Partial thioamide scan on the lipopeptaibiotic trichogin GA IV. Effects on folding and bioactivity

Article abstract of DOI:10.3762/bjoc.8.129

Backbone modification is a common chemical tool to control the conformation of linear peptides and to explore potentially useful effects on their biochemical and biophysical properties. The thioamide, ψ[CS-NH], group is a nearly isosteric structural mimic

Full text of DOI:10.3762/bjoc.8.129

Partial thioamide scan on the lipopeptaibiotic
trichogin GA IV. Effects on folding and bioactivity
Marta De Zotti1, Barbara Biondi1, Cristina Peggion1, Matteo De Poli1,
Haleh Fathi1, Simona Oancea2, Claudio Toniolo1 and Fernando Formaggio*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 1161–1171.
1ICB, Padova Unit, CNR, Department of Chemistry, University of
Padova, 35131 Padova, Italy and 2Department of Biochemistry and
Toxicology, Lucian Blaga University of Sibiu, 550012 Sibiu, Romania
doi:10.3762/bjoc.8.129
Received: 27 April 2012
Accepted: 21 June 2012
Published: 24 July 2012
Email:
Fernando Formaggio* - fernando.formaggio@unipd.it
This article is part of the Thematic Series "Antibiotic and cytotoxic
peptides".
* Corresponding author
Keywords:
Guest Editor: N. Sewald
bioactivity; conformation; peptaibiotic; peptide synthesis; peptides;
thiopeptides
© 2012 De Zotti et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Backbone modification is a common chemical tool to control the conformation of linear peptides and to explore potentially useful
effects on their biochemical and biophysical properties. The thioamide, ψ[CS-NH], group is a nearly isosteric structural mimic of
the amide (peptide) functionality. In this paper, we describe the solution synthesis, chemical characterization, preferred
conformation, and membrane and biological activities of three, carefully selected, peptide analogues of the lipopeptaibiotic
[Leu11-OMe] trichogin GA IV. In each analogue, a single thioamide replacement was incorporated. Sequence positions near the
N-terminus, at the center, and near the C-terminus were investigated. Our results indicate that (i) a thioamide linkage is well
tolerated in the overall helical conformation of the [Leu11-OMe] lipopeptide analogue and (ii) this backbone modification is
compatible with the preservation of its typical membrane leakage and antibiotic properties, although somewhat attenuated.
Introduction
Since their first incorporation into peptides [1], backbone amide A ψ[CS-NH] thioamide group is one of the closest mimics of an
surrogates have attracted remarkable attention from organic and amide (peptide) linkage. However, it exhibits significantly
medicinal chemists. Not only may these modifications impart to different chemical and physical properties, some of which are of
a peptide an increased resistance to enzymatic hydrolysis as great potential interest to peptide chemists [2-44]. Among these
well as higher receptor affinity and specificity, but they may properties, we highlight the following: (i) The thioamide NH
also influence its preferred secondary structure.
group is more acidic than that of its oxygenated counterpart and
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Beilstein J. Org. Chem. 2012, 8, 1161–1171.
consequently it is a stronger H-bonding donor. (ii) Its cis/trans Nα-blocking fatty acyl moiety plays a major role in its
isomerization can be phototriggered by irradiation at about membrane permeability and antibiotic activity.
260 nm [absorption maximum of the -C(=S)NH- π→π* elec-
tronic transition]. (iii) It may act as a minimalist, effective In each analogue, the single ψ[CS-NH] group was strategically
quencher for any type of protein and nonprotein fluorophores. incorporated either at an internal position of the amino-acid
sequence, ψ[CS-NH]5, or near each of the two ends, ψ[CS-
Recently, based on the aforementioned characteristics of the NH]2 and ψ[CS-NH]9. Moreover, the preferred conformations
ψ[CS-NH] thioamide bond, we started a program aimed at of these analogues were investigated by circular dichroism
exploring how thiopeptide groups may affect folding and the (CD), FT-IR absorption, and NMR. Finally, we carried out fluo-
related biophysical/biochemical activities of the membrane- rescence leakage experiments in model membranes and antibac-
active peptaibiotics [45]. This class of compounds represents a terial assays on a large set of both Gram-positive and Gram-
subject of long-standing, relevant, interest to our research negative strains.
