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Supplementary data Design and Syntheses of Gramicidin s analogues, Cyclo

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Supplementary data
Design and Syntheses of Gramicidin S Analogues,

Cyclo(-X-Leu-X-D-Phe-Pro-)2 (X= His, Lys, Orn, Dab and Dap)
Makoto Tamakia, Kenta Fujinumaa, Takuji Haradaa, Kazumasa Takanashia,

Mitsuno Shindob, Masahiro Kimurab and Yoshiki Uchida*b

aDepartment of Chemistry, Toho University, Funabashi, Chiba 274-8510, Japan

bDepartment of Health and Nutrition, Osaka Shoin Women’s University, Higashi-Osaka, Osaka 577-8550, Japan

Table of Contents for the Supplementary data (total 7 pages)
Page 1: Title, authors and their affiliations and Table of Contents.

Page 2: HPLC analyses of 1-5.

Page 3-4: MS spectra of 1-5.

Page 5: CD spectra of 1-5.

Page 5: Determination of antibiotic activity (MIC) of 1-5.

Page 5-6: Determination of hemolytic activity (MHC) of 1-5.

1. HPLC analyses of GS peptides

HPLC analyses of GR peptides were achieved using analytical reverse phase HPLC system, equipped with an PU-980 intelligent HPLC pump, an UV-970 intelligent UV/VIS detector, an CO-965 column oven, and a Mightysil RP-18 GP column (4.0 x 150 mm, 5 m particle size, Kanto Chemical CO., INC Japan). Chromatographies were carried out by linear gradients of 50-65 % MeOH/0.1%TFAaq over 30 min for 1 and 45-60 % MeOH/0.1%TFAaq over 30 min for 2-5 with a flow rate of 1 ml/min at 30 ºC. The column eluent was monitored at 220 nm.

HPLC profiles of the GS peptides (1-5) were shown in next figures.

グループ化 2

2. MS spectra of 1-5

Low-resolution mass spectra (LR-MS) of 1-5 were obtained by using fast-atom bombardment (FAB) mass spectrometry (matrix: thioglycerin) on a JEOL600H mass spectrometer.






3. CD spectra of 1-5

The CD spectra were obtained by use of a JASCO spectra- polarimeter; model J-820 (JASCO LTD., Tokyo, Japan), using a 0.5-mm quartz cell at room temperature. The CD spectroscopy of 1-5 was carried out with a methanol solution at a concentration of 1.1-1.5 x 10-4 M.

4. Determination of antibiotic activity (MIC) of the cyclic peptides

Bacillus subtilis NBRC 3513, Bacillus megaterium ATCC 19213, Staphylococcus epidermidis NBRC 12993, Staphylococcus aureus NBRC 12732, Escherichia coli NBRC l2734 and Pseudomonas aeruginosa NBRC 3080 were grown overnight at 37 °C on nutrient agar medium and harvested in sterile saline. Densities of bacterial suspensions were determined at 600 nm, using a standard curve relating absorbance to number of colony forming units (CFU).

MICs of the synthetic peptides against several bacterial strains were assayed by the microplate dilution method as follows: 100 l of serial dilution of the synthetic peptide was added to a mixture of 10 l of bacterial suspension (approximately 106 CFU ml-1) and 90 l of Mueller-Hinton broth (Difco Laboratories, NJ, USA) in each well of a flat-bottomed microplate (Corning Laboratory Sciences Company, NY, USA). The highest peptide concentration tested was 100 gml-1. The plates were then incubated overnight at 37 °C for MIC evaluation. MIC was expressed as the lowest final concentration (g/m1) at which no growth was observed. The experiments were performed four times for each peptide.

5. Determination of hemolytic activity (MHC) of the cyclic peptides

The hemolytic activity of the peptide was determined using sheep red blood cells (RBCs). Freshly collected sheep blood in Alsever’s solution (preserved blood) was purchased from Nippon Bio-Test Laboratories Inc, and used as the source of the RBCs. The RBCs were isolated from preserved blood by centrifugation at 3000 rpm for 5min at 4 oC and then washed three times with HEPES-NaOH 90 buffered saline (HBS; 150 mM NaCl/5 mM HEPES-NaOH pH 7.4) just prior to the assay. The cyclic peptides were dissolved in water or DMSO to produce 1 mM cyclic peptide stock solution and stored -20 oC. Then, each concentration of cyclic peptide was prepared by dilution of the cyclic peptide stock solution in HBS. To each micro tube added 200 ml of erythrocytes (1% hematocrit in HBS) and 100 ml of each concentration of cyclic peptide. The micro tubes were incubated at 37 oC for 30 min and centrifuged for 5 min at 3000 rpm and 4 oC. The supernatant of each micro tube transferred to a polystyrene microtiter plate, and the absorbance was measured at 540 nm by a microtiter plate reader. HBS (no peptide) and triton X-100 (added 2 ml of 10% triton X-100) were used as references. The hemolytic activity was calculated as follows:{Abs peptide-Abs HBS}/{Abs triton X-100-AbsHBS}x100. The experiments were carried out three times for each peptide.

Figure. Dose dependence curves of hemolysis (%) against sheep erythrocytes induced by 1-5 and

GS. The experiments were carried out three times for each peptide.

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