd, cFu, and tFd were converged to conformations tBd, tBe, cDd, cFd, cFe, and tFd, respectively. Three initial conformations cAu, tAu, and tFu were converged to conformations cDd, tBe, and tFd, respectively. On the basis of X-ray structures of the azPro dipeptide,10 the enantiomers (i.e., mirror images) of six local minima were generated by switching the signs of the torsion angles φ and ψ and optimized to yield the additional three local minima tB*u, tB*e′, and tF*u. The 2-D potential energy surfaces (PESs) of the conformations for the azPro dipeptide with trans and cis imide bonds were calculated along the torsion angles ψ and χ1 at the HF/ 6-31+G(d) level, in which adiabatic optimizations were performed at each value of ψ with an interval of 15° for -180° e ψ e 180° and of χ1 with an interval of 5° for -60° e χ1 e 60°. Five local minima tBd, tB*u, cDd, cFd, and cFe were used as initial structures of these adiabatic optimizations. At the HF/6-31+G(d) level, the two conformations optimized adiabatically from the conformation cDd with ω′ ) +116° and -65° for the Ac-azPro peptide bond (Figure 1), which were defined as two syn/exo and anti/exo structures in ref 12, respectively, were used as initial structures to locate the transition states ts1 and ts3, respectively, as done for Ac-ProNHMe.21,26 After minimizations at the HF/6-31+G(d) level, a single transition state ts1 with an envelope conformation was located at ω′ ≈ +123° for the azPro dipeptide. Local minima and transition state for the azPro dipeptide optimized at the HF/ 6-31+G(d) level were used as initial points for optimizations at the hybrid density functional B3LYP/6-311++G(d,p) level of theory. We employed the conductor-like polarizable continuum model (CPCM) SCRF method,36,37 implemented in the Gaussian 03 package,33 to compute solvation free energies (∆Gsolv) at the HF/6-31+G(d) level with the UAKS cavities, which are the united atom topological model (UATM) radii optimized at the density functional PBE0/6-31G(d) level of theory.38,39 The solvation free energy is the sum of the electrostatic free energy and the nonelectrostatic energy terms.40 The latter is composed of the cavitation, dispersion, and repulsion energy terms. For CPCM-UAKS calculations, the default average areas of 0.2 Å2 for tesserae were used. The solvents considered here are nonpolar chloroform and polar water, whose dielectric constants are 4.9 and 78.4 at 25 °C, respectively. Single-point solvation free energy calculations were carried out on each grid point of the PES along the backbone torsion angle ψ in the gas phase to get the PESs in chloroform and water (Figure S1 of the Supporting Information). Recently, the CPCM-UAKS calculations for a number of neutral and charged organic molecules at the HF/6-31+G(d)//HF/6-31+G(d) and HF/6-31+G(d)//B3LYP/ 6-31+G(d) levels provided hydration free energies in agreement with available experimental data.41 All local minima and transition state for the azPro dipeptide optimized at the HF/6-31+G(d) level in the gas phase were used

Conformational Preferences of azPro Residue as starting structures for optimizations at the CPCM HF/631+G(d) level in chloroform and water. The B3LYP/6311++G(d,p) single-point energies were calculated for all local minima and transition states of the azPro dipeptide located at the CPCM HF/6-31+G(d) level in solution. Vibrational frequencies were calculated for all stationary points at the HF level in the gas phase and in solution and the B3LYP level in the gas phase, which were used to compute enthalpies and Gibbs free energies with the scale factors of 0.8942 and 0.9843 at HF and B3LYP levels, respectively, at 25 °C and 1 atm. A scale factor of 0.89 at the HF/6-31+G(d) level was chosen to reproduce experimental frequencies for the amide I band of N-methylacetamide in Ar and N2 matrixes.42 A scale factor of 0.98 at the B3LYP/6-311++G(d,p) level reproduced well some experimental frequencies of proline in an Ar matrix.43 The zero-point energy correction and the thermal energy corrections were used to calculate the enthalpy (H) and entropy (S) of each conformation.44,45 The analysis uses the standard thermodynamic expressions for an ideal gas in the canonical ensemble. Each transition state was confirmed by checking whether it had one imaginary frequency after frequency calculations at the HF and B3LYP levels. The relative total free energy (∆G) for each conformation in solution was computed by taking the sum of the relative conformational free energy (∆Ee), the thermal contributions, and the entropic contribution. The relative conformational free energy (∆Ee) is the sum of the conformational electronic energy (∆Ee,s) and the relative solvation free energy (∆∆Gsolv) in solution. The relative total free energies are used here to interpret the conformational preferences and cis-trans isomerization in the gas phase and in solution. Results and Discussion Potential Energy Surfaces. The 2-D PESs of the azPro dipeptide with trans and cis imide bonds calculated along the torsion angles ψ and χ1 at the HF/6-31+G(d) level in the gas phase are shown in Figure 2. On the PES of the cis-azPro dipeptide, there are two local minima cDd and cFd at ψ ≈ +20° and -175° with the down puckering, respectively. The third local minimum cFe with the envelope puckering locates near the local minimum cFd. The conformational transitions seem to occur between conformations cDd and cFd and between conformations cFd and cFe. The barriers for transitions cDd f cFd and vice versa are estimated to be 15.3 and 10.6 kcal/mol at ψ ≈ -75°, respectively. At ψ ≈ -180°, the low barriers of 0.7 and 0.2 kcal/mol are computed for transitions cFd f cFe and vice versa, respectively. For the trans-azPro dipeptide, the PES shows two minima tBd and tFd at ψ ≈ 40° and 170° with the down puckering, respectively and their mirror images tB*u and tF*u at ψ ≈ -40° and -170° with the up puckering, respectively. There are other local minima tBe and tB*e′ with the envelope puckering located at ψ ≈ +30° and -30°, respectively. The PES indicates that conformational transitions appear to be feasible for pairs of conformations tBd and tBe at ψ ≈ 30°, conformations tB*u and tB*e′ at ψ ≈ -30°, conformations tBd and tFd at ψ ≈ 120°, conformations tB*u and tF*u at ψ ≈ -120°, and conformations tBe and tB*e′ at ψ ≈ 0°. At ψ ≈ (120°, the barriers for transitions tBd f tFd (or tB*u f tF*u) and tFd f tBd (or tF*u f tB*u) are estimated to be 6.3 and 1.5 kcal/mol, respectively. The barriers for transitions tBd f tBe (or tB*u f tB*e′) and tBe f tBd (or tB*e′ f tB*u) are calculated to be 1.1 and 0.1 kcal/mol at ψ ≈ (30°, respectively. The barriers at ψ ≈ 0° are estimated to be 1.8 kcal/mol for both transitions

