Effects of D-Leu Residues on the Helical Secondary Structures of L-Leu-Based Nonapeptides

Abstract

The influence of D-Leu residues on the helical structures of L-Leu-based-nonapeptides was investigated. Specifically, the preferred conformations of four diastereomeric nonapeptides, Boc-(L-Leu-L-Leu-Aib)₃-OMe (1); Boc-(L-Leu-L-Leu-Aib)₂-L-Leu-D-Leu-Aib-OMe (2), which contained one D-Leu residue; Boc-L-Leu-D-Leu-Aib-L-Leu-L-Leu-Aib-L-Leu-D-Leu-Aib-OMe (3), which contained two D-Leu residues; and Boc-(L-Leu-D-Leu-Aib)₃-OMe (4), were analyzed in solution and in the crystalline state. Peptide 1 formed a right-handed (P) 3₁₀-helix in solution. Peptides 2 and 3 both formed (P) 3₁₀-helices in solution and (P) α-helices in the crystalline state. Peptide 4 formed a (P) α-helix both in solution and in the crystalline state.

Helices are present throughout nature and play important roles in many daily functions. In the human body, microhelices are ubiquitous, e.g., they are found in DNA and protein molecules, and the helices in proteins play important roles in the recognition of biomolecules such as DNA, RNA, and other proteins. Therefore, the de novo design of peptides that fold into well-defined helical structures has been attracted great interest of many chemists. Non-proteinogenic amino acids such as α,α-disubstituted α-amino acids,^1–4) cyclic β-amino acids,^5–9) and cross-linked side chains^10,11) are often utilized as tools for peptide-helix stabilization. In particular, α-aminoisobutyric acid (Aib) has been widely incorporated into short natural L-peptides to promote the formation and the stabilization of helical structures.¹²⁾ Recently, we have reported that the introduction of Aib residues into the leucine (Leu)-based L-peptide stabilized its right-handed (P) 3₁₀-helix Boc-(L-Leu-L-Leu-Aib)₃-OMe (1),¹³⁾ whereas the insertion of achiral Aib residues into alternating Leu-based LD-peptides stabilized their α-helices, but not their 3₁₀-helices. Specifically, we reported that the nonapeptide Boc-(L-Leu-D-Leu-Aib)₃-OMe (4) formed a (P) α-helix both in solution and in the crystalline state.^14,15) As part of our ongoing research, we investigated the influence of D-Leu residues on the helical structures of L-Leu-based nonapeptides. We designed and synthesized four diastereomeric nonapeptides, Boc-(L-Leu-L-Leu-Aib)₃-OMe (1),¹³⁾ Boc-(L-Leu-L-Leu-Aib)₂-L-Leu-D-Leu-Aib-OMe (2), which contained one D-Leu residue, Boc-L-Leu-D-Leu-Aib-L-Leu-L-Leu-Aib-L-Leu-D-Leu-Aib-OMe (3), which contained two D-Leu residues, and Boc-(L-Leu-D-Leu-Aib)₃-OMe (4),¹⁴⁾ which contained equal amounts of L-Leu and D-Leu residues, and then analyzed their preferred conformations in solution and in the crystalline state (Fig. 1).

Fig. 1. Structures of the Peptides Examined in This Study

Results and Discussion

Synthesis of Peptides

The peptides 1 and 4 were synthesized in accordance with the methods outlined in refs. 13 and 14, respectively. The nonapeptides Boc-(L-Leu-L-Leu-Aib)₂-L-Leu-D-Leu-Aib-OMe (2) and Boc-L-Leu-D-Leu-Aib-L-Leu-L-Leu-Aib-L-Leu-D-Leu-Aib-OMe (3) were synthesized using conventional solution-phase methods involving a fragment condensation strategy in which 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) hydrochloride and 1-hydroxybenzotriazole (HOBt) hydrate were employed as coupling reagents. All of the compounds were purified by column chromatography on silica gel.