group. In this paper, we describe our results on the syntheses by
Results and Discussion
solution methods and characterizations of three thioamide-
containing [Leu11-OMe] analogues of the short helical lipopep- Peptide synthesis
taibiotic trichogin GA IV [46-50] (Figure 1). We already found The total syntheses of the three monothionated ψ[CS-NH]
that the replacement of the native C-terminal 1,2-amino alcohol trichogin GA IV analogues at positions 2, 5, and 9, respectively,
leucinol (Lol) by the corresponding α-amino methyl ester were accomplished by using the solution-phase method
(Leu-OMe) alters only slightly the biophysical and biological (Schemes 1–3). Both step-by-step and segment condensation
properties of trichogin GA IV. Conversely, we showed that the approaches were utilized. The former strategy was used in the
Figure 1: List of primary structures and abbreviations for the peptides studied in this work. The [Leu11-OMe] trichogin GA IV is termed tric-OMe and
its thionated derivatives at position 2, position 5, and position 9 are termed ψ[CS-NH]2, ψ[CS-NH]5, and ψ[CS-NH]9, respectively.
Scheme 1: Synthesis of ψ[CS-NH]2. 1: Coupling in the presence of EDC/HOBt. 2: Deprotection by using TFA/DCM. 3: Deprotection by catalytic
hydrogenation with Pd/C. 4: Thionation with Lawesson's reagent in THF. 5: Coupling with n-Oct-OH in the presence of EDC/HOBt. 6: Saponification
with NaOH/MeOH. 7: Coupling in the presence of EDC/HOAt.
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Scheme 2: Synthesis of ψ[CS-NH]5. 1: Coupling in the presence of EDC/HOBt. 2: Deprotection by using TFA/DCM. 3: Deprotection by catalytic
hydrogenation with Pd/C. 4: Coupling with Fmoc-OSu in 1,4-dioxane. 5: Thionation with Lawesson's reagent in THF. 6: Coupling with n-Oct-OH in the
presence of EDC/HOBt. 7: Coupling in the presence of EDC/HOAt. 8: Deprotection with DEA in DCM.
Scheme 3: Synthesis of ψ[CS-NH]9. 1: Deprotection by catalytic hydrogenation with Pd/C. 2: Coupling with n-Oct-OH in the presence of EDC/HOBt.
3: Coupling in the presence of EDC/HOBt. 4: Deprotection by using TFA/DCM. 5: Thionation with Lawesson's reagent in THF. 6: Coupling in the pres-
ence of EDC/HOAt.
preparation of Boc-Gly5-Gly-Leu-Aib-Gly-Ile-Leu11-OMe be appropriate for the formation of the other peptide bonds. The
(segment 5–11, for the synthesis of ψ[CS-NH]2), Boc-Gly9-Ile- yields of the segment condensation reactions were good
Leu11-OMe (segment 9–11), and n-Oct-Aib1-Gly-Leu-Aib4-Ot- (75–83%). An important feature, which makes trichogin GA IV
Bu (segment 1–4, for the synthesis of ψ[CS-NH]5), and n-Oct- a versatile synthetic platform, is the presence of as many as
Aib1-Gly-Leu-Aib-Gly-Gly-Leu-Aib8-Ot-Bu (segment 1–8, for seven achiral (Aib or Gly) residues spread across its sequence,
the synthesis of ψ[CS-NH]9). The latter strategy, which permits which allows one to design suitable segments each with an
a faster preparation of multiple analogues, afforded (i) ψ[CS- achiral residue at its C-terminus, thus reducing dramatically the
NH]2 by condensation of the thionated 1–4 and the 5–11 usually considerable risk of epimerization during the coupling
segments; (ii) ψ[CS-NH]5 by condensation of the thionated 5–8 reactions.
and the 9–11 segments followed by condensation of the 1–4 and
the resulting, thionated, 5–11 segments; and (iii) ψ[CS-NH]9 by For the best choice of the step involving treatment with the
condensation of the 1–8 and the thionated 9–11 segments.
