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Supplementary Material

Non-hemolytic short peptidomimetics as a new class of potent and broad-spectrum antimicrobial agents

Ravichandran N. Murugan,‡a Binu Jacob,‡b Eun-Hee Kim, ‡a Mija Ahn,a Hoik Sohn,c Ji-Hyung Seo,a Chaejoon Cheong,a Jae-Kyung Hyun,d Kyung S. Lee,e Song Yub Shin b* and Jeong Kyu Bang a*

aDivision of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chung-Buk 363-883, Republic of Korea. Fax: +82-43-240-5059; Tel: +82-43-240-5023; E-mail: bangjk@kbsi.re.kr

bDepartment of Bio-Materials, Graduate School and Department of Cellular & Molecular Medicine, School of Medicine, Chosun University, Gwangju 501-759, Republic of Korea

Fax: +82-62-233-6337; Tel: +82-62-230-6769; E-mail: syshin@chosun.ac.kr

cDepartment of Chemisty and Biochemistry, College of Natural Science, University of Texas at Austin, Austin, TX 78712, U.S.A.

dDivision of Electron Micorscopic Research, Korea Basic Science Institute, 113 Gwahanno, Daejeon 305-333,Republic of Korea.

eLaboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, U. S. A.

1. General Materials and methods

Rink amide 4-methylbenzhydrylamine (MBHA) resin and 9-fluorenylmethoxycarbonyl (Fmoc) amino acids were obtained from Calbiochem-Novabiochem (La Jolla, CA). Other reagents used for peptide synthesis included trifluoroacetic acid (TFA; Sigma), piperidine (Merck), 1-O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate (HBTU, Novabiochem), dicyclohexylcarbodiimide (DCC; Fluka), N-hydroxybenzotriazole hydrate (HOBT; Aldrich). Biolab DMEM and fetal bovine serum (FBS) were obtained by HyClone (Seoulin, Bioscience, Korea). The buffers were prepared in double glass-distilled water.

[pic]

Figure S1. The structures of Trp/Arg-rich peptides tested for their antimicrobial activity.

2 Synthesis and characterization of SAMPs

All peptides were prepared by Fmoc SPPS methods using Rink amide resin with an initial loading of 0.61 mmol/g, unless otherwise noted. Resins were swollen in N,N-dimethylforamide (DMF) for 45 min prior to synthesis. For sequence extension, the Fmoc-Arg(Pbf)-OH (5 eq.) was activated by treatment with 1-O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate (HBTU) (5.0 eq.) and hydroxybenzotriazole (HOBt) (5.0 eq.) and N,N-diisopropylethylamine (DIEA) (5.0 eq.) in DMF (2 mL) for 2 min. This solution was added to the free amine on resin, and the coupling reaction was allowed to proceed for 1h with Vortex stirring. After washing with DMF, Fmoc deprotection was achieved with 20% piperidine in DMF (1 × 10 min, 2 × 3 min). The resin was washed once again, and the process was repeated for the next amino acid and finally the resin was washed with DMF, methanol, dichloromethane and ether, and then dried under vacuum. Linear peptides were cleaved from the resin with 5% triisopropylsilane (TIS) and 5% H2O in trifluoroacetic acid (TFA, approximately 2 mL of TFA per 100 mg of resin) for 2 h. The cleavage mixture was mixed with cold ether to precipitate the peptide and then filtered. Purification of crude peptide was carried out on the preparative Vydac C18 column (15 (m, 20 mm ( 250 mm) using an appropriate 10(90% water/acetonitrile gradient in the presence of 0.05% TFA(A: water buffer, B: acetonitrile buffer). The final purity of the peptides (>98%) was assessed by RP-HPLC on an analytical Vydac C18 column (4.6 mm × 250 mm, 300 Å, 5 (m particle size). The molecular masses of purified peptides were determined using matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) (Shimadzu, Japan). Optical rotation values were determined with an automatic digital AUTOPOL IV (A12628) polarimeter.

[pic]

Scheme S1. Synthetic protocol of Fmoc-His-OH derivatives and trimeric SAMP-1 using solid phase peptide synthesis (SPPS). (a) DCC/HOBt, MeOH, overnight; (b) i. R1-OH or R2-OH, Tf2O, DIEA, DCM; ii. TFA, TIS, DCM, 2 h; (c) 2 N HCl/1,4-Dioxane; (d) i. 20% piperidine in DMF, 15 min; ii. Fmoc-Arg(pbf)-OH, HBTU, HOBt, DIEA, 1 h; iii. 20% piperidine in DMF, 15 min; (e) HBTU, HOBt, 1, DIEA, overnight; (f) TFA/TIS/H2O (90:5:5), 2 h.

General procedure for the preparation of N (α)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(π)-,N(τ)-bisalkyl-L-histidine.