J. Phys. Chem. B, Vol. 111, No. 19, 2007 5379

Figure 2. Potential energy surfaces of the azPro dipeptide with cis and trans peptide bonds at the HF/6-31+G(d) level along the backbone torsion angle ψ and the endocyclic torsion angle χ1 in the gas phase. Local minima are represented by +. Local minima a-i correspond to conformations cDd, tBd, tB*u, tBe, tB*e′, cFd, cFe, tFd, and tF*u, respectively (see footnote b of Table 1).

tBe f tB*e′ and vice versa. These calculated results indicate that most of the conformational transitions are feasible, except for the transitions tBd f tFd and tB*u f tF*u. Thus, the shapes of PESs and local minima for the azPro dipeptide with trans and cis peptide bonds in the gas phase are quite different from those of the Pro dipeptide.21 Potential energy surfaces of the azPro dipeptide with the down puckering (χ1 ≈ +40°) along the backbone torsion angle ψ at the HF/6-31+G(d) level in the gas phase and at the CPCM HF/ 6-31+G(d) level in chloroform and water are shown in Figure S1 of the Supporting Information. The overall shapes of the PESs in the gas phase and water are quite similar to those at the MP2/6-31+G(d,p)//HF/6-31+G(d,p) level in refs 30 and 31. The barrier heights at ψ ≈ -90° and 120° for the conformational transition tBd f tFd are calculated to be 15.0 and 6.3 kcal/mol in the gas phase, 13.8 and 6.7 kcal/mol in chloroform, and 15.1 and 8.7 kcal/mol in water, respectively. The barrier heights at ψ ≈ -75° and +120° for the conformational transition cDd f cFd are calculated to be 15.3 and 8.3 kcal/mol in the gas phase, 14.4 and 8.4 kcal/mol in chloroform, and 14.7 and 9.5 kcal/ mol in water, respectively. Thus, the barrier heights for these conformational transitions are not significantly changed for both trans and cis conformations, as the solvent polarity increases. In particular, the electronic energy difference (∆Ee(t/c)) between conformations cDd and tBd are calculated to be 3.41, 3.06, and 1.63 kcal/mol in the gas phase, chloroform, and water, respectively, which indicates the decrease of ∆Ee(t/c) with the increase of solvent polarity. However, the values of ∆Ee(t/c) are computed to 3.15 kcal/mol at the MP2/6-31+G(d,p)//HF/6-31+G(d,p)

5380 J. Phys. Chem. B, Vol. 111, No. 19, 2007

Kang and Byun

TABLE 1: Backbone Torsion Angles, Endocyclic Torsion Angles, and Thermodynamic Properties of Ac-azPro-NHMe Optimized at the HF/6-31+G(d) and B3LYP/6-311++G(d,p) Levels in the Gas Phase backbone torsion anglesa

endocyclic torsion anglesa

conformerb

ω′

φ

ψ

ω

cDd (a) tBd (b) tB*u (c) tBe (d) tB*e′ (e) cFd (f) cFe (g) tFd (h) tF*u (i) ts1

19.8 -167.4 167.5 -165.8 165.8 11.3 20.1 177.5 -177.5 122.6

-116.6 -92.7 92.7 -92.6 92.7 -97.5 -101.2 -79.6 79.6 -124.4

21.3 40.8 -40.8 31.4 -31.4 -175.6 -172.1 169.9 -169.9 17.2

-179.8 171.5 -171.5 171.1 -171.1 -170.7 -170.0 -173.4 173.4 -176.4

cDd tBd tB*u tBe tB*e′ cFd cFe tFd tF*u ts1

19.6 -168.9 168.9 -169.0 169.0 12.0 17.5 178.7 -178.7 121.8

-115.2 -86.5 86.5 -85.2 85.3 -95.6 -95.4 -81.0 81.0 -127.5

21.6 41.8 -41.8 36.2 -36.2 -175.4 -170.6 169.5 -169.5 18.5

179.9 172.9 -172.9 172.6 -172.7 -171.3 -170.2 -174.6 174.6 -176.5

χ0

χ1

thermodynamic properties

χ2

χ3

χ4

∆Eec

∆Hd

∆Ge

-28.4 -34.6 34.6 18.3 -18.1 -31.0 14.3 -28.3 28.3 7.0

10.2 19.1 -19.0 -33.3 33.2 13.1 -30.5 8.7 -8.7 -24.2

12.8 3.7 -3.8 37.4 -37.4 10.7 37.6 15.5 -15.5 32.9

0.00 3.41 3.41 4.41 4.41 4.69 5.22 8.05 8.05 13.21

0.00 3.54 3.54 4.55 4.55 4.68 5.20 8.03 8.03 12.37

0.00 4.19 4.19 4.88 4.88 4.51 5.04 7.69 7.69 13.74

B3LYP/6-311++G(d,p) -30.2 36.3 -28.7 -23.3 36.1 -35.1 23.3 -36.1 35.1 -23.8 1.4 20.0 23.8 -1.3 -20.1 -29.7 36.5 -30.3 -27.3 6.1 15.5 -31.2 36.9 -29.5 31.2 -36.9 29.5 -28.9 13.6 6.7

11.4 21.0 -21.0 -33.4 33.4 13.1 -30.6 11.4 -11.4 -23.7

11.0 0.6 -0.6 35.9 -35.8 9.8 36.4 11.9 -11.9 32.1

0.00 1.96 1.96 2.96 2.96 4.57 4.87 7.88 7.88 14.46

0.00 2.06 2.06 3.10 3.10 4.53 4.85 7.84 7.84 13.43

0.00 2.84 2.84 3.57 3.56 4.12 4.68 7.33 7.33 14.86

HF/6-31+G(d) -32.1 37.4 -26.4 37.8 26.5 -37.8 -26.6 4.2 26.7 -4.3 -31.1 37.8 -29.3 8.1 -34.2 37.8 34.2 -37.8 -29.6 13.8

a Defined in Figure 1; units in degrees. b See the text for definition. For example, the first letter code cDd is the down-puckered conformation D with the cis prolyl peptide bond. Local minima are also denoted by bold alphabets in parentheses, which are located on the PES of Figure 2. c Relative electronic energies in kcal/mol. d Relative enthalpy changes in kcal/mol at 25 °C. e Relative Gibbs free energy changes in kcal/mol at 25 °C.