Lawesson's reagent [53], we decided to select the longest
segment of our sequences, which, in principle, could permit
For the difficult coupling steps, in particular those involving the complete regioselectivity between the peptide bond to be
segment condensations, the N-[3-(dimethylamino)-propyl]-N'- thionated and the other peptide bonds (in addition to the known
ethylcarbodiimide (EDC)/7-aza-1-hydroxy-1,2,3-benzotriazole regioselectivity versus the urethane and ester bonds) [53-56]. In
(HOAt) [51] activating method was used, while the EDC/1- this reaction, regioselectivity is known to be heavily governed
hydroxy-1,2,3-benzotriazole (HOBt) [52] method turned out to by the steric hindrance of the two residues around the peptide
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bond. Therefore, we synthesized Boc-Gly2ψ[CS-NH]Leu-Aib4-
OMe (for the preparation of ψ[CS-NH]2), Fmoc-Gly5ψ[CS-
NH]Gly-Leu-Aib8-Ot-Bu (for the preparation of ψ[CS-NH]5),
and Boc-Gly9ψ[CS-NH]Ile-Leu11-OMe (for the preparation of
ψ[CS-NH]9). Thionation of a peptide as short as a dipeptide is
not recommended because a further synthetic step at the
C-terminus of this compound would not take place satisfacto-
rily, owing to the easy formation of a poorly reactive 1,3-thiazo-
lidin-5-one. All thionation steps were conducted by use of the
Lawesson's reagent under mild conditions (in tetrahydrofuran at
room temperature) [53-56]. The only synthetic problem was
identified in the preparation of the Fmoc-Gly5ψ[CS-NH]Gly-
Leu-Aib8-Ot-Bu, in which case the thionation selectivity on the
Gly-Gly peptide bond was incomplete. Indeed, a limited
production (about 10%) of the isomeric Fmoc-Gly-Gly6ψ[CS-
NH]Leu-Aib-Ot-Bu, with its slightly more hindered, thionated
Gly-Leu peptide bond, was observed. In this case, the desired
monothionated peptide was separated from its isomer by means
of flash chromatography.
Figure 2: RP-HPLC profiles obtained for [Leu11-OMe] trichogin GA IV
(tric-OMe) and its ψ[CS-NH]2, ψ[CS-NH]5, and ψ[CS-NH]9
analogues.
GA IV analogues synthesized in this work was performed by
For details of the synthetic procedures and characterizations of using CD, FT-IR absorption, and 2D NMR investigations in
these analogues (and the synthetic precursor segments as well), different solvents.
see the Experimental section and Supporting Information File 1.
Figure 2 gives the RP-HPLC profiles of the three mono- The far- (250–195 nm) and near-UV (400–250 nm) CD spectra
thionated trichogin GA IV analogues.
of the three peptides, recorded in 2,2,2-trifluoroethanol (TFE)
solution, are illustrated in Figure 3. In the near-UV region, in
which the peptide chromophore is known not to absorb, both
Conformational analysis
A detailed analysis of the spectroscopic properties and con- ψ[CS-NH]2 and ψ[CS-NH]9 display two well-separated Cotton
formational preferences of the three monothionated trichogin effects at about 335 and 265 nm. The longer wavelength band is
Figure 3: Far-UV (panel I) and near-UV (panel II) CD spectra of [Leu11-OMe] trichogin GA IV (tric-OMe) and its ψ[CS-NH]2 (2), ψ[CS-NH]5 (5), and
ψ[CS-NH]9 (9) analogues in TFE solution. Peptide concentration: 1 mM.
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Beilstein J. Org. Chem. 2012, 8, 1161–1171.
positive and weak, while the shorter wavelength band is nega- known to contribute heavily in this spectral region. However,
tive and much more intense. Interestingly, the positive band is we can safely state that the shape of the CD spectrum of the
absent and the negative band is much weaker in ψ[CS-NH]5. [Leu11-OMe] trichogin GA IV prototypical peptide [50,59] is
The spectral positions of these Cotton effects correspond to a most closely paralleled by that of its ψ[CS-NH]5 analogue,
very intense maximum (265 nm) and an extremely weak although the former is significantly more intense. This finding
maximum (320 nm) found in the corresponding region of the is not surprising in view of the above-mentioned low influence
UV absorption spectra (not shown) of our peptides in the same of the thioamide chromophore on the spectrum of this mono-
solvent. We assign the two bands to the π→π* and n→π* tran- thionated compound. It may also suggest that the overall con-
sitions, respectively, of thioamide chromophore [29,30,32- formation of the lipopeptaibiotic is disturbed by this specific
36,40,41,43]. We attribute the overall much less intense CD oxygen-to-sulfur exchange only to a limited extent.
