Gramicidin S analogs having six basic amino acid residues

Main

Gramicidin S (GS), cyclo(-Val^1,1′-Orn^2,2′-Leu^3,3′-D-Phe^4,4′-Pro^5,5′-)₂,^{1, 2, 3} is a potent cyclopeptide antibiotic isolated from Bacillus brevis. Its secondary structure has been established as an antiparallel β-sheet conformation with amphiphilicity.^{4, 5} The conformation is characteristically featured with the orientation of side chains in such a way that the charged Orn side chains are situated on one side of the molecule and the hydrophobic Val and Leu side chains are situated on the other side. The side-chain arrangement is apparently held together by a rigid conformation containing two D-Phe-Pro type II′ β-turns. It has been proposed that the principal modes of antibiotic actions result from an interaction of GS with the cell membrane of the target microorganisms. GS then adopts an antiparallel β-sheet conformation with amphiphilicity, which disrupts cell membrane.⁶ Thus, the hydrophobic Val and Leu residues have been considered to be essential for exhibiting the strong activity of GS. Therefore, syntheses of antimicrobially active analogs of GS containing amino acid residues with hydrophilic side chain in place of Val and Leu residues have not been reported yet.^{2, 3, 7, 8, 9}

In this account, we designed and synthesized novel GS analogs, cyclo(-Orn^1,1′-Orn^2,2′-Orn^3,3′-D-Phe^4,4′-Pro^5,5′-)₂ (1) and cyclo(-Lys^1,1′-Orn^2,2′-Lys^3,3′-D-Phe^4,4′-Pro^5,5′-)₂ (2), which have four basic amino acid residues (Orn and Lys residues) in place of four hydrophobic amino acid residues (Val and Leu residues), to investigate the role of amphiphilic β-sheet structure of GS for the antibiotic activity.

Syntheses of 1 and 2 were performed by a solid-phase method using oxime resin.¹⁰ The formation of the cyclic peptides by the dimerization–cyclization of H-D-Phe-Pro-X-Orn(Z)-X-oxime on resin (X=Orn(Z), and Lys(Z)) (Z-=benzyloxycarbonyl-) gave cyclo(-X^1,1′-Orn(Z)^2,2′-X^3,3′-D-Phe^4,4′-Pro^5,5′-)₂ in 83 and 92% yields, respectively. The removal of all the masking groups by 25% HBr/AcOH produced the corresponding antibiotics 1 and 2 in 53 and 67% yields, respectively. The homogeneities of 1 and 2 were confirmed by thin-layer chromatography, high performance liquid chromatography, FAB-MS and ¹H NMR spectrometry.

To investigate the secondary structures of 1 and 2, CD and ¹H NMR spectra of 1 and 2 were measured. Compounds 1, 2 and GS showed almost identical CD spectra in methanol (Figure 1), suggesting that 1 and 2 have a β-sheet structure similar to that of GS.

Figure 1

CD spectra of 1, 2 and GS in methanol.

The ¹H NMR (400 MHz) spectra of 1 and 2 were measured at 30 °C in DMSO-d6 (peptide concentration: ∼17 mg ml⁻¹). All protons were assigned by means of H–H COSY (correlation spectroscopy), TOCSY (total correlation spectroscopy) and ROESY (rotating frame nuclear Overhauser enhancement spectroscopy). Only one ^αNH resonance appeared for X^1,1′, Orn^2,2′, X^3,3′ and D-Phe^4,4′ residues (1: X=Orn, 2: X=Lys), indicating that 1 and 2 have conformations with C₂ symmetry in the NMR time average. In the ¹H NMR spectrum of 1, temperature coefficient values of ^αNH groups for Orn^1,1′, Orn^2,2′, Orn^3,3′ and D-Phe^4,4′ were 2.8, 6.3, 2.8 and 5.7 ppb K⁻¹, respectively. These results indicated that Orn^{1,1′ α}NH and Orn^{3,3′ α}NH are shielded from the solvent and involved in two stable intramolecular hydrogen bonds, whereas Orn^{2,2′ α}NH and D-Phe^{4,4′ α}NH are exposed to the solvent. The ${\text{J}}_{{\text{NH-}}^{\text{α}}\text{CH}}$ values of Orn^1,1′, Orn^2,2′, Orn^3,3′ and D-Phe^4,4′ residues were 8.1, 8.8, 7.6 and 2.0 Hz, respectively. The ${\text{J}}_{{\text{NH-}}^{\text{α}}\text{CH}}$ values observed for Orn^1,1′, Orn^2,2′ and Orn^3,3′ residues were strongly indicative of an extended β-sheet conformation.¹¹ On the other hand, ${\text{J}}_{{\text{NH-}}^{\text{α}}\text{CH}}$ value of D-Phe residue was indicative of a β-turn conformation.¹¹ The chemical shift perturbation¹² (ΔδH_α=observed δH_α−random coil δH_α) of the ^αH of Orn^1,1′, Orn^2,2′ and Orn^3,3′ showed positive values (>0.1 p.p.m.). On the other hand, the D-Phe^4,4′ and Pro^5,5′ residues showed negative values. The chemical shift perturbation of the ^αH of 1 agreed well with those of GS.¹³ The results suggested that Orn^1,1′-Orn^2,2′-Orn^3,3′ sequences in 1 have a similar β-sheet conformation to that of GS sequences. Next, for detailed analysis, the spatial ROE correlations were measured. (Figure 2) ROE spatial correlations between Pro^{5,5′ α}CH and Orn^{1,1′ α}NH, Orn^{1,1′ α}CH and Orn^{2,2′ α}NH, Orn^{2,2′ α}CH and Orn^{3,3′ α}NH, Orn^{3,3′ α}CH and D-Phe^{4,4′ α}NH, and D-Phe^{4,4′ α}CH and Pro^{5,5′ δ}CH₂ were observed. The results indicated that amide bonds in 1 are all-trans conformation. The signal of D-Phe ^βCH₂ was two multiplets, indicating that they are nonequivalent and fixed in certain arrangement. The chemical shifts of H resonances for the diastereotopic β-, γ- and δ-CH₂ of Pro residues were separated by 0.29, 0.00 and 0.83 p.p.m., respectively, suggesting that the aromatic ring of D-Phe residue orients in close proximity to Pro ^δCH. Similar results were obtained from the NMR studies of 2. The NMR data indicated that 1 and 2 have GS-like antiparallel β-sheet structures with a type II′ β-turn around D-Phe-Pro as shown in Figure 2, and that the hydrophobic side chains of Val and Leu residues are not necessary for holding the rigid β-sheet conformation of GS.

Figure 2

Proposed secondary structures of 1 (Y^1,1′ and Z^3,3′=-(CH₂)₃NH₂), 2 (Y^1,1′ and Z^3,3′=-(CH₂)₄NH₂) and GS (Y^1,1′=-CH(CH₃)₂, Z^3,3′=-CH₂CH(NH₃)₂) with ROE spatial correlations.

The antibiotic activities and hemolytic activities of 1, 2 and GS were summarized in Table 1. The difference of antibiotic activities among 1, 2 and GS reflects the characters of side chains of the amino acid residues at positions 1, 1′, 3 and 3′ because the antibiotics have similar β-sheet structure to each other. The antibiotic activities of 1 and 2 were 1/4 and 1/8 of GS against Bacillus subtilis NBRC 3513 and Bacillus megaterium ATCC 19213, respectively, and less against Staphylococcus epidermidis NBRC 12933 and Staphylococcus aureus NBRC 12732. On the other hand, 1 and 2 showed no activity against Gram-negative microorganisms tested. The results indicated that the presence of hydrophobic side chains of Val and Leu residues are important for exhibiting the strong activity of GS. In addition, it is interesting to note that 1 and 2 showed some selectivity against different microorganisms. Then, 1 and 2 showed almost no hemolytic activity toward sheep red blood cells (Table 1),¹⁴ indicating that the replacement of the Val and Leu hydrophobic side chains into the Orn and Lys basic side chains could result in substantial reduction of hemolytic activities.

Table 1 Antibiotic activities^a and hemolytic activity^b of 1, 2 and GS

In these studies, we reported the structure–activity relationship of GS analogs 1 and 2 containing Orn and Lys residues with basic side chains in place of four hydrophobic Val and Leu residues. Currently, we are investigating the design and syntheses of other antimicrobially active analogs of GS without the hydrophobic Val and Leu residues on one side of the molecule on the basis of these studies in order to find new types of drug candidates with high antimicrobial and low hemolytic activities.

Experimental section

Melting points were measured on Mel-Temp II melting point apparatus (Laboratory Devices, Cambridge, MA, USA) and are uncorrected. Low-resolution mass spectra (LR-MS) were obtained by using FAB mass spectrometry on a JEOL600H mass spectrometer (Jeol, Tokyo, Japan). CD spectra were recorded on a Jasco J-820 spectropolarimeter (Jasco, Tokyo, Japan) using a quarts cell of 0.5-mm pathlength. The CD spectra in methanol were measured at a peptide concentration of 1.10 × 10⁻⁴ M at room temperature. ¹H NMR spectra were measured in DMSO-d₆ at 30 °C (peptide concentration ca. 17 mg ml⁻¹) on a JEOL JNM-ECP400 spectrometer (Jeol) using standard pulse sequences and software. The chemical shifts were determined with respect to internal TMS (tetramethylsilane).

cyclo(-Orn-Orn-Orn-D-Phe-Pro-)₂ 6HBr (1)

A protected linear precursor oxime, H-D-Phe-Pro-Orn(Z)-Orn(Z)-Orn(Z)-oxime, was prepared by using Boc-solid phase peptide synthesis on resin (Loading of oxime group: 0. 35 mmol g⁻¹ resins). The formation of the cyclic peptide by the dimerization–cyclization of H-D-Phe-Pro-Orn(Z)-Orn(Z)-Orn(Z)-oxime on resin was performed in 1,4-dioxane with two equiv. of triethylamine and acetic acid for 1 day at room temperature.¹⁰ The cyclizations gave cyclo(-Orn(Z)-Orn(Z)-Orn(Z)-D-Phe-Pro-)₂ in yield of 83%. The removal of all the masking groups was performed by 25% HBr/acetic acid. The product was purified by gel filtration on a Sephadex LH-20 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and by reprecipitation from methanol–ether to give a white powder of 1 in 53% yield.

Mp 234–235 °C. LR-FAB-MS (matrix: m-NBA (m-nitro benzyl alcohol)) Calcd for C₅₈H₉₂N₁₆O₁₀ [M]⁺=1173, Found m/z 1174 ([M+H]⁺, 0.90%), 1196 ([M+Na]⁺, 0.32%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.30 (d, 2H, NH_α D-Phe^4,4′, ³J_NH-Hα=2.0 Hz), 8.75 (d, 2H, NH_α Orn^2,2′, ³J_NH-Hα=8.8 Hz), 8.16 (d, 2H, NH_α Orn^3,3′, ³J_NH-Hα=7.6 Hz), 7.78 (m, 4H, NH_δ Orn^1,1′, m, 4H, NH_δ Orn^3,3′), 7.69 (m, 4H, NH_δ Orn^2,2′), 7.33–7.27 (m, 10H, H_ar D-Phe^4,4′), 7.31 (d, 2H, NH_α Orn^1,1′, ³J_NH-Hα=8.1 Hz), 4.82 (m, 2H, H_α Orn^2,2′), 4.55 (m, 2H, H_α Orn^1,1′), 4.49 (m, 2H, H_α Orn^3,3′), 4.43 (m, 2H, H_α D-Phe^4,4′), 4.35 (m, 2H, H_α Pro^5,5′), 3.52 (m, 2H, H_δ Pro^5,5′), 3.00 (m, 2H H_β D-Phe^4,4′), 2.87 (m, 2H H_β D-Phe^4,4′), 2.80 (m, 4H, H_δ Orn^1,1′, m, 4H, H_δ Orn^2,2′, m, 4H, H_δ Orn^3,3′), 2.69 (m, 2H, H_δ Pro^5,5′), 1.97 (m, 2H, H_β Pro^5,5′), 1.74 (m, 2H, H_β Orn^1,1′), 1.70 (m, 2H, H_β Orn^2,2′), 1.69 (m, 2H, H_β Orn^1,1′), 1.61 (m, 2H, H_β Orn^2,2′, m, 2H, H_β Orn^3,3′, m, 2H, H_β Pro^5,5′, m, 4H, H_γ Pro^5,5′), 1.47 (m, 2H, H_β Orn^3,3′, m, 4H, H_γ Orn^3,3′), 1.45 (m, 4H, H_γ Orn^1,1′, m, 4H, H_γ Orn^2,2′).

cyclo(-Lys-Orn-Lys-D-Phe-Pro-)₂ 6HBr (2)

cyclo(-Lys(Z)-Orn(Z)-Lys(Z)-D-Phe-Pro-)₂ was synthesized in 92% yield as has been described for the preparation of cyclo(-Orn(Z)-Orn(Z)-Orn(Z)-D-Phe-Pro-)₂. The removal of all the masking groups by 25% HBr/acetic acid produced 2 in 67% yield.

Mp. 231.5–233.0 °C. LR-FAB-MS (matrix: m-NBA) Calcd for C₆₂H₁₀₀N₁₆O₁₀ [M]⁺=1229, Found m/z 1230 ([M+H]⁺, 1.01%), 1252 ([M+Na]⁺, 0.33%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.22 (d, 2H, NH_α D-Phe^4,4′, ³J_NH-Hα=1.6 Hz), 8.69 (d, 2H, NH_α Orn^2,2′, ³J_NH-Hα=7.8 Hz), 8.16 (d, 2H, NH_α Lys^3,3′, ³J_NH-Hα=8.1 Hz), 7.84 (m, 4H, NH_ɛ Lys^1,1′, m, 4H, NH_δ Orn^2,2′, m, 4H, NH_ɛ Lys^3,3′), 7.32–7.27 (m, 10H, H_ar D-Phe^4,4′), 7.28 (d, 2H, NH_α Lys^1,1′, ³J_NH-Hα=8.3 Hz), 4.79 (m, 2H, H_α Orn^2,2′), 4.50 (m, 2H, H_α Lys^1,1′), 4.47 (m, 2H, H_α Lys^3,3′), 4.41 (m, 2H, H_α D-Phe^4,4′), 4.32 (m, 2H, H_α Pro^5,5′), 3.59 (m, 2H, H_δ Pro^5,5′), 3.00 (m, 2H H_β D-Phe^4,4′), 2.89 (m, 2H H_β D-Phe^4,4′), 2.83 (m, 4H, H_δ Orn^2,2′), 2.74 (m, 4H, H_ɛ Lys^3,3′), 2.68 (m, 4H, H_ɛ Lys^1,1′), 2.52 (m, 2H, H_δ Pro^5,5′), 1.98 (m, 2H, H_β Pro^5,5′), 1.74 (m, 2H, H_β Orn^2,2′), 1.71 (m, 4H, H_β Lys^1,1′), 1.69 (m, 2H, H_β Orn^2,2′), 1.57 (m, 4H, H_γ Lys^1,1′, m, 4H, H_γ Orn^2,2′, m, 4H, H_β Lys^3,3′, m, 2H, H_β Pro^5,5′, m, 4H, H_γ Pro^5,5′), 1.33 (m, 4H, H_γ Lys^3,3′), 1.27 (m, 4H, H_δ Lys^1,1′), 1.19 (m, 4H, H_δ Lys^3,3′).