Compound B: (Scheme S1)

A 100-mL Schlenk flask was charged with solid Fmoc-His(Trt)-OH (10 g, 16.14 mmol), HOBt (3.27 g, 24.20 mmol) and degassed by vacuum-Ar purge cycle. Dry THF (80 mL) was added to dissolve all solids and the solution was cooled to -13 °C with an ice-acetone bath. To this mixture, a dry THF solution (80 mL) of DCC (3.33 g, 16.14 mmol) was added along with MeOH (12.40 ml, 513.0 mmol) dropwise. The reaction was allowed to warm slowly to room temperature while stirring overnight, during which time solid dicyclohexylurea was observed to precipitate. This was removed by vacuum filtration through a medium-porosity frit and the solvent was removed by rotary evaporation. The oily residue was redissolved in dichloromethane, washed three times with saturated NaHCO3 (aq), three times with water, and the organic layer was dried over Mg2SO4. The solvent is removed by rotary evaporation and a crude yellow solid was isolated after drying. The compound was purified by column chromatography on silica gel, eluted with 1% MeOH-DCM (10.34 g, quantitative yield) Rf = 0.33. The compound was purified by column chromatography on silica gel, eluted with 1% MeOH in DCM. (6.89 g, 83.6 % yield). [α]D26.5ºC 8.99 (c 0.5, CHCl3).

1H-NMR (400 MHz, CDCl3): δ 7.75 (d, 2H, J = 7.5 Hz), 7.62 (t, 2H, J = 7.5 Hz), 7.53 (s, 1H), 7.42-7.26 (m, 13H), 7.13-7.10 (m, 6H), 6.57 (s, 1H), 6.53 (d, 1H, J = 8.2 Hz), 4.63 (m, 1H), 4.38-4.22 (m, 3H), 3.63 (s, 3H), 3.13-3.03 (m, 2H).

13C NMR (100 MHz, CDCl3): δ 172.0, 156.2, 144.1, 143.9, 142.2, 141.2, 138.8, 136.2, 129.7, 128.12,, 128.09, 127.6, 127.1, 125.4, 125.3, 119.9, 119.7, 75.4, 67.2, 54.3, 52.2, 47.2, 30.1. All the values are in agreement with the reported values.1

Synthesis of N (α)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(π)-,N(τ)-bis(3-cyclohexyl propyl)-L-histidine methylester (Compound C1)

To a stirred solution of triflic anhydride (532 μL, 3.16 mmol) in DCM (15 ml) under Ar at −75 °C was added a solution of 3-cyclohexyl-1-propanol (494 μL, 3.16 mmol) and diisopropylethylamine (DIEA) (552 μL, 3.16 mmol) in DCM (30 ml) dropwise over 10 min. Stirring was continued at −75 °C (20 min), then a solution of Fmoc-His(Trt)-OMe (1.0 g, 1.58 mmol) in DCM (15 mL) was added dropwise, and the mixture was allowed to gradually warm to room temperature over a period of 18 h. The mixture was quenched using aqueous NaHCO3 and stirred vigorously (30 min). The organic layer was diluted with DCM and washed with aqueous NaHCO3 and brine, then dried (Mg2SO4), and concentrated to viscous oil. To a solution of the resulting gum in DCM (15 mL) was added trifluoroacetic acid (1.18 mL, 15.8 mmol) and triisopropylsilane (TIS) (356 μL, 1.74 mmol), and the mixture was stirred at room temperature until reaction was complete as shown by TLC (2 h). The solvent was removed in vacuo, and the residue was purified by silica gel column chromatography using 1% to 5% MeOH in DCM. Yield: Major: Dialkylated Product-C1 (0.712 g, 1.11 mmol, 70%) [α]D26.5ºC -3.80 (c 0.5, CHCl3). Minor: Monoalkylated Product-C2 (0.230 g, 0.36 mmol, 28%) [α]D26.5ºC 7.20 (c 0.5, CHCl3). *Although the insitu generated triflates will remove the trityl protecting group from the histidine, we treated again with TFA in order to make sure the complete removal of trityl group from either minor monoalkylated or unknown product for the better purification process.2

Compound C1 (Major): lH NMR (400 MHz, CDCl3) δ 8.93 (s, 1H), 7.74 (d, J = 7.2 Hz, 2H), 7.57-7.69 (m ,2H), 7.38 (t, J = 7.46 Hz, 2H), 7.23-7.34 (m, 2H), 6.64 (d, J = 7.9 Hz, 1H), 4.55 (m, 1H), 4.32 (t, J = 6.5 Hz, 1H), 4.15-4.27 (m, 2H), 4.03-4.14 (m, 2H) 3.99 (t, J = 7.4 Hz, 2H), 3.78 (s, 3H), 3.18-3.34 (m, 2H), 1.78-1.90 (m, 2H), 1.52-1.77 (m, 15H), 0.98-1.24 (m, 12H), 0.68-0.90 (m, 3H).