spectrum of ψ[CS-NH]5 in this region primarily to a thioamide
chromophore positioned in this peptide between the two achiral The preferred conformations of the three monothionated
Gly5 and Gly6 residues, i.e., it is significantly far apart from the [Leu11-OMe] trichogin GA IV analogues were more safely
nearest chiral center (the Leu7 α-carbon).
analyzed by means of FT-IR absorption and 2D NMR. In this
study, we used CDCl3, a solvent of low polarity. In the
In the far-UV region the CD spectra of all three analogues show 3D-structurally informative 3550–3200 cm−1 region at 1.0 mM
multiple (negative/positive) Cotton effects of weak and medium concentration, the FT-IR spectra of [Leu11-OMe] trichogin GA
intensities. Here, the bands (shoulders) of ψ[CS-NH]9 are the IV and its analogues are similar and dominated by a broad and
weakest. In our view, it is risky to attempt to extract any conclu- intense absorption at 3316–3328 cm−1 (Figure 4), assigned to
sive conformational information from the CD properties of the N–H stretching mode of the largely prevailing H-bonded
these three isomeric peptides between 195 and 250 nm because, peptide groups [60,61]. Additional, very weak bands are visible
in addition to the expected Cotton effects of the peptide chro- in the 3450–3400 cm−1 region, mostly attributed to free
mophores [57,58], the thioamide chromophore [34,35] is also (solvated) peptide NH groups.
Figure 4: FT-IR absorption spectra (3550–3200 cm−1 region) in CDCl3 solution of [Leu11-OMe] trichogin GA IV (tric-OMe) and its ψ[CS-NH]2 (2),
ψ[CS-NH]5 (5), and ψ[CS-NH]9 (9) analogues. Peptide concentration: 1 mM.
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Beilstein J. Org. Chem. 2012, 8, 1161–1171.
A nonnegligible dilution effect is observed in the spectra of the
four peptides between 1.0 and 0.1 mM concentration (not
shown). We ascribe this difference to the well-known tendency
of trichogin GA IV and its analogues to self-associate above
1.0 mM concentration in CDCl3 solution. We interpret the
spectra at 0.1 mM concentration as arising almost exclusively
from intramolecular C=O(S)···H–N interactions. It is clear that
under these experimental conditions the preferred conformation
of [Leu11-OMe] trichogin GA IV [46-50] and its three
analogues, all rich in the helix-supporting Aib residues, is
highly folded and largely stabilized by intramolecular H-bonds.
In the amide I (C=O stretching) region, the maximum of the
absorption band of the four peptides is located between 1662
and 1656 cm−1 (not shown), close to the canonical positions of
this band [61,62] in α- and 310-helical peptides [63].
Figure 5: Region of the amide NH protons in the H/H-ROESY spec-
trum of ψ[CS-NH]9 (400 MHz, 1 mM in CD3CN solution, 298 K).
A 2D NMR investigation was performed in CD3CN solution for The conformational properties of ψ[CS-NH]9 were further
all of the ψ[CS-NH]-containing [Leu11-OMe] trichogin GA IV investigated by simulated annealing (SA) and restrained molec-
analogues. The proton resonances were fully assigned following ular dynamics (MD) calculations. A total of 69 interproton dis-
the Wüthrich procedure [64]. Regions of the ROESY spectrum tance restraints, derived from the related ROESY spectrum
acquired for ψ[CS-NH]9 are shown in Figures 5–7. The (Table S-I, Supporting Information File 1), were used in the SA
patterns of NH(i)–NH(i+1) connectivities (e.g., Figure 5) protocol. The structures possessing violations to the NOE
strongly suggest the onset of an overall helical structure for all restraints lower than 0.5 Å were selected out of the 150 gener-
analogues. More relevant 3D-structural information was ated structures. The superposition of the 18 structures with a
extracted from the fingerprint regions of the ROESY spectra total energy <144 kcal/mol is shown in Figure S-I, Supporting
(e.g., Figure 6 and Figure 7), which show evidence for dα(β),N Information File 1. All these structures converge to a well-
(i, i+2), (i, i+3), and (i, i+4) medium-range connectivities, diag- defined, right-handed, mixed 310-/α-helical conformation
nostic of the presence of mixed 310-/α-helical conformations.
