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V. Cerovsky et al.
of CH2Cl2/TFE/HOAc (4 mL; 7:2:1) at RT for 60 and then again for
15 min. The combined solution was concentrated under vacuum,
and the residue triturated with a mixture of tert-butyl methyl ether
and n-hexane yielding the protected peptide (165 mg). The purity
and identity of the peptide was checked by HPLC and MS after
deprotection of the sample (about 1 mg). ESI-MS: m/z calcd for
C31H54N8O13S: 778.35, found: 778.3. The mixture of Boc-1–8 side-
chain-protected peptide fragment (82 mg, 0.064 mmol) and HOBt
(11 mg, 0.08 mmol) in NMP (0.4 mL) was mixed with 32-residue
peptide–resin in the syringe and then the resin was soaked with
2m solution of DIPC in DMF (56 mL, 0.112 mmol) and agitated for
24 h. After washing the resin, the condensation reaction was re-
peated under the same conditions. The deprotection and cleavage
of the 40-residue peptide from the resin was done as described in
method A, yielding crude product (87 mg). This product was puri-
fied by HPLC prior to oxidative folding (13 mg).
by HPLC as a single symmetrical peak. Its identity was confirmed
by ESI-MS: m/z calcd for C178H296N66O57S6: 4462.06, found: 4462.0.
Determination of antimicrobial activity: A simple qualitative esti-
mate of antimicrobial properties was undertaken by using the
drop–diffusion test on Petri dishes by the double-layer tech-
nique.[34] The Petri dishes (90 mm in diameter) contained 20 mL of
LB agar (Sigma). Melted “soft” agar (2 mL), prepared from LB broth
(Sigma) and 0.5% agar (Difco) containing about 107 colony-forming
units of various bacteria (M. luteus, B. subtilis, S. aureus) was poured
over the surface. Fresh bacterial cultures were always prepared in
the LB broth and added when the melted soft agar cooled to
about 458C. Antimicrobial-activity-containing materials (evaporated
HPLC fractions) were diluted in H2O (10 mL) and dropped (2 mL)
onto the surface of the solidified upper layer. To compare the activ-
ity of lucifensin with its analogues (Figure 7) their amounts were
determined from the HPLC peak areas. The plates were incubated
at 378C. Clear zones of inhibition appeared within a few hours and
remained clear for days. The potency was semiquantitatively esti-
mated according to the diameter and clarity of the zones of inhibi-
tion. Quantitative minimum inhibitory concentrations (MICs) were
established by observing bacterial growth in multiwell plates.[35,36]
Mid-exponential phase bacteria were added to individual wells
containing solutions of the peptides at different concentrations in
LB broth (final volume 0.2 mL, final peptide concentration in the
range of 0.5 to 100 mm). The plates were incubated at 378C for
20 h while being continuously shaken in a Bioscreen C instrument
(Helsinki, Finland). The absorbance was measured at 540 nm every
15 min and each peptide was tested at least three times in dupli-
cate. Routinely, 5ꢃ103–10ꢃ103 CFU of bacteria were used per well.
Tetracycline in a concentration range of 0.5–50 mm was tested as a
standard.
Method C, Automated peptide synthesis: Crude linear 40-residue
peptides of lucifensin (Figure 1), Luc[C3–C30], Luc[C16–C36] and
Luc[C20–C38] (Figure 4A, B, and C, respectively), were synthesized
on 100 or 200 mg of preloaded Fmoc-Asn(Trt)-Wang Resin in an
Applied Biosystems 433A peptide synthesizer by using the HBTU/
HOBt/DIPEA activation protocol of Fmoc chemistry. The protected
amino acids were coupled in tenfold excess in NMP as solvent. The
resin substitution was reduced as described above prior to placing
it into the instrument. The deprotection and cleavage as described
in method A, yielded on average 160 mg of crude product per
100 mg of the resin. Measured molecular masses of the pure pep-
tides were in a good agreement with their calculated values
(Table S1).