level in the gas phase30 and 3.37 kcal/mol at the MP2/6-31+G(d,p)//HF/6-31+G(d,p) level with PCM solvation free energies in water.31 Preferred Conformations in the Gas Phase. Table 1 lists the backbone torsion angles, endocyclic torsion angles, and thermodynamic properties of local minima and transition states for the azPro dipeptide optimized at the HF/6-31+G(d) and B3LYP/6-311++G(d,p) levels in the gas phase. The conformations and thermodynamic properties of local minima and transition states optimized at both levels are quite similar, although the relative values of thermodynamic properties are somewhat larger at the HF level than those at the B3LYP level, except for transition state ts1. The conformational stabilities of the azPro dipeptide are calculated to be in the orders cDd > tBd ) tB*u > tBe ) tB*e′ ≈ cFd > cFe > tFd ) tF*u at the HF/6-31+G(d) level and cDd > tBd ) tB*u > tBe ) tB*e′ > cFd ≈ cFe > tFd ) tF*u at the B3LYP/6-311++G(d,p) level by relative electronic energies (∆Ee) and relative free energies (∆G) in the gas phase. The representative conformations cDd, tBd, and tB*u with cis and trans azaprolyl peptide bonds, respectively, and the transition state ts1 optimized at the B3LYP/ 6-311++G(d,p) level in the gas phase are shown in Figure 3. The most preferred conformation of the azPro dipeptide is calculated to be cDd with the cis azaprolyl peptide bond and the distorted down-puckering at both levels in the gas phase. The stability of the cis conformation was ascribed to the C5 hydrogen bond between the azaprolyl N and the following N-H of the carboxyl group.10 The distances of this hydrogen bond are calculated to be 2.26 and 2.25 Å at the HF/6-31+G(d) and B3LYP/6-311++G(d,p) levels, respectively. In particular, this type of hydrogen bond has been known to play a role in stabilizing the transition states for the cis-trans isomerization of the prolyl peptide bond.12,15,18-26 However, it should be noted that there are remarkable shifts in the backbone torsion angles φ and ψ by -27° and +30° from those of the conformation

Figure 3. Representative conformations cDd, tBd, and tB*u with cis and trans azaprolyl peptide bonds, respectively, and transition state ts1 optimized at the B3LYP/6-311++G(d,p) level in the gas phase. Hydrogen bonds are represented by dotted lines, and their distances are 2.25, 1.96, 1.96, and 2.20 Å for conformations cDd, tBd, tB*u, and ts1, respectively.

cAd of the Pro dipeptide at the HF/6-31+G(d) level, respectively, and the corresponding shifts are -24° and +27° at the B3LYP/6-311++G(d,p) level, respectively.26 The second preferred conformation tBd with the trans azaprolyl peptide bond and the down puckering has a C7 hydrogen bond between CdO of the amino group and N-H of the carboxyl group, which plays a role in determining the lowest energy conformations for the Pro dipeptide and its derivatives.13-22,24,26 The distances of this hydrogen bond for the azPro dipeptide are calculated to be 2.11 and 1.96 Å at the

Conformational Preferences of azPro Residue


TABLE 2: Backbone Torsion Angles, Endocyclic Torsion Angles, and Thermodynamic Properties of Ac-azPro-NHMe Optimized at the HF/6-31+G(d) Level Using the CPCM Method in Solution backbone torsion anglesa

endocyclic torsion anglesa

conformerb

ω′

φ

ψ

ω


17.7 -167.6 167.7 -166.3 166.1 8.7 16.2 179.7 -180.0 119.3

-113.5 -92.9 92.9 -105.4 105.3 -95.8 -99.6 -82.9 83.3 -123.0

18.3 34.8 -34.7 13.9 -14.1 -173.8 -172.2 177.7 -178.1 14.3

178.5 171.5 -171.6 173.0 -172.0 -171.9 -171.0 -174.8 174.9 -178.2


15.4 -168.2 168.2 -169.2 168.7 7.8 13.3 -175.0 174.9 117.0

-111.5 -102.1 102.2 -101.4 103.4 -96.9 -98.3 -89.5 89.5 -121.9

16.2 13.5 -13.5 11.9 -12.0 -174.6 -173.0 -177.9 177.8 11.1

178.3 176.0 -176.1 177.7 -177.0 -173.7 -173.8 -174.8 174.7 -179.2

-32.3 -28.0 28.1 -29.1 30.6 -30.5 -29.1 -28.6 28.6 -28.1

a-e

χ0

χ1

thermodynamic properties

χ2

χ3

χ4

∆Eec

∆Hd

∆Ge

chloroform -31.4 37.9 -26.8 38.0 26.8 -38.0 -31.9 11.3 32.0 -11.4 -31.1 38.1 -29.5 8.3 -32.7 38.4 32.6 -38.6 -28.9 13.0

-30.0 -34.6 34.6 12.5 -12.4 -31.4 14.3 -30.4 30.8 7.5

12.2 18.9 -18.9 -30.6 30.5 13.6 -30.5 11.5 -12.0 -24.3

11.2 4.1 -4.0 38.6 -38.6 10.4 37.7 12.8 -12.3 32.5

0.00 3.06 2.99 3.49 3.49 2.59 3.27 5.47 5.47 13.65

0.00 3.18 3.11 3.51 3.51 2.56 3.26 5.42 5.42 12.82

0.00 3.45 3.38 2.88 2.91 2.32 2.94 4.97 4.95 13.97

water 38.3 38.6 -38.6 7.0 -8.9 38.6 7.2 38.3 -38.3 12.3

-29.9 -34.3 34.2 16.6 -14.9 -32.8 15.7 -33.8 33.8 7.9

11.6 18.1 -18.0 -33.0 32.1 15.2 -31.7 17.2 -17.2 -24.3

12.2 5.4 -5.5 38.6 -38.9 9.0 38.1 6.5 -6.5 32.1

0.00 1.63 1.63 1.71 1.69 1.44 2.25 2.79 2.79 14.81

0.00 1.63 1.64 1.72 1.71 1.37 2.20 2.72 2.73 14.04

0.00 1.56 1.57 1.45 1.40 1.15 1.69 2.38 2.40 15.20

See footnotes a-e of Table 1.