Conformational Analyses of the Peptides in Solution (Fourier Transform (FT)-IR and Circular Dichroism (CD) Spectra)

The IR spectra of peptides 2, 3 and 4 were measured in the NH-stretching region (3200–3500 cm⁻¹) at a peptide concentration of 2.0 mM in CDCl₃ solution. In the spectra of 2, 3 and 4, the weak bands in the 3434 and 3439 cm⁻¹ regions were assigned to solvated free peptide NH groups, and the strong band at 3334 cm⁻¹ was assigned to peptide NH groups with N-H···O=C intramolecular H-bonds. These spectra were very similar to those of helical peptides in solution.^3,16) Peptide 1 exhibited similar spectra to peptides 2, 3 and 4¹³⁾ (Fig. 2).

Fig. 2. FT-IR Spectra of Peptides 2 (a), 3 (b) and 4 (c) in CDCl₃ Solution

Peptide concentration: 2.0 mM.

The CD spectra of peptides 1–4 were measured in 2,2,2-trifluoroethanol (TFE) and TFE and phosphate buffered saline (PBS) buffer (1 : 1) solution (peptide concentration: 0.1 mM). The spectra of the peptides displayed negative maxima at around 206 and 224 nm (208 nm and 224 nm for the peptide 4), indicating that they possessed a right-handed (P) helical screw sense, as shown in Fig. 3A.¹²⁾ Based on the R values (θ₂₂₄/θ₂₀₆) of the peptides, the homochiral peptide 1 and the heterochiral peptides 2 and 3 formed 3₁₀-helices as their dominant conformations (R=0.38 for 1, R=0.44 for 2, R=0.34 for 3), whereas the LD-peptide 4 preferentially formed an α-helix (R=θ₂₂₄/θ₂₀₉=0.71).¹⁷⁾ Based on the intensity values of the peptides’ CD spectra, it was considered that peptide 1, which is entirely composed of L-Leu residues, formed the most right-handed (P) helical structure, and the peptides’ CD intensities decreased linearly as the number of D-Leu residues in the nonapeptide sequence increased. Whereas, the peptide 4 having the alternating L-Leu-D-Leu-Aib segment preferentially formed a right-handed (P) α-helix rather than a 3₁₀-helix.¹⁵⁾ Thus, as the number of D-Leu residues in a helical L-peptide sequence increased, the right-handed (P) 3₁₀-helices are gradually destabilized and tends to change their dominant conformations from 3₁₀-helices to α-helices. Even in a mixture of TFE and PBS buffer (1 : 1) solution, all peptides formed (P) helical structures, respectively, as shown in Fig. 3B.

Fig. 3. CD Spectra of Nonapeptides 1–4 in TFE (a) and TFE/PBS (pH 7.2)=1 : 1 Solution (b)

Peptide concentration: 0.1 mM.

X-Ray Diffraction Analysis

Peptides 2 and 3 produced suitable crystals for X-ray crystallographic analysis after the slow evaporation of N,N-dimethylacetamide (DMA)/H₂O and MeOH/MeCN, respectively, at room temperature. The crystal and diffraction parameters of 2 and 3 are summarized in Table 1. Data collection was performed using a Bruker AXS SMART APEX imaging plate diffractometer and graphite-monochromated MoKα radiation. The structures of 2 and 3 were solved by the direct method using SHELXS 97¹⁸⁾ and expanded using the Fourier technique.¹⁹⁾ All non-H atoms were given anisotropic thermal parameters, some H atoms were refined isotropically, and the remaining H atoms were placed at the calculated positions.²⁰⁾ The crystal and diffraction parameters of the peptides, as well as relevant backbone and side chain torsion angles and intra- and intermolecular hydrogen-bond parameters are summarized in Tables 2 and 3.