Oxidative folding: The lyophilized linear peptides prepurified by
HPLC were dissolved (at concentration 1 mg/4 mL) in 0.1m
NH4OAc buffer, pH 7.8 (prepared by bubbling gaseous NH3 into
0.1m acetic acid) and stirred under open air at RT. The time course
of the disulfide bond formation was monitored by HPLC. Typically,
after 4–6 h of the folding reaction the solvent was removed by lyo-
philization and the desired folded peptides were further purified
by preparative HPLC by using a gradient of solvents ranging from
80% of solvent A to 50% of solvent B at a flow rate 3 mLminÀ1
over 60 min. The final products were then lyophilized. Typically, we
obtained 3–4 mg of final folded peptides starting from 10 mg of
corresponding linear precursors.
CD spectra measurement: The circular dichroism (CD) experi-
ments were carried out on a Jasco 815 spectropolarimeter (Tokyo,
Japan). All peptide samples were measured in H2O, in 50% (v/v)
TFE, and in the presence of SDS at concentrations of 0.16 mm to
16 mm (below and above the critical micelle concentration) with
the final peptide concentration 0.25 mgmLÀ1. The spectra were
collected from 190 nm to 300 nm as averages over four scans at
RT by using a 0.1 cm path length. A 0.5 nm step resolution,
10 nmminÀ1 speed, 32 s response time, and 1 nm bandwidth were
generally used. Following baseline correction, the final spectra
were expressed as molar ellipticity q [degcm2 dmolÀ1] per residue.
The secondary structure representation was calculated by using
the circular dichroism analysis program Dichroweb.[26]
Determination of disulfide bridges: Lucifensin and Luc[des1–10,
Ala30] (about 0.05 mg) were dissolved in 0.1m Mes buffer, pH 6.5
containing 2 mm CaCl2 (50 mL). After addition of of thermolysin
stock solution (1 mL; 1 mgmLÀ1), the mixture was incubated at
358C for 16 h. The reaction was quenched by adding 10% TFA
(2 mL) and then the entire mixture was subjected to fractionation
by HPLC on a Vydac C-18 column (250ꢃ4.6 mm). The material was
at first eluted with 2% MeCN/H2O/0.1% TFA for 10 min and then
with a linear gradient of solvents from 2% to 35% MeCN/H2O/
0.1% TFA over 60 min at a 1 mLminÀ1 flow rate. The elution was
monitored by absorption at 220, 254, and 280 nm. The fractions
were collected, the solvent evaporated in a Speed-Vac, and the
peptide fragments were analyzed by MS to identify their sequen-
ces.
Transmission electron microscopy: Bacillus subtilis cells treated
with lucifensin for either 10 or 60 min and untreated control cells
were used for negative staining examination. Bacteria were ad-
sorbed on parlodion-carbon-coated copper grids for 5 min. After
short washing, the samples were negatively stained by floating on
a drop of 0.25% phosphotungstic acid (PTA) with 0.01% BSA in
dH2O for 30 s. Excess stain was blotted off with a piece of filter
paper and samples were air-dried. A JEOL JEM/1200EX transmission
electron microscope operating at 60 kV was used for analysis.
Acknowledgements
Alkylation of linear lucifensin peptide: HPLC-purified linear luci-
fensin peptide was dissolved in a solution of iodoacetamide (1 mg)
in 0.05m NH4HCO3 (50 mL). The mixture was shaken at RT in dark-
ness for 1 h and then the reaction was quenched by 10% TFA
(3 mL). The fully alkylated peptide (all 6 Cys residues) was isolated
This work was supported by the Czech Science Foundation, Grant
No. 203/08/0536, and by Research Project No. Z40550506 of the
Institute of Organic Chemistry and Biochemistry, Academy of Sci-
ences of the Czech Republic. We thank our technical assistant
1360
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1352 – 1361