HF/6-31+G(d) and B3LYP/6-311++G(d,p) levels, respectively, which are similar to the values of 2.11 and 1.98 Å for the conformation tCd of the Pro dipeptide at the HF and B3LYP levels, respectively.26 This hydrogen-bond distance is estimated to be 1.98 Å at the MP2/6-31+G(d,p) level.30 As found for the conformation cDd, there are remarkable shifts in the backbone torsion angle ψ by -34° and -29° from those of the conformation tCd for the Pro dipeptide at the HF/6-31+G(d) and B3LYP/ 6-311++G(d,p) levels, respectively, whereas the shifts in the backbone torsion angle φ are only -7° and -3° at the same levels, respectively.26 In particular, it was suggested that the trans conformation would impose overlapping of the NR residual lone electron pair with that of the preceding carbonyl oxygen.10,30 Thus, the higher stability of the cis conformation over the trans conformation can be attributed to the formation of a C5 hydrogen bond in the cis conformation and the repulsions between lone electron pairs in the trans conformation. The calculated thermodynamic properties indicate that the relative conformational free energy (∆G) is governed by the relative conformational electronic energy (∆Ee) for each conformation at both the HF and B3LYP levels. The differences in ∆Ee and ∆G for the trans conformation tBd relative to the cis conformation cDd are calculated to be 3.41 and 4.19 kcal/ mol at the HF/6-31+G(d) level, respectively, and 1.96 and 2.84 kcal/mol at the B3LYP/6-311++G(d,p) level, respectively. The corresponding differences in ∆Ee are estimated to be 3.15 and 1.83 kcal/mol at MP2/6-31+G(d,p) and B3LYP/6-31+G(d,p) levels, respectively.30 Preferred Conformations in Solution. Table 2 lists the backbone torsion angles, endocyclic torsion angles, and thermodynamic properties of local minima and transition states for the azPro dipeptide optimized at the CPCM HF/6-31+G(d) level in chloroform and water. All local minima in the gas phase are also retained as local minima in chloroform and water. On going from the gas phase to chloroform, there are some shifts in the backbone torsion angles φ and ψ for the local minima and transition state. Their shifts are (4° in φ and (8° in ψ. However, there are large shifts in φ and ψ by -13° and -18° for the conformation tBe and by 13° and 17° for the conforma-

tion tB*e′, respectively. On going from chloroform to water, the shifts are calculated to be ( 7° in φ and ( 4° in ψ. In addition, there are large shifts in φ and ψ by -9° and -21° for the conformation tBd and by +9° and +21° for the conformation tB*u, respectively. In particular, all values of ∆Ee and ∆G decrease as the solvent polarity increases. The conformational stabilities of the azPro dipeptide are calculated by ∆G to be in the orders cDd > cFd > tBe ) tB*e′ ≈ cFe > tBd ) tB*u > tFd ) tF*u in chloroform and cDd > cFd > tBe ) tB*e′ ≈ tBd ) tB*u ≈ cFe > tFd ) tF*u in water. This indicates that the cis conformation cFd becomes more preferred and the trans conformations such as tBd, tB*u, tBe, and tB*e′ become less preferred in chloroform and water. The most preferred conformation of the azPro dipeptide is calculated to be cDd in chloroform and water, as found in the gas phase. The distances of the C5 hydrogen bond between the azaprolyl N and the following N-H of the carboxyl group of the conformation cDd are calculated to be 2.27 and 2.28 Å in chloroform and water, respectively, which are almost the same as those in the gas phase. The conformations tBe and tB*e′ with the envelope puckering are found to be the most preferred trans conformations in solution, whose ∆G are 2.88 and 2.91 kcal/ mol in chloroform and 1.45 and 1.40 kcal/mol in water, respectively. Although the conformations tBe and tB*e′ have in common a C7 hydrogen bond with the distance of 2.19 Å as similar to the conformations tBd and tB*u in the gas phase, the distance becomes longer than 3 Å in solution because of the large shifts in φ and ψ, as described above. The thermodynamic properties of the azPro dipeptide corrected by the single-point energies at the B3LYP/6-311++G(d,p)//CPCM HF/6-31+G(d) level in chloroform and water are listed in Table 3, which are used in the analysis of the conformational populations and the cis-trans isomerization below. It should be noted that the values of ∆Ee and ∆G decrease more at this corrected level than those at the CPCM HF/6-31+G(d) level in solution. The conformational stabilities by ∆G at this corrected level are similar to the orders by ∆G at the CPCM HF/6-31+G(d) level in chloroform and water, as described above, except that the relative stability of the


Kang and Byun

TABLE 3: Thermodynamic Properties of Ac-azPro-NHMe at the B3LYP/6-311++G(d,p)//CPCM HF/6-31+G(d) Level in Solutiona chloroform

