Total Synthesis of Septocylindrin B and C-Terminus Modified Analogues

et al. (2012) Total Synthesis of Septocylindrin B and C-Terminus Modified Analogues. PLoS ONE 7(12): e51708. doi:10.1371/journal.pone.0051708 Total Synthesis of Septocylindrin B and C-Terminus Modified Analogues Jo Nelissen 0 Koen Nuyts 0 Marta De Zotti 0 Rob Lavigne 0 Chris Lamberigts 0 Wim M. De Borggraeve 0 Chandra Verma, Bioinformatics Institute, Singapore 0 1 Molecular Design and Synthesis, Department of Chemistry, University of Leuven , Heverlee , Belgium , 2 Department of Chemistry, University of Padova , Padova , Italy , 3 Laboratory of Gene Technology, Biosystems Department, University of Leuven , Heverlee , Belgium 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 C-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. - Funding: JN thanks the Institute for the Promotion of Innovation through Science and Technology in Flanders (Belgium, I.W.T., www.iwt.be) for financial support. KN thanks KU Leuven for financial support (Project OT/11/047/TBA). CL is supported by the IOF Knowledge Platform grant (Functional peptidomics IOF/KP/09/ 003) of the KU Leuven. This work was also partly supported by a grant of the FWO Vlaanderen (G.0599.11). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. In 2007 two new, 20 amino acid long, Phaol-terminated peptaibols, Septocylindrin A and Septocylindrin B were isolated from Septocylindrum sp. by Summers et al (Figure 1 and 2) [1]. They exhibit significant antibacterial and antifungal activity and are closely related to Alamethicin, one of the most extensively studied peptaibols. Peptaibols are a large class of natural peptides with three characteristic features: a high a-aminoisobutyric acid (Aib) content, an acylated N-terminus and a 1,2-amino alcohol at the C-terminus [2]. They are known for their membranemodifying and pore-forming abilities, thereby exhibiting antibacterial and antifungal activity [3]. They promote voltage-dependent ion channel formation in lipid bilayers. The mechanism of this action is yet to be fully understood. Previous studies suggest that simple physico-chemical properties of membrane-active peptides, like aggregation and water/membrane partition, probably play a fundamental role in determining the overall activity [4]. Development of bacterial resistance against this kind of peptides is much less likely in view of their mechanism of action. As a consequence, these peptides are attractive candidates for clinical development. Phaol (Figure 1) is the amino alcohol present at the C-terminus of a number of peptaibols, like Aibellin [5] and Septocylindrin [1]. In view of a total synthesis of these peptides, orthogonally protected Phaol is needed in large amounts. In 2011, Nelissen et al. [6] presented a procedure for the large scale synthesis of orthogonally protected Phaol and analogues thereof. The goal of the present work is to exploit these newly synthesized amino alcohol moieties to accomplish the total synthesis of Septocylindrin B and analogues via a segment condensation approach. These compounds will then be used to determine the structure-activity relationship (SAR). Results and Discussion Synthesis of Septocylindrin B Septocylindrin B is synthesized via a segment condensation approach, which is depicted in Figure 2. Septocylindrin B is synthesized by converting the Glu(OMe) residues to Gln residues by aminolysis. The presence of sterically hindered a,a-dialkyl amino acid residues (Aib, U), renders this peptide, like all other peptaibols, less attractive for normal solid phase peptide synthesis. Peggion et al [7] published a flexible segment condensation approach for the synthesis of ALM F50/5, one of the components of the complex, natural Alamethicin mixture. They split the peptide into four segments, taking into account the acid sensitive Aib-Pro bond [8], the high tendency of the H-Aib-Pro- dipeptide sequence to cyclize to the corresponding diketopiperazine and the epimerization risk involved in a segment condensation. In particular, to avoid the latter event, each segment was planned so as to have a non-racemizable, achiral Aib residue at the Cterminus. A similar approach was used to synthesize Septocylindrin B. We further optimize this approach by splitting the Cterminal segment into two parts: C and D. In this way, the synthesis of Septocylindrin analogues with a modified C-terminus is more time efficient, as only the appropriate segment D has to be synthesized, while ABC-OtBu can be prepared only once on a large scale. All segments were synthesized using HOAt and EDC as coupling reagents and NMM as a base. Cbz was used for the protection of all N-termini and tBu was used for the protection of all C-termini. The Cbz group was cleaved off by catalytic hydrogenation, while the tBu group was removed using TFA with the exception of the peptide segments containing the acid sensitive Aib-Pro bond. In this case, the Lewis acid ZnBr2 was used [9]. The secondary amine and the alcohol of the Phaol residue were both protected with a Boc group, as described by Nelissen et al [6], and deprotected using BiCl3 [10]. The glutamic acid residues were used as c-methyl esters (residue Q* in Figure 2) and subsequently converted to glutamine by treatment with NH3. The single coupling steps in the synthesis of segment A resulted in good yields: about 85% each. Also the synthesis of segment B did not pose problems. The coupling reactions proceeded with yields between 76% and 88%. Similarly, the C segment was synthesized with yields between 68% to 88% per coupling step. The synthesis of the D segment also proceeded smoothly: 91% yield for the synthesis of Cbz-Q*-Phaol(Boc)2 and 79% for that of Cbz-Q*Q*-Phaol(Boc)2. The condensation approach for the completed fragments is depicted in Figure 3. Firstly, the tBu group of the A segment is cleaved off using TFA to couple it to H-B-OtBu (obtained via Cbz hydrogenolysis of the corresponding segment), using HOAt and EDC, yielding 89% of Cbz-AB-OtBu. The tBu group of this peptide was removed using TFA, after which it was coupled to the Cbz deprotected C segment, H-C-OtBu, yielding 41% of CbzABC-OtBu. This peptide was hydrogenolytically deprotected and coupled to Ac-Aib-OH, yielding 67% of ABC-OtBu. This peptide was deprotected with ZnBr2, yielding 76% of ABC-OH. This molecule was coupled to the hydrogenolytically deprotected D segment, H-Glu(OMe)-Glu(OMe)-Phaol-(Boc)2, yielding 30% of ABCD. This peptide was then Boc deprotected with BiCl3 and transformed into Septocylindrin B by treatment with ammoni (...truncated)