Table 1. Crystal and Diffraction Parameters of Peptides 2 and 3

	2	3
Formula	C54H99N9O12, C4H9NO	C54H99N9O12, CH4O, C2H3N
Crystal dimensions [mm]	0.50×0.40×0.03	0.45×0.30×0.15
Crystal system	Orthorhombic	Orthorhombic
Lattice parameters:
a [Å]	12.6227	14.7729
b [Å]	13.7970	20.2473
c [Å]	38.891	45.357
α [°]	90	90
β [°]	90	90
γ [°]	90	90
V [Å3]	6773.0	13567
Spacer group	P212121	P212121
Z value	4	4
Dcalc [g/cm3]	1.13	1.08
µ (MoKα) [cm−1]	0.80	0.86
No. of observations	6824 (I>2σ(I))	12914 (I>2σ(I))
No. of variables	730	1399
R1, Rw	0.0561, 0.1566	0.0557, 0.1567
Solvent	DMA/H2O	MeOH/MeCN

Table 2. Selected Torsion Angles (φ, ψ, ω, and χ) [°] for Peptides 2 and 3

Residue	Torsion angle
	φ	ψ	ω	χ
Boc-(L-Leu-L-Leu-Aib)2-L-Leu-D-Leu-Aib-OMe (2)
L-Leu(1)	−53.7	−44.3	−176.1	−178.1
L-Leu(2)	−53.2	−47.1	−177.6	174.7
Aib(3)	−57.7	−42.8	−174.5	—
L-Leu(4)	−77.6	−33.2	172.8	−53.8
L-Leu(5)	−56.7	−43.8	179.0	172.3
Aib(6)	−55.5	−29.8	−178.1	—
L-Leu(7)	−96.2	10.5	172.6	−61.7
D-Leu(8)	92.3	11.9	173.7	62.2
Aib(9)	−48.7	−40.3	174.9	—
Boc-L-Leu-D-Leu-Aib-L-Leu-L-Leu-Aib-L-Leu-D-Leu-Aib-OMe (3)
Molecule A
L-Leu(1A)	−64.4	−35.5	−178.4	177.2
D-Leu(2A)	−52.7	−57.4	−178.8	−29.1
Aib(3A)	−57.5	−44.8	−177.0	—
L-Leu(4A)	−60.4	−40.2	177.5	−61.7
L-Leu(5A)	−66.1	−44.1	−178.8	−67.3
Aib(6A)	−54.6	−35.2	−177.5	—
L-Leu(7A)	−86.4	−8.1	−169.8	−66.6
D-Leu(8A)	55.8	39.5	175.2	174.0
Aib(9A)	−54.2	−28.3	176.8	—
Molecule B
L-Leu(1B)	−66.1	−43.8	−176.6	176.5
D-Leu(2B)	−53.0	−53.6	−178.6	63.7
Aib(3B)	−55.7	−47.1	−176.7	—
L-Leu(4B)	−59.7	−44.4	−177.1	−66.1
L-Leu(5B)	−70.1	−40.0	179.4	−64.1
Aib(6B)	−54.9	−35.2	−177.1	—
L-Leu(7B)	−88.5	−9.5	−170.8	−75.3
D-Leu(8B)	66.0	25.5	169.8	77.4
Aib(9B)	49.8	−136.8	−179.5	—