water

conformerb

∆Ee,spc

∆∆Gsd

∆Eee

∆Hf

∆Gg

∆Ee,spc

∆∆Gsd

∆Eee

∆Hf

∆Gg


0.00 2.39 2.39 4.93 4.86 4.46 4.81 7.83 7.89 14.50

0.00 -0.47 -0.53 -1.91 -1.84 -2.22 -2.02 -2.99 -3.05 0.51

0.00 1.92 1.86 3.02 3.02 2.24 2.79 4.84 4.84 15.01

0.00 2.05 1.98 3.03 3.04 2.21 2.77 4.79 4.79 14.18

0.00 2.32 2.25 2.41 2.44 1.97 2.45 4.33 4.32 15.34

0.00 5.25 5.25 5.26 5.19 4.47 4.88 8.20 8.21 14.37

0.00 -4.65 -4.65 -4.34 -4.25 -3.50 -3.21 -6.43 -6.43 1.89

0.00 0.60 0.61 0.91 0.95 0.97 1.67 1.77 1.78 16.26

0.00 0.60 0.61 0.92 0.96 0.90 1.62 1.70 1.71 15.48

0.00 0.53 0.54 0.65 0.65 0.69 1.11 1.36 1.38 16.65

a Units in kcal/mol. b See footnote b of Table 1. c Relative single-point energies at the B3LYP/6-311++G(d,p) level for the geometries optimized at the HF/6-31+G(d) level using the CPCM method in solution. d Relative solvation free energies for the geometries optimized at the HF/631+G(d) level using the CPCM method in solution. e Relative electronic energies obtained from the sum of ∆Ee,sp and ∆∆Gs in solution. f Relative enthalpy changes obtained from the sum of ∆Ee of this Table and ∆∆H in solution; ∆∆H ) ∆H - ∆Ee at the HF/6-31+G(d) level of Table 2. g Relative Gibbs free energy changes obtained from the sum of ∆H of this table and -T∆S in solution; -T∆S ) ∆G - ∆H at the HF/6-31+G(d) level of Table 2.

TABLE 4: Lengths of CO-N and Nr-CO Bonds and Nonplanarities at Prolyl N and Nr Atoms for Representative Conformers of Ac-azPro-NHMe Optimized at the HF/6-31+G(d) and B3LYP/6-311++G(d,p) Levels in the Gas Phase and in Solution gas phase HF/6-31+G(d) conformera cDd cisd,e tBd transd,e ts1e

r1b 1.383 1.371 (1.359 1.358 1.361 (1.353 1.451 (1.434

r2b

δ1c

1.400 -12.6 1.400 -8.0 1.530 -1.2) 1.404 -3.6 1.404 -3.8 1.534 -2.1) 1.384 -33.0 1.529 -23.5)

chloroform

B3LYP/6-311++G(d,p) δ2c -19.0 -18.8

r1b

1.397 1.385 (1.373 -19.0 1.367 -19.3 1.370 (1.365 -10.9 1.479 (1.456

r2b

δ1c

1.422 -11.9 1.421 -7.0 1.541 -1.4) 1.429 -2.3 1.429 -2.0 1.547 -1.9) 1.406 -34.9 1.540 -25.9)

water

HF/6-31+G(d)

δ2c -20.1 -19.1

r1b

1.374 1.362 (1.351 -19.2 1.355 -20.0 1.362 (1.348 -12.6 1.449 (1.432

r2b

δ1c

1.397 -11.2 1.397 -7.3 1.530 -1.0) 1.396 -4.0 1.394 -6.0 1.531 -1.5) 1.381 -32.6 1.528 -22.6)

HF/6-31+G(d) δ2c -19.0 -18.2

r1b

1.363 1.354 (1.342 -17.8 1.363 -17.6 1.359 (1.341 -11.0 1.448 (1.428

r2b

δ1c

1.394 -9.6 1.395 -6.9 1.528 -1.0) 1.391 -7.8 1.390 -7.2 1.527 -1.1) 1.378 -32.5 1.526 -22.0)

δ2c -19.0 -18.3 -17.8 -17.0 -10.9

a See footnote b of Table 1. b Units in Å; r and r are the lengths for the bonds CO-N and NR-CO for the azPro dipeptide, respectively. In 1 2 particular, r2 is the CR-CO bond length for the Pro dipeptide. c Units in degrees; δ1 and δ2 are the nonplanarities at the prolyl N and NR atoms, respectively. The nonplanarity is defined as S - 360°, where S is the sum of three bond angles around the prolyl N or NR atoms; see refs 24 and 26. d The values for the “cis” and “trans” conformers are the average values for all cis and trans conformers, respectively, optimized at each level of theory in the gas phase and in solution. e The values in parentheses correspond to those for the Pro dipeptide; from refs 21 and 26.

conformation cFd decreases and it becomes comparable to conformations tBe and tB*e′ in water. Comparison of Structural Parameters. The important structural parameters of local minima and transition states for the azPro dipeptide are compared to those of the Pro dipeptide in the gas phase and in solution. The structural parameters for the Pro dipeptide are taken from refs 21 and 26. Based on X-ray structures of the azPro residue,10 only the lengths of CO-N and NR-CO bonds and the nonplanarities at the azaprolyl N and NR atoms for representative conformers optimized at the HF/6-31+G(d) and B3LYP/6-311++G(d,p) levels in the gas phase and in solution are listed in Table 4. The bond lengths r1(CO-N) for conformations cDd and tBd and transition state ts1 as well as the average bond lengths of cis and trans conformations of the azPro residue become somewhat longer by 0.01-0.02 Å than those of the Pro dipeptide at the HF/6-31+G(d) and B3LYP/6-311++G(d,p) levels in the gas phase and in solution. However, the bond lengths r2(NRCO) for the azPro dipeptide become shorter by 0.12-0.15 Å than the values of the r2(CR-CO) of the Pro dipeptide at both levels in the gas phase and in solution. These calculated changes in the two bond lengths are consistent with the results obtained from analysis of X-ray structures of the azPro residues.10 Recently, we estimated the degree of nonplanarity (i.e., the pyramidal sp3 character) of the imide nitrogen by the quantity δ defined as S - 360°, where S is the sum of three bond angles (i.e., C′-N-Cγ, C′-N-CR, and Cγ-N-CR for the prolyl ring)

around the nitrogen.24,26 As the quantity δ becomes more negative, the degree of nonplanarity of the nitrogen increases more. For trimethylamine, the value of δ can be estimated to be -27.3° from the value of 110.9° for the bond angle C-NC.46 This indicates that the nitrogen of trimethylamine has a pyramidal geometry. The values of δ2 at the NR for the azPro dipeptide are calculated to be -17° to -20° for all cis and trans conformations and -11° for the transitions state ts1 at the HF and B3LYP levels in the gas phase and in solution. This implies that the NR’s of cis and trans conformations for the azPro dipeptide have more the pyramidal sp3 character than the transition state, although the magnitudes are smaller than that of trimethylamine. The values of δ1 at the azaprolyl N are calculated to be -7° to -13° for cis conformations and -2° to -8° for trans conformations for the azPro dipeptide at both levels in the gas phase and in solution. However, the values of δ1 for the Pro dipeptide are calculated to be only -1° to -2° for cis and trans conformations and -22° to -26° for transition states at both levels in the gas phase and in solution (see the values in parentheses of Table 4). This indicates that the cis conformations of the azPro dipeptide have more the pyramidal sp3 character by ∼6° in δ1 than those of the Pro dipeptide in the gas phase and in solution, whereas the trans conformations of the azPro dipeptide have similar degrees of nonplanarity to those of the Pro dipeptide in the gas phase and a little more the pyramidal