throughout the sequence with a backbone average pairwise root-
Figure 6: Fingerprint region of the H/H-ROESY spectrum of ψ[CS-NH]9 (400 MHz, 1 mM in CD3CN solution, 298 K). The CαHi → NHi+2, and CαHi →
NHi+3 cross peaks are highlighted in color.
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Beilstein J. Org. Chem. 2012, 8, 1161–1171.
Figure 7: Fingerprint region of the H/H-ROESY spectrum of ψ[CS-NH]9 (400 MHz, 1 mM in CD3CN solution, 298 K). The CβHi → NHi+2, CβHi →
NHi+3, and CβHi → NHi+4 cross peaks are highlighted in color.
mean-square deviation of 0.49 ± 0.18 Å (deviations from ideal- In conclusion, the MD calculations based on the restraints
ized geometry and mean energies for the 18 lowest energy derived from the ROESY spectrum of ψ[CS-NH]9 reveal the
structures are listed in Table S-I, Supporting Information marked preference of this analogue for a mixed α-/310- helical
File 1). Due to heavily overlapped ROE signals, the helical conformation, with a clear α-helical character at the C-terminus
structures originated from the MD calculations appear not to on which the thioamide bond is present. The lowest-energy 3D
superimpose perfectly in correspondence with the first three structure, shown in Figure 8, exhibits the slightly amphipathic
residues of the sequence. Even with this experimental limit, the character typical of the native peptide, with the poorly
residues belonging to the central and C-terminal parts of the hydrophilic Gly residues aligned on the same face of the helix.
sequence showed values of torsion angles corresponding to
those characteristic of right-handed helices (Table S-II, Membrane and biological activities
Supporting Information File 1). The helical structures are stabi- The membrane permeability properties of [Leu11-OMe]
lized by intramolecular H-bonds throughout the sequence. trichogin GA IV and its monothionated analogues were tested
H-bonds both of the α- (i←i+4) and 310- (i←i+3) helical types by measuring the induced leakage of 5(6)-carboxyfluorescein
are present, although with a large predominance of the latter (CF) entrapped in small unilamellar vesicles (SUVs) [65]. For
type (mean distance: 1.81 ± 0.17 Å; mean angle: 27.4 ± 3.7°), at this investigation, the overall negatively charged 1,2-dioleoyl-
the N-terminus. Conversely, only the former type (mean dis- sn-glycero-3-phosphoethanolamine (DOPE)/1,2-dioleoyl-sn-
tance: 1.53 ± 0.02 Å; mean angle: 27.3 ± 2.9°) was detected at glycero-3-phospho-(1'-rac-glycerol) (DOPG) model membrane
the C-terminus.
was exploited. The extent of permeation induced by the three
Figure 8: Ribbon representation of the lowest energy (138.7 kcal/mol) 3D structure obtained for ψ[CS-NH]9. All amino acid side chains are shown.
The yellow atom on Gly9 refers to sulfur.
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Beilstein J. Org. Chem. 2012, 8, 1161–1171.
analogues is similar, although 30–40% lower than that of the (0.6 mmol) was added. The reaction mixture was stirred at rt
parent peptaibiotic.
overnight. After removal of the solvent under reduced pressure,
the crude product was purified by flash chromatography with a
The antimicrobial activities were assessed on a large set of mixture of ethyl acetate and petroleum ether as eluant.