Table 3. Intra- and Intermolecular H-Bond Parameters for Peptides 2 and 3

Peptidea)	Donor D–H	Acceptor A	Distance [Å] D…A	Angle [°] D–H…A	Symmetry operations
Boc-(L-Leu-L-Leu-Aib)2-L-Leu-D-Leu-Aib-OMe (2)
	N4–H	O0	3.47b)	156	x, y, z
	N5–H	O1	2.91	165	x, y, z
	N6–H	O2	3.20	161	x, y, z
	N7–H	O4	3.01	134	x, y, z
	N8–H	O5	2.89	163	x, y, z
	N9–H	O4	3.01	163	x, y, z
	N1–H	ODa, c)	2.90	140	x, 1+y, z
	N2–H	O7′	2.81	162	x, 1+y, z
Boc-L-Leu-D-Leu-Aib-L-Leu-L-Leu-Aib-L-Leu-D-Leu-Aib-OMe (3)
Molecule A	N4a–H	O0a	3.14	165	x, y, z
	N5a–H	O1a	2.89	160	x, y, z
	N6a–H	O2a	3.09	165	x, y, z
	N7–H	O3a	3.18	149	x, y, z
	N8a–H	O5a	2.94	163	x, y, z
	N9a–H	O4a	2.93	163	x, y, z
Molecule B
	N4b–H	O0b	3.25	163	x, y, z
	N5b–H	O1b	2.95	161	x, y, z
	N6b–H	O2b	3.02	169	x, y, z
	N7b–H	O3b	3.31b)	148	x, y, z
	N8b–H	O5b	2.99	159	x, y, z
	N9b–H	O4b	2.95	141	x, y, z
	N1b–H	OMc)	2.85	154	x, y, z
	N2b–H	O7a	2.74	122	x, y, z
	OM–Hc)	O6a	2.74	166	x, y, z
	N3a–H	O7b′	3.04	157	−1+x, −1+y, z

a) The amino acid numbering of the residues begins at the N-terminus of the peptide chain. b) The D…A distance is a bit long for a hydrogen bond. c) O_D: DMA, O_M: MeOH.

A right-handed (P) α-helix with a dimethylacetamide molecule was existed in the asymmetric unit of Boc-(L-Leu-L-Leu-Aib)₂-L-Leu-D-Leu-Aib-OMe (2) (Fig. 4). The mean φ and ψ torsion angles of the amino acid residues (1–6) were −59.1° and −40.2°, respectively, which are close to those of an ideal (P) α-helix (−60° and −45°, respectively). However, the D-Leu (8) residue was flipped,^21,22) and its φ and ψ torsion angles were positive (φ=+92.3°, ψ=+11.9°). Two i←i+3 type, two i←i+4 type, and one i←i+5 type hydrogen bond were found in the α-helix of 2. Hydrogen bonds were detected between the H-N(4) and C(0)=O(1) [N(4)…O(0)=3.47 Å; a bit long for a hydrogen bond], between the H-N(5) and C(1)=O(1) [N(5)…O(1)=2.91 Å], between the H-N(6) and C(2)=O(2) [N(6)…O(2)=3.20 Å], between the H-N(7) and C(4)=O(4) [N(7)…O(4)=3.01 Å], between the H-N(8) and C(5)=O(5) [N(8)…O(5)=2.89 Å], and between the H-N(9) and C(4)=O(4) [N(9)…O(4)=3.01 Å]. In packing mode, successive helical molecules were connected by an intermolecular hydrogen bond to form head-to-tail aligned chains.

Fig. 4. X-Ray Diffraction Structure of the Nonapeptide 2 as Viewed (a) Perpendicular to and (b) along Its Helical Axis

The asymmetric unit of Boc-L-Leu-D-Leu-Aib-L-Leu-L-Leu-Aib-L-Leu-D-Leu-Aib-OMe (3) contained two right-handed (P) α-helical structures, in which the D-Leu (8) residue was flipped (Fig. 5). The conformations of molecules A and B were well matched, except for small differences in the conformations of their side chains and C-terminus peptide backbones (Fig. 6). The mean φ and ψ torsion angles of the α-helical residues (1–7) were −59.3° and −42.9°, respectively, for A, and −59.9° and −44.0°, respectively, for B. Regarding the intramolecular H-bonds in molecule A, one i←i+3 type, four i←i+4 type, and one i←i+5 type hydrogen bond were formed. The hydrogen bonds were located between the H-N(4a) and C(0a)=O(0a) [N(4a)…O(0a)=3.14 Å], between the H-N(5a) and C(1a)=O(1a) [N(5a)…O(1a)=2.89 Å], between the H-N(6a) and C(2a)=O(2a) [N(6a)…O(2a)=3.09 Å], between the H-N(7a) and C(3a)=O(3a) [N(7a)…O(3a)=3.18 Å], between the H-N(8a) and C(5a)=O(5a) [N(8a)…O(5a)=2.94 Å], and between the H-N(9a) and C(4a)=O(4a) [N(9a)…O(4a)=2.93 Å]. In molecule B, the same types of H-bonds as shown in molecule A, were detected between the H-N(4b) and C(0b)=O(0b) [N(4b)…O(0b)=3.25 Å], between the H-N(5b) and C(1b)=O(1b) [N(5b)…O(1b)=2.95 Å], between the H-N(6b) and C(2b)=O(2b) [N(6b)…O(2b)=3.02 Å], between the H-N(7b) and C(3b)=O(3b) [N(7b)…O(3b)=3.31 Å; a bit long for a H-bond], between the H-N(8b) and C(5b)=O(5b) [N(8b)…O(5b)=2.99 Å], and between the H-N(9b) and C(4b)=O(4b) [N(9b)…O(4b)=2.95 Å]. In packing mode, molecules A and B were alternately connected by intermolecular hydrogen bonds.