13C NMR (100 MHz, CDCl3) δ 170.5, 156.4, 143.8, 143.5, 141.2, 135.6, 131.4, 127.8, 127.17, 127.15, 125.5, 125.4, 122.2, 120.4, 119.97, 119.96, 119.0, 67.4, 53.1, 50.3, 47.6, 46.9, 37.0, 36.9, 33.7, 33.6, 33,04, 33.00, 27.4, 27.3, 26.42, 26.40, 26.13, 26.09.

MS (MALDI-TOF) m/z 640.3 [M].

Compound C2 (Minor): lH NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 7.75 (d, J = 7.6 Hz, 2H), 7.58 (d, J = 7.3 Hz, 2H), 7.39 (t, J = 7.32 Hz, 2H), 7.26-7.34 (m, 2H), 6.22 (d, J = 7.1Hz, 1H), 4.59 (m, 1H), 4.37 (d, J = 6.9 Hz, 2H), 4.19 (t, J = 6.9 Hz, 1H), 3.84 (t, J = 6.9 Hz, 2H), 3.75 (s, 3H), 3.09-3.19 (m, 2H), 1.72-1.51 (m, 7H), 1.02-1.31 (m, 6H) 0.68-0.90 (m, 2H).

13C NMR (100 MHz, CDCl3) δ 171.3, 155.9, 143.7, 143.6, 141.3, 136.8, 132.3, 127.8, 127.1, 126.9, 125.3, 125.1, 120.0, 119.96, 119.0, 67.4, 52.8, 50.6, 47.0, 45.7, 37.2, 34.1, 33.1, 28.1, 26.6, 26.5, 26.2.

MS (MALDI-TOF) m/z 516.2 [M+H]+.

Synthesis of N (α)-[(9H-Fluoren-9-ylmethoxy) carbonyl]-N(π)-,N(τ)-bis(3-cyclohexyl propyl)-L-histidine (Compound D1)

1:1 mixture of 2 N HCl (30 ml) and 1,4-Dioxane (30 ml) solution were added to the C1 (0.889 g, 1.39 mmol) and reflux at 100 0C for 3 h. The mixture was brought to room temperature, and the solvent was removed by Rota vapor. The resulting aqueous mixture was extracted with DCM, washed with brine, then dried (Mg2SO4), and concentrated to viscous oil. The organic extract was dried (Mg2SO4) and concentrated in vacuo, and the residue was purified by silica gel flash chromatography from 5% to 20% MeOH in DCM to provide Acid as a light yellow gum (0.601 g, 0.96 mmol, 69% yield). [α]D26.5ºC 23.38 (c 0.5, CHCl3).

lH NMR (400 MHz, CDCl3) δ 8.78 (s, 1H), 7.75 (d, J =7.44 Hz, 2H), 7.52-7.70 (m, 2H), 7.38 (t, J =7.35 Hz, 2H), 7.26-7.31 (m, 2 H), 6.69 (d, J = 5.94 Hz, 1H), 4.38-4.53 (m, 1H), 4.21-4.34 (m, 2H), 4.11-4.21 (m, 1H), 4.01-4.11 (m, 2H), 3.85-4,01 (m, 2H), 3.10-3.33 (m, 2H), 1.68-1.82 (m, 4H), 1.56 (s, 10H), 0.95-1.31 (m, 12H), 0.64-0.95 (m, 4H).

13C NMR (100 MHz, CDCl3) δ 173.3, 156.5, 144.4, 144.1, 141.7, 135.8, 133.0, 128.1, 127.4, 125.6, 125.5, 120.5, 120.4, 67.1, 55.0, 50.4, 47.8, 47.6, 37.5, 37.4, 34.2, 34.1, 33.5, 28.1, 28.0, 27.0, 26.8, 26.5

MS (MALDI-TOF) m/z 626.33[M] .

Synthesis of N (α)-[(9H-Fluoren-9-ylmethoxy) carbonyl]- N(π)-(3-cyclohexyl propyl) -L-histidine methyl ester (Compound C2)

The title compound was synthesized in a manner identical to compound C1 using 1.1 equiv. of 3-cyclohexyl 1-propanol (274 μl, 1.75 mmol). Materials: Fmoc-His(Trt)-OMe (1 g, 1.58 mmol), Tf2O (295 μl, 1.75 mml), DIEA (307 μl, 1.75 mmol), TFA (1.2 ml, 15.80 mmol), and TIS (356 μl, 1.74 mmol). The compound is purified by column chromatography on silica gel, eluted with 1-5% MeOH-DCM. 0.711 