Gram-positive and Gram-negative bacterial strains. We found
that [Leu11-OMe] trichogin GA IV is active on the Gram-posi- Peptides Boc-Ile-Leu-OMe [66], Z-Leu-Aib-OMe [67], Z-Leu-
tive bacteria Staphylococcus aureus and Streptococcus pyro- Aib-Ot-Bu [68], Boc-Gly-Leu-Aib-OMe [69], Boc-Gly-Ile-Leu-
genes, whereas the spectrum of action of its three analogues is OMe [66], Boc-Leu-Aib-Gly-Ile-Leu-OMe [66], Boc-Gly-Gly-
more restricted, as they are not active on the latter strain. The Leu-Aib-Gly-Ile-Leu-OMe [66], Z-Gly-Gly-Leu-Aib-Ot-Bu
activities of the analogues on S. aureus are comparable, but [59], and Z-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Ot-Bu [59] are
reduced by about 40% with respect to that of the parent known compounds. Chemical characterizations and selected
peptaibiotic.
NMR spectra for the other peptides reported in Schemes 1–3 are
given in Supporting Information File 1.
Conclusion
In this work, we performed a partial thioamide scan of the short Circular dichroism
lipopeptaibiotic [Leu11-OMe] trichogin GA IV by synthesizing The CD spectra were measured on a Jasco (Tokyo, Japan)
and studying three thioamide-containing analogues. The model J-715 spectropolarimeter equipped with a Haake thermo-
syntheses were accomplished in solution by using a combina- stat (Thermo Fisher Scientific, Waltham, MA). Baselines were
tion of the step-by-step and segment condensation methods. corrected by subtracting the solvent contribution. Fused quartz
Appropriate tri- and tetrapeptide segments were monothionated cells of 1.0 mm and 10.0 mm path lengths (Hellma, Mühlheim,
by treating the all-amide precursors with the Lawesson's Germany) were used. The values are expressed in terms of [θ]T,
reagent. Our CD, FT-IR absorption, and NMR conformational the total molar ellipticity (deg·cm2·dmol−1). Spectrograde TFE
investigations show that all analogues maintain the mixed 310-/ and 99.9% MeOH (Acros Organic, Geel, Belgium) were
α-helical structure and the self-association propensity of the employed as solvents.
native lipopeptide. They also preserve, at least to a great extent,
its well-established capability to interact with model phospho- FT-IR absorption
lipid membranes and to exhibit activity against Gram-positive The FT-IR absorption spectra were recorded at 293 K by using
bacterial strains.
a Perkin-Elmer model 1720X FT-IR spectrophotometer,
nitrogen-flushed, equipped with a sample-shuttle device, at
2 cm−1 nominal resolution, averaging 100 scans. Solvent (base-
Experimental
All reagents and solvents, of analytical grade and purchased line) spectra were recorded under the same conditions. For
from commercial sources, were used without further purifica- spectral elaboration, the software SPECTRACALC provided by
tion. Melting points were measured by means of a capillary tube Galactic (Salem, MA) was employed. Cells with path lengths of
immersed in an oil bath (Tottoli apparatus, Büchi) and are 1.0 and 10 mm (with CaF2 windows) were used. Spectrograde
uncorrected. Optical rotations [α]D20 (given in units of deuterated chloroform (99.8%, d) was purchased from Merck
10−1 deg·cm2·g−1) were measured at 20 °C on a Perkin-Elmer (Darmstadt, Germany).
PE241 polarimeter, with a 1 dm path length cell, at the D-wave-
length of sodium (589 nm). The concentration of each com- Nuclear magnetic resonance
pound (c) is given in mg/cL. Mass spectroscopy (electrospray All 1H NMR experiments were performed on a Bruker
ionization, ESIMS) was performed by using a PerSeptive AVANCE DMX-600, DRX-400 or AC200 spectrometer oper-
Biosystem Mariner instrument (Framingham, MA). Analytical ating at 600, 400 or 200 MHz, respectively, using the TOPSPIN
TLC and preparative column chromatography were performed software package. Splitting patterns are abbreviated as (s)
on Kieselgel F 254 and Kieselgel 60 (0.040–0.063 mm) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet. The
(Merck), respectively. The retention factor (Rf) values were homonuclear 2D spectra of the three monothionated [Leu11-
determined by using three solvent mixtures as eluants: Rf1: OMe] trichogin GA IV analogues were recorded at 298 K, with
chloroform/ethanol 9:1; Rf2: 1-butanol/acetic acid/water 3:1:1; CD3CN as solvent. All spectra were acquired by recording 512
Rf3: toluene/ethanol 7:1.
