Total synthesis of zervamicin IIB and its deuterium-labelled analogues

Abstract

For the first time the total synthesis of the peptaibol zervamicin IIB is described. Synthesis of this peptaibol was achieved by the Fmoc/tert-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered alpha-aminoisobutyric acid residues BOP/DMAP activation was applied. The fmoc group was removed by reaction with 0.1 M NaOH in dioxane/methanol/water (30/9/1, v/v/v). Peptide fragments were coupled by means of a new coupling reagent, CF3-PyBOP. Using the strategy developed, zervamicin IIB and two analogues specifically deuterium-labelled at different positions of the glutamine-11 residue have been synthesized in 40% overall yield based on the isotopically labelled amino acid and with 98 +/- 2% of isotope enrichment. FAB mass spectroscopy, 600 MHz 1H-NMR spectroscopy and high-performance liquid chromatography provided convincing evidence that the synthetic products, zervamicin IIB and its deuterium-labelled analogues, fully correspond to the naturally occurring zervamicin IIB.

Synthesis of the15N-Gln11 labeled peptaibol antibiotic zervamicin-IIB

“…We used a new methodology for the Fmoc group removal, which has been developed in previous work [1]. Fragment 10-16 contains two acid-labile peptide bonds: Aib12-Hyp 13 and Aibla-Pro 15.…”

Isotopic Mass-Dependent MS-Patch-Clamp and Isotopic Mass-Independent ESR-Patch-Clamp

“…For example, from the beginning of the ESR-technique development, biophysicists have accepted the spin labeling technique for the simplest ion channels, such as spin-labeled derivatives of gramicidin peptides [162] and spin-labeled gramicidin itself [163], labeled valinomycin and its analogs [164] (along with the NMR observations of the nuclear Overhauser effect of transfer of the nuclear spin polarization from one nuclear spin population to another one via cross-relaxation [165,166]; the same method has been applied to the gramicidin [167][168][169]), cecropin [170][171][172][173], zervamicin [174,175] (early labeled by deuterium [176], 13 C and 15 N for NMR measurements [177]), alamethicin [178][179][180][181][182], etc. It is noteworthy that gramicidin as well as valinomycin [183] are well known as the simple ion channels [184][185][186][187][188], which can be studied by spin labeling and magnetic resonance methods [189,190] (as well as ion-channel-forming valinomycin [191][192][193]); zervamicin is also well known as the ion-channel-forming agent, ion channel peptide and a good model for the membrane ion channels [194][195][196] with a well-studied gating mechanisms [197][198][199] which can operate not only in the native membranes, but also in the artificial micelles and lipid bilayers…”

Is it possible to design isotopic mass-dependent ms-patch-clamp and isotopic mass-independent esr-patch-clamp?

“…For example, from the beginning of the ESR-technique development, biophysicists have accepted the spin labeling technique for the simplest ion channels, such as spin-labeled derivatives of gramicidin peptides (Ivanov, 1973) and spin-labeled gramicidin itself (Dzikovski, 2011), labeled valinomycin and its analogs (Ivanov, 1974) (along with the NMR observations of the nuclear Overhauser effect of transfer of the nuclear spin polarization from one nuclear spin population to another one via cross-relaxation (Glickson, 1976;Krishna, 1978); the same method has been applied to the gramicidin (Jones, 1978;Huang, 1981;Barsukov, 1987), cecropin (Mchaourab, 1993;Mchaourab, 1994;Hung, 1999;Bhargava, 2004), zervamicin (Milov, 2002; Milov, 2010) (early labeled by deuterium (Ogrel, 1997), 13 C and 15 N for NMR measurements (Ovchinnikova, 2003)), alamethicin (Archer, 1991;Crisma, 2007;Marsh, 2007;Bartucci, 2009;Marsh, 2009), etc. It is noteworthy that gramicidin as well as valinomycin (Gliozzi, 1996) are well known as the simple ion channels (Hu, 1993; Haldar, 2012; Wang, 2013; Basu, 2014; Chaudhuri, 2014), which can be studied by spin labeling and magnetic resonance methods (Planque, 1998; Dzikovsky, 2004), as well as ion-channel-forming valinomycin (Eastman, 1974;Meers, 1988;Kriz, 2006); zervamicin is also well known as the ion-channel-forming agent, ion channel peptide and a good model for the membrane ion channels (Agarwalla, 1992;Sansom, 1993;) with a well-studied gating mechanisms (Karle, 1991;Ballesteras, 1992;Karle, 1994) which can operate not only in the native membranes, but also in the artificial micelles and lipid bilayers (Shenkarev, 2002), and can be studied using spin labeling ESR approaches as well as other equivalent mechanisms (Barranger-Mathys, 1996; Perozo, 2001; Dellisanti, 2013); colicin (also studied by spin labeling and ESR (Todd, 1989; Shin, 1993; Pulagam, 2013)) also well known as the ion channel-formin...…”