Fig. 5. X-Ray Diffraction Structure of the Nonapeptide 3 as Viewed (a) Perpendicular to and (b) along Its Helical Axis

Fig. 6. Superimposed Structures of Molecules A (Light Gray) and B (Gray) as Viewed (a) Perpendicular to and (b) along Their Helical Axes

Conclusion

Four diastereomeric nonapeptides, Boc-(L-Leu-L-Leu-Aib)₃-OMe (1),¹³⁾ Boc-(L-Leu-L-Leu-Aib)₂-L-Leu-D-Leu-Aib-OMe (2), Boc-L-Leu-D-Leu-Aib-L-Leu-L-Leu-Aib-L-Leu-D-Leu-Aib-OMe (3), and Boc-(L-Leu-D-Leu-Aib)₃-OMe (4),¹⁴⁾ were synthesized to investigate the influence of D-Leu residues on the helical structures of L-Leu-based nonapeptides. In solution, the homochiral peptide 1, which only contained L-Leu residues, formed the most righ-handed (P) helical structure. Increasing the number of D-Leu residues in the L-Leu-based nonapeptide sequence decreased the right-handedness of the resultant helical peptides. Peptides 1, 2, and 3 formed 3₁₀-helices as their dominant conformations, whereas the dominant conformation of peptide 4, which contained alternating L-Leu-D-Leu fragments, was an α-helix. In short peptide sequences, Aib residues are able to stabilize 3₁₀-helices, but not α-helices, and therefore, peptides 1, 2, and 3 formed 3₁₀-helices in solution. On the other hand, peptide 4, which was composed of the well-regulated L-Leu-D-Leu-Aib segment, formed a right-handed (P) α-helix rather than a 3₁₀-helix.¹⁵⁾

In the crystalline state, peptide 2, which contained one D-Leu residue, and peptide 3, which contained two D-Leu residues, were folded into (P) α-helices with flipped C-terminal D-Leu (8) residues. In packing mode, peptides 3 and 4 probably tend to form α-helices, rather than 3₁₀-helices, in order to avoid amino acid side-chain competition between the i and i+3 positions. The incorporation of increasing numbers of D-Leu residues into L-Leu-based helical nonapeptides gradually destabilizes the right-handedness of their helices and tends to change their dominant conformations from 3₁₀-helices to α-helices. These findings could have important implications for the construction of well-defined short helical peptides.

Experimental

General

Optical rotation [α]_D was assessed using a 1.0 dm cell in MeOH. ¹H- and ¹³C-NMR spectra were recorded at 400 MHz and 100 MHz in CDCl₃ using tetramethylsilane as an internal standard. Fourier transform infrared (FT-IR) spectra were recorded on a JASCO FT/IR-4100 spectrometer at a resolution of 1 cm⁻¹ over a mean of 128 scans using the solution (CDCl₃) method and NaCl cells with a path length of 0.1 mm. CD spectra were recorded with a JASCO J-720W spectropolarimeter using a cell with a 1.0-mm path length. The resultant data are expressed in terms of [θ]_R, the total molar ellipticity (deg cm² dmol⁻¹). 2,2,2-Trifluoroethanol (TFE) and a mixture of TFE/PBS (1 : 1) solution were used as a solvent. The data collection for the X-ray diffraction analysis was performed on Rigaku RAXIS-RAPID and Bruker AXS SMART APEX imaging plate diffractometers using graphite-monochromated MoKα radiation.