Conformational Preferences of azPro Residue


TABLE 5: Populations of Backbone Conformations for Ac-azPro-NHMe and Ac-Pro-NHMe at the B3LYP/6-311++G(d,p) Level in the Gas Phase and in Solutiona solvent

D

B

B*

gas phase chloroform water

97.8 88.6 31.8

1.1 3.3 23.6

1.1 3.4 23.4


C

97.4 54.9 0.0

cF

tF*

cisb

Ac-azPro-NHMe 0.0 0.1 3.2

0.1 4.6 14.9

0.0 0.1 3.1

97.9 93.2 46.7

∼75c

Ac-Pro-NHMed 2.5 0.0 12.2 27.4 10.1 68.3

0.0 5.5 21.6

2.5 9.0 23.3

14,e 15 ( 4f 24 ( 4,f 27 ( 3,g 28h

A

tF

exptl cisb

a

Units in %. The values for each dipeptide were computed using the relative Gibbs free energy (∆G) of each local minimum in Tables 1 and 3. cis Ac-azPro or Ac-Pro peptide bonds. c Estimated on the basis of the absorption intensities of the NH group following the azPro residue of t-Boc-Ala-azPro-Ala-NHiPr and t-Boc-Ala-azPro-Ala-NHMe in dichloromethane; from ref 11. d The calculated values for Ac-Pro-NHMe in the gas phase and in solution; from ref 26. e From ref 50. f From ref 51. g From ref 52. h From ref 53. b

character by -3° and -5° in δ1 than those of the Pro dipeptide in chloroform and water, respectively. In particular, the values of δ1 for the transition state ts1 of the azPro dipeptide are estimated to be -33° in the gas phase and in solution. This implies that the azaprolyl N of the transition state for the azPro dipeptide has more the pyramidal character by about -10° in δ1 than the prolyl N of the transition state for the Pro dipeptide. The puckering amplitudes of the prolyl ring for local minima and transition states of the azPro dipeptide optimized at the HF/ 6-31+G(d) and B3LYP/6-311++G(d,p) levels in the gas phase and at the CPCM HF/6-31+G(d) level in chloroform and water are listed in Table S1 of the Supporting Information. In this Table, three kinds of puckering amplitudes, qR of Han and Kang,47 qz by Cremer and Pople,48 and χm of Altona and Sundaralingam,49 are employed to describe the degree of puckering of the azaprolyl ring. The puckering amplitude qR is the maximum angle between the mean plane and the line joining the center of mass and each atom of the ring. The puckering amplitude qz is the maximum z-displacement perpendicular to the mean plane of the ring. The puckering amplitude χm is the maximum value attainable by endocyclic torsion angles of the ring. The calculated puckering amplitudes for the azPro dipeptide indicate that the transition state ts1 is less puckered than local minima in both the gas phase and solution, which is opposite to the results for the Pro dipeptide.21,26 Recently, we reported that these three puckering amplitudes showed the same trend of puckering along the prolyl cis-trans isomerization for the Pro dipeptide although their absolute values are different.22 Comparison with X-ray Structures. The X-ray structure of the azPro dipeptide corresponds to the conformation cDd with the backbone torsion angles of ω′ ) 15°, φ ) -111°, and ψ ) 25° and the distorted down-puckering,10 which are in good agreement with our calculated values of the most preferred conformation cDd in the gas phase and in solution (Tables 1 and 2). In addition, the endocyclic torsion angles χ0-χ4 of the X-ray structure of the azPro dipeptide are consistent with our calculated values for the conformation cDd. However, an X-ray structure of Z-azPro-NHiPr has the conformation cD*e with the backbone torsion angles of ω′ ) -19°, φ ) 111°, and ψ ) -23° and the envelope puckering,8 which is not a local minimum for the azPro dipeptide but has similar values of the backbone torsion angles φ and ψ and the endocyclic torsion angles χ0-χ4 to the conformation tB*u in the gas phase and in solution. These conformational differences may be ascribed to the different terminal groups as well as the packing and intermolecular hydrogen bonds in crystal that cannot be considered in the isolated azPro dipeptide.

Population of Backbone Conformations. The populations of the backbone conformations for the azPro dipeptide are listed in Table 5. Each population was computed using the normalized Boltzmann weight by the relative free energy at the B3LYP/ 6-311++G(d,p) level in the gas phase and at the B3LYP/6311++G(d,p)//CPCM HF/6-31+G(d) level in chloroform and water. In the gas phase, the population of the conformation D for the azPro dipeptide is found to be dominant, which is ascribed to the conformation cDd with the cis azaprolyl peptide bond and the C5 hydrogen bond between the azaprolyl N and the following N-H of the carboxyl group. Populations of other conformations are computed to be negligible. The cis population is calculated to be 97.9% for the azPro dipeptide due to the population of the conformation cDd, whereas the cis population is only 2.5% for the Pro dipeptide.26 As the solvent polarity increases, i.e., from the gas phase to chloroform to water, the populations of conformation D decrease and the populations of conformations B and B* and polyproline I (PPI)-like conformation cF increase. The cis population for the azPro dipeptide decreases due to the decrease in the population of the conformation cDd with the increase of solvent polarity, which is opposite to the results for the Pro dipeptide.26 In particular, the cis population (46.7%) for the azPro dipeptide is larger than twice of that for the Pro dipeptide (23.3%) in water, which is due to the populations of the conformation cDd (31.8%) and PPI-like conformation cF (14.9%). These calculated results indicate that the azPro residue has quite different backbone populations from those for the Pro residue in the gas phase and in solution. The calculated cis population of 93.2% for the azPro dipeptide in chloroform is reasonably consistent with the value of ∼75% from NMR experiments for t-BocAla-azPro-Ala-NHiPr and t-Boc-Ala-azPro-Ala-NHMe in dichloromethane.11 cis-trans Isomerization. Relative energies of cis conformers and rotational barriers to cis-trans isomerization of the Ac-X peptide bond for azPro and Pro dipeptides in the gas phase and in solution calculated at the B3LYP/6-311++G(d,p) level in the gas phase and in solution are listed in Table 6. The most preferred cis and trans conformations are calculated to be cDd and tBd/tB*u for the azPro dipeptide, respectively, in the gas phase and in solution, as described above. Although the free energy difference (∆Gc/t) between cis and trans conformations for the azPro dipeptide is diminished with the increase of solvent polarity, as found for the Pro dipeptide, the cis population decreases for the azPro dipeptide but increases for the Pro dipeptide. A single transition state ts1 with the envelope puckering is located for the cis-trans isomerization of the Ac-azPro peptide


Kang and Byun

TABLE 6: Rotational Barriers and Relative Energies of cis Conformers for Ac-azPro-NHMe and Ac-Pro-NHMe Calculated at the B3LYP/6-311++G(d,p) Level in the Gas Phase and in Solutiona rotational barrierb,c

relative energyb,d

∆Gtcq

solvent gas phase chloroform water

12.02 13.02 16.12


19.15 19.32 21.61 (20.4,f 21.1,g 21.1h)

∆Gctq Ac-azPro-NHMe 14.86 15.34 16.65 Ac-Pro-NHMee 16.99 17.67 20.93 (19.8,f 20.7,g 20.2h)

∆Gc/t -2.84 -2.25 -0.53 2.16 1.64 0.68 (0.6,f,i 0.4,g 0.9h)

a The values for each dipeptide were computed using the relative Gibbs free energy (∆G) of each local minimum in Tables 1 and 3. b Units in kcal/mol. The lowest electronic energy or Gibbs free energy for each of trans, cis, and transition state conformations was used for these calculations. Free energies were calculated at 25 °C. c ∆Gtcq and ∆Gctq represent the barriers for the trans-to-cis and cis-to-trans rotations for the Ac-azPro or Ac-Pro peptide bonds. d ∆Gc/t is the relative electronic energy or Gibbs free energy of the cis conformer to the trans conformer. e The calculated values for Ac-Pro-NHMe in the gas phase, chloroform, and water; from ref 26. f From ref 52. g For Ac-Pro-OMe; from ref 54. h For Ac-Pro-OMe; from ref 55. i from ref 53.

bond with ω′ ≈ +120° in the gas phase and in solution, which is quite similar to the transition state ts1 for the Pro dipeptide.21,26 This also corresponds to the syn/exo conformation according to the definition in ref 12. This indicates that the cis-trans isomerization proceeds in common through only the clockwise rotation with ω′ ≈ +120° about the azaprolyl peptide bonds in the gas phase and in solution, as seen for prolyl and alanyl peptide bonds.26 At the B3LYP/6-311++G(d,p) level in the gas phase, the rotational barriers (∆Gtcq and ∆Gctq) to the trans-to-cis and cis-to-trans isomerizations for the azaprolyl peptide bond are estimated to be 12.02 and 14.86 kcal/mol, respectively. At the B3LYP/6-311++G(d,p)//CPCM HF/6-31+G(d) level, the values of ∆Gtcq and ∆Gctq are calculated to be 13.02 and 15.34 kcal/mol in chloroform, respectively. The corresponding values are computed to be 16.12 and 16.65 kcal/mol in water, respectively. Thus, the values of ∆Gtcq and ∆Gctq are lowered by 5.5-7.1 and 2.1-4.3 kcal/mol for the azPro dipeptide, respectively, from those of the Pro dipeptide in the gas phase and in solution.26 In particular, the rotational barriers for the azPro dipeptide increase as the solvent polarity increases, as seen for the Pro dipeptide. By analysis of the contributions to rotational barriers, the cistrans isomerizations for the azaprolyl peptide bond are proven to be entirely enthalpy driven in the gas phase and in solution, to which the electronic energies have contributed considerably, as seen for the Ala dipeptide,26 the Pro dipeptide,15,18,21,26 and Pro derivatives.19,24,25 This is consistent with the experimental results on proline-containing peptides, kinetically determined as a function of temperature.56 The kinetic and spectroscopic results have been interpreted as the evidence that indicates the existence of an intramolecular hydrogen bond between the prolyl nitrogen and the following amide N-H group for the transition state structure, which is capable of catalyzing the prolyl isomerization by up to 260fold in model peptides.57 The strength of this hydrogen bond is explained in terms of the hydrogen-bond distance and the pyramidality of imide nitrogen in the gas phase and water.24,26 The hydrogen-bond distances d(N‚‚‚H-NNHMe) between the imide nitrogen and the following hydrogen of NHMe group for the transition state ts1 structures of azPro and Pro dipeptides are computed to be 2.20 and 2.1726 Å at the HF/6-31+G(d) level in the gas phase, respectively. The corresponding distances for azPro and Pro dipeptides at the CPCM HF/6-31+G(d) level are computed to be 2.24 and 2.2826 Å in chloroform and 2.27 and 2.3226 Å in water, respectively. Thus, the distance

