Total Synthesis of Septocylindrin B and C-Terminus Modified Analogues

<div><p>The total synthesis is reported of the peptaibol Septocylindrin B which is related to the well documented channel forming peptaibol antibiotic Alamethicin. Several analogues were synthesized with a modified <em>C</em>-terminus, to investigate the SAR of the terminal residue Phaol. All these peptides were tested for their membrane perturbation properties by fluorescent dye leakage assay and for their antibacterial activity.</p> </div

Synthetic strategy for the synthesis of ABCD-ethanolamine with Z = Cbz.

<p>(i) Cbz-removal with H₂, Pd-C and MeOH followed by coupling via EDC/HOAt. (ii) O<i>t</i>Bu-removal with ZnBr₂ .iii) O<i>t</i>Bu-removal of the <i>C</i>-component with TFA/CH₂Cl₂ and Cbz-removal of the <i>N</i>-component with H₂, Pd-C and MeOH, followed by the coupling via EDC/HOAt. (iv) Cbz-removal with H₂, Pd-C and MeOH followed by coupling via PyBOP, followed by deprotection using TBAF.</p

Release of CF from PC∶Ch (7∶3) liposomes after 20 minutes at different ratios R-1 = [peptide]/[lipid].

<p>Alamethicin F50 is used as a reference.</p

Synthetic strategy for the synthesis of Septocylindrin B with Z = Cbz.

<p>(i) Cbz-removal with H₂, Pd-C and MeOH followed by coupling via EDC/HOAt. (ii) O<i>t</i>Bu-removal of the <i>C</i>-component with TFA/CH₂Cl₂ and Cbz-removal of the <i>N</i>-component with H₂, Pd-C and MeOH, followed by coupling using EDC/HOAt. (iii) O<i>t</i>Bu-removal using ZnBr₂ or TFA and Cbz-removal of the <i>N</i>-component with H₂, Pd-C and MeOH, followed by coupling using EDC/HOAt, after which the peptide is Boc deprotected using ZnBr₂. (iv) aminolysis.</p

Synthetic approach for Septocylindrin B.

<p>A' = residue 2–5, A = residue 1–5, B = 6–13, C = residue 14–17, D = residue 18–20.</p

Synthetic strategy for the synthesis of ABCD-Phaol-N6 with Z = Cbz.

<p>(i) Cbz-removal with H₂, Pd-C and MeOH followed by coupling via EDC/HOAt. (ii) O<i>t</i>Bu-removal of the <i>C</i>-component with TFA/CH₂Cl₂ and Cbz-removal of the <i>N</i>-component with H₂, Pd-C and MeOH, followed by coupling using EDC/HOAt. (iii) O<i>t</i>Bu removal using ZnBr₂ (iv) Cbz-removal of the <i>N</i>-component with H₂, Pd-C and MeOH, followed by coupling via HOAt/EDC (v) Boc-deprotection using BiCl₃.</p

Phaol [2-{[(2<i>S</i>)-2-amino-3-phenylpropyl]amino}ethanol].

<p>R-Phaol, R = Ac-UPUAUAQUVUGLUPVUUQQ for Septocylindrin B.</p

Antibacterial activity (MIC, µM) of Septocylindrin B and selected analogues.^a

a<p>For HPLC traces of selected analogues see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051708#pone.0051708.s004" target="_blank">Figures S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051708#pone.0051708.s005" target="_blank">S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051708#pone.0051708.s006" target="_blank">S6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051708#pone.0051708.s007" target="_blank">S7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051708#pone.0051708.s008" target="_blank">S8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051708#pone.0051708.s009" target="_blank">S9</a>.</p>b<p>Data from ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051708#pone.0051708-Summers1" target="_blank">[1]</a>.</p>c<p>Not available.</p>d<p>A reduced growth is clearly visible, but no MIC end point ≤100 µM.</p

Other glycines substitutions that destabilize BH4-Bcl-2’s

<div><p><b>α-helical structure abolish its IP₃R-inhibitory properties</b>.</p> <p>(A, left) Panel showing the values of ΔΔG, in kcal/mol, resulting from the <i>in </i><i>silico</i> analysis (Eris automated estimator) of the II/GG and VIL/GGG substitutions. Positive ΔΔG values indicate destabilizing mutations (A, right). Predicted-secondary structure assignments for the isolated BH4 domain mutated as described above. Each panel shows (from top to bottom) the primary structure, the secondary-structure predictions (C = random coil, S = strand [black/bold]) and the level of confidence of the predictions (confidence scores from 0 to 9). The key residues involved in the regulation of IP ₃Rs are depicted in black/bold while the position of the exchanged residues is indicated by the red G residues in the primary structure. (B) CD spectra of synthetic Bcl-2-BH4 (black line) in comparison with the ones for its G-substituted counterparts [II/GG (purple trace), VIL/GGG (green trace)]. The ellipticity is calculated per mole of amino-acid residue. Both mutant peptides showed a relative decrease in α-helical conformation as assessed by spectra analysis with the CONTIN/LL deconvolution method (see provided change in α-helical percentage for each condition. For the percentages of the other secondary structure features see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073386#pone.0073386.s001" target="_blank">Table S1</a> 1). (C) Representative unidirectional ⁴⁵Ca²⁺ fluxes in permeabilized MEF cells plotted as fractional loss (% / 2 min) as a function of time. Ca²⁺ release was activated 10 min after starting the experiment by applying 3 µM IP₃ (arrow) in the absence or presence of 50 µM of the different BH4-domain peptides (the traces are color coded as in B). The gray bar indicates the peptide-incubation period. Data points represent means ± SD (D) IICR was quantified as the difference of the fractional loss after 2 min of incubation with IP₃ and the fractional loss before the IP₃ addition in the presence of vehicle (DMSO), Bcl-2-BH4 and the respective mutant peptides. The 100% value corresponds to IICR in the presence of the vehicle. All values were normalized to this control. Data points represent means ± SEM. * indicates a statistically significant difference from vehicle control.</p></div

A Bcl-2-BH4 double-glycine variant (I14G/V15G) has a decreased

<div><p><b>α-helical content</b>. </p> <p>(A) Primary structure of the Bcl-2-BH4 peptide. The key residues involved in the regulation of IP ₃Rs are depicted in black/bold. The residues considered for the glycine substitution (I14 and V15) are depicted in red. The α-helical structure underneath represents the best predictive model obtained from I-TASSER web server and drawn using Pymol. The labels for the key peptide residues follow the same color code as in the primary structure. (B) Predicted-secondary structure assignments for the isolated BH4 domain of Bcl-2 (upper panel) and for its IV/GG counterpart (lower panel). Each panel shows the amino acid sequence, the secondary-structure predictions (H = α-helix; C = random coil) and the level of confidence of the predictions (confidence scores from 0 to 9). Residues 14 and 15 of the BH4 domain are highlighted by a semi-transparent red square. (C) CD spectra of synthetic Bcl-2-BH4 (black line) and Bcl-2-BH4 IV/GG peptides (red line). The ellipticity is calculated per mole of amino-acid residue. Bcl-2-BH4-IV/GG peptide lost the native α-helical conformation to adopt a more β-sheet-like structure (210 nm-ellipticity minimum). For the percentages of the other secondary structure features see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073386#pone.0073386.s001" target="_blank">Table S1</a>.</p></div