Boc-(L-Leu-L-Leu-Aib)₂-L-Leu-D-Leu-Aib-OMe (2)

Colorless crystals; mp 225–227°C; [α]²¹_D=−11.8 (c=0.5, CHCl₃); IR (ATR): ν 3434, 3334, 2961, 2871, 1736, 1662 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ: 7.74 (d, J=6.0 Hz, 1H), 7.58 (d, J=7.6 Hz, 1H), 7.55 (br s, 1H), 7.31 (d, J=6.0 Hz, 1H), 7.25 (br s, 1H), 7.13 (br s, 1H), 7.10 (d, J=8.0 Hz, 1H), 6.77 (br s, 1H), 5.44 (br s, 1H), 4.30–4.41 (m, 2H), 3.80–4.11 (m, 4H), 3.68 (s, 3H), 1.39–1.88 (m, 45H), 0.79–1.00 (m, 36H); ¹³C-NMR (100 MHz, CDCl₃) δ: 176.3, 175.6, 175.2, 175.1, 174.6, 173.7, 173.4, 173.0, 175.2, 81.9, 57.4, 57.1, 55.9, 55.7, 55.3, 55.1, 54.9, 52.6, 52.4, 40.6, 40.4, 40.1, 40.0, 39.4, 28.5, 27.6, 27.2, 25.7, 25.4, 25.3, 25.2, 25.1, 24.7, 23.8, 23.5, 23.4, 23.3, 23.0, 22.2, 21.9, 21.7, 21.6, 21.1, 21.0; [high resolution electrospray ionization (HR-ESI)(+)]: m/z Calcd for C₅₄H₉₉N₉O₁₂Na [M+Na]⁺ 1088.7311: found 1088.7313.

Boc-L-Leu-D-Leu-Aib-L-Leu-L-Leu-Aib-L-Leu-D-Leu-Aib-OMe (3)

Colorless crystals; mp 237–239°C; [α]²¹_D=+2.2 (c=0.5, CHCl₃); IR (ATR): ν 3439, 3334, 2960, 2871, 1735, 1662 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ: 7.60–7.71 (m, 4H), 7.51 (br s, 1H), 7.43 (br s, 1H), 7.23 (br s, 1H), 7.18 (br s, 1H), 5.59 (br s, 1H), 4.20–4.32 (m, 2H), 4.08 (m, 1H), 3.98 (m, 1H), 3.84 (m, 1H), 3.73 (m, 1H), 3.67 (s, 3H), 1.40–1.96 (m, 45H), 0.78–1.09 (m, 36H); ¹³C-NMR (100 MHz, CDCl₃) δ: 176.7, 175.8, 175.5, 174.9, 174.8, 173.9, 173.3, 172.8, 157.1, 81.5, 57.6, 57.3, 56.1, 55.9, 55.2, 55.1, 53.9, 53.3, 53.0, 52.6, 40.9, 40.1, 39.7, 39.5, 38.0, 28.8, 27.9, 27.0, 25.9, 25.5, 25.2, 25.1, 24.9, 24.0, 23.7, 23.6, 23.5, 23.1, 22.3, 22.0; [HR-ESI(+)]: m/z Calcd for C₅₄H₉₉N₉O₁₂Na [M+Na]⁺ 1088.7311: found 1088.7309.

Acknowledgments

This study was supported, in part, by JSPS KAKENHI Grant No. 26460169 (Y.D.) and by a Grant-in-Aid from the Tokyo Biochemical Research Foundation (Y.D.).

Conflict of Interest

The authors declare no conflict of interest.

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