d(N‚‚‚H-NNHMe) for the transition state ts1 of the azPro dipeptide is shorter by ∼0.04 Å than those for the Pro dipeptide in solution. We described the degree of the pyramidal sp3 character of the imide nitrogen by the quantity δ1 computed from three bond angles around the nitrogen above. The values of δ1 for the transition state ts1 of azPro and Pro dipeptides are estimated to be about -33° and -23° in the gas phase and in solution. These results indicate that the imide nitrogen of the ts1 structure for the azPro dipeptide has more the pyramidal character and is potent to form a hydrogen bond than that for the Pro dipeptide. From these analyses of two structural quantities for the imide cis-trans isomerization, the pertinent distance d(N‚‚‚H-NNHMe) and the pyramidality of imide nitrogen can describe the role of this hydrogen bond in stabilizing the transition state structure and the lower rotational barriers for the azPro dipeptide than those for the Pro dipeptide in the gas phase and in solution. Conclusions The incorporation of the NR atom into the prolyl ring results in the different puckering, backbone population, and barriers to prolyl cis-trans isomerization from those of the Pro dipeptide. The most preferred conformation of the azPro dipeptide is calculated to be cDd with the cis azaprolyl peptide bond and the distorted down-puckering in the gas phase and in solution. The higher stability of the cis conformation over the trans conformation can be attributed to (a) the formation of an intramolecular hydrogen bond between the prolyl nitrogen and the amide hydrogen following the azPro residue in the cis conformation and (b) the unfavorable interactions between electron lone pairs of the acetyl carbonyl oxygen and the prolyl NR in the trans conformation. The lengths of CO-N and NR-CO bonds for local minima and transition state of the azPro residue become somewhat longer by 0.01-0.02 Å and shorter by 0.12-0.15 Å than those of the Pro dipeptide, respectively, in the gas phase and in solution, which are consistent with the results obtained from analysis of X-ray structures of azPro residues. As the solvent polarity increases, the populations of the conformation D decrease and the populations of conformations B and B* and polyproline I-like conformation cF increase. The cis population for the azPro dipeptide decreases due to the decrease in the population of the conformation cDd with the increase of solvent polarity, which is opposite to the results for the Pro dipeptide.

Conformational Preferences of azPro Residue The barriers to prolyl cis-trans isomerization for the azPro dipeptide increase with the increase of solvent polarity and the cis-trans isomerization proceeds through only the clockwise rotation with ω′ ≈ +120° about the prolyl peptide bond for the azPro dipeptide in the gas phase and in solution, as seen for the Pro dipeptide. The pertinent distance d(N‚‚‚H-NNHMe) and the pyramidality of imide nitrogen can describe the role of this hydrogen bond in stabilizing the transition state structure and the lower rotational barriers for the azPro dipeptide than those for the Pro dipeptide in the gas phase and in solution. Supporting Information Available: Potential energy surfaces of the azPro dipeptide with the down puckering along the backbone torsion angle ψ at the HF/6-31+G(d) level in the gas phase and at the CPCM HF/6-31+G(d) level in chloroform and water, and puckering amplitudes of the azPro dipeptide optimized at the HF/6-31+G(d) and B3LYP/6-311++G(d,p) levels in the gas phase and in solution. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Gante, J. Synthesis 1989, 405. (2) Gante, J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1699. (3) Zega, A.; Urleb, U. Acta Chim. SloV. 2002, 49, 649. (4) Borloo, M.; Augustyns, K.; Belyaev, A.; de Meester, I.; Lambeir, A.-M.; Goossens, F.; Bollaert, W.; Rajan, P.; Scharpe´, S.; Haemers, A. Lett. Pept. Sci. 1995, 2, 198. (5) Bac, A.; Rivoal, K.; Cung, M. T.; Boussard, G.; Marraud, M.; Soudan, B.; Tetaert, D.; Degand, P. Lett. Pept. Sci. 1997, 4, 251. (6) Zhang, W.-J.; Berglund, A.; Kao, J. L.-F.; Couty, J.-P.; Gershengorn, M. C.; Marshall, G. R. J. Am. Chem. Soc. 2003, 125, 1221. (7) Wilkinson, D. E.; Thomas, B. E., IV; Limburg, D. C.; Holmes, A.; Sauer, H.; Ross, D. T.; Soni, R.; Chen, Y.; Guo, H.; Howorth, P.; Valentine, H.; Spicer, D.; Fuller, M.; Steiner, J. P.; Hamilton, G. S.; Wu, Y.-Q. Bioorg. Med. Chem. 2003, 11, 4815. (8) Lecoq, A.; Boussard, G.; Marraud, M.; Aubry, A. Biopolymers 1993, 33, 1051. (9) Didierjean, C.; Aubry, A.; Zouikri, M.; Boussard, G.; Marraud, M. Acta Crystallogr., Sect. C 1995, 51, 688. (10) Didierjean, C.; Duca, V. D.; Benedetti, E.; Aubry, A.; Zouikri, M.; Marraud, M.; Boussard, G. J. Pept. Res. 1997, 50, 451. (11) Zouikri, M.; Vicherat, A.; Aubry, A.; Marraud, M.; Boussard, G. J. Pept. Res. 1998, 52, 19. (12) Fischer, S.; Dunbrack, R. L., Jr.; Karplus, M. J. Am. Chem. Soc. 1994, 116, 11931. (13) Kang, Y. K. J. Phys. Chem. 1996, 100, 11589. (14) McDonald, D. Q.; Still, W. C. J. Org. Chem. 1996, 61, 1385. (15) Jhon, J. S.; Kang, Y. K. J. Phys. Chem. A 1999, 103, 5436. (16) Improta, R.; Benzi, C.; Barone, V. J. Am. Chem. Soc. 2001, 123, 12568. (17) Benzi, C.; Improta, R.; Scalmani, G.; Barone, V. J. Comput. Chem. 2002, 23, 341. (18) Kang, Y. K.; Park, H. S. THEOCHEM 2002, 593, 55. (19) Kang, Y. K. J. Phys. Chem. B 2002, 106, 2074. (20) Rankin, K. N.; Boyd, R. J. J. Phys. Chem. A 2002, 106, 11168. (21) Kang, Y. K. THEOCHEM 2004, 675, 37. (22) Kang, Y. K.; Choi, H. Y. Biophys. Chem. 2004, 111, 135. (23) Kang, Y. K.; Park, H. S. Biophys. Chem. 2005, 113, 93. (24) Song, I. K.; Kang, Y. K. J. Phys. Chem. B 2006, 110, 1915. (25) Kang, Y. K.; Jhon, J. S.; Park, H. S. J. Phys. Chem. B 2006, 110, 17645. (26) Kang, Y. K. J. Phys. Chem. B 2006, 110, 21338. (27) Kang, Y. K. J. Phys. Chem. B 2004, 108, 5463. (28) Kang, Y. K.; Park, H. S. THEOCHEM 2005, 718, 17. (29) Song, I. K.; Kang, Y. K. J. Phys. Chem. B 2005, 109, 16982. (30) Che, Y.; Marshall, G. R. J. Org. Chem. 2004, 69, 9030. (31) Che, Y.; Marshall, G. R. Biopolymers 2006, 81, 392. (32) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.;

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