experiments, each one consisting of 64–80 scans and 2000 data
points. The spin systems of protein amino acid residues were
identified by using standard DQF-COSY [70] and CLEAN-
General procedure for thionation of peptides
To a solution of the peptide (1 mmol) in anhydrous tetrahydro- TOCSY [71,72] spectra. In the latter case, the spin-lock pulse
furan under nitrogen atmosphere, the Lawesson's reagent sequence was 70 ms long. A ROESY experiment was exploited
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Beilstein J. Org. Chem. 2012, 8, 1161–1171.
for sequence-specific assignment. The mixing time of the presence of peptide, and FT = total fluorescence intensity deter-
ROESY experiment acquired for ψ[CS-NH]9 (peptide concen- mined by disrupting the vesicles by the addition of 50 μL of a
tration: 1.00 mM in CD3CN) and used for interproton distance Triton X-100 solution. The experiments were stopped at
determination was 200 ms. Interproton distances were obtained 20 min.
by integration of the ROESY spectra using SPARKY 3.111.
The calibration was based on the average of the integration Antibacterial activity
values of the cross peaks due to the interactions between the Peptide antibacterial activity was tested against Gram-positive
sequential amide protons, set to a distance of 2.80 Å. When and Gram-negative bacteria by the standardized disk diffusion
peaks could not be integrated because of partial overlap, a dis- Bauer–Kirby method [75] using the Müller–Hinton culture
tance corresponding to the maximum limit of detection of the medium at pH 7.2–7.4 as recommended by the National
experiment (4.0 Å) was assigned to the corresponding proton Committee for Clinical Laboratory Standards [76]. The antibac-
pair.
terial activity assay was performed in a similar way to that
described in reference [77]. The activity of peptides was tested
MD calculations were carried out using the SA protocol of the against clinical isolates of bacteria and reference bacterial
XPLOR-NIH 2.9.6 program [73]. For distances involving strains: Staphylococcus aureus American Type Culture Collec-
equivalent or non-stereo-assigned protons, an r−6 averaging was tion strains (ATCC) 25923, Streptococcus pyogenes ATCC
used. The MD calculations involved a minimization stage of 19615, Escherichia coli ATCC 25922, Pseudomonas aerugi-
100 cycles, followed by SA and refinement stages. The SA nosa ATCC 27853, Klebsiella pneumoniae ATCC 13883,
consisted of 30 ps of dynamics at 1,500 K (10,000 cycles, in Salmonella entereditis 13076, and Proteus mirabilis ATCC
3 fs steps) and of 30 ps of cooling from 1,500 to 100 K in 50 K 10975. The well-known antibiotics bacitracin and tetracyclin
decrements (15,000 cycles, in 2 fs steps). The SA procedure, in (10 μg/disk) were used as controls.
which the weights of ROE and nonbonded terms were gradu-
ally increased, was followed by 200 cycles of energy minimiza-
Supporting Information
tion. In the SA refinement stage, the system was cooled from
1,000 to 100 K in 50 K decrements (20,000 cycles, in 1 fs
Supporting Information File 1
steps). Finally, the calculations were completed with 200 cycles
of energy minimization by using a NOE force constant of
50 kcal/mol. The generated structures were visualized with the
MOLMOL [74] (version 2K.2) program.
Chemical characterization data for the peptides reported in
Schemes 1–3.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-129-S1.pdf]
Small unilamellar vesicles (SUVs) preparation
DOPE and DOPG were purchased from Avanti Polar Lipids,
Inc. (Alabaster, AL). The preparation of SUVs was performed
in a similar way as described in reference [59].
References
1. Spatola, A. F. Peptide backbone modifications: Structure–activity
analysis of peptides containing amide bond surrogates. In Chemistry
and Biochemistry of Amino Acids, Peptides and Proteins;
Weinstein, B., Ed.; Dekker: New York, 1983; pp 267–357.
2. La Cour, T. F. M.; Hansen, H. A. S.; Clausen, K.; Lawesson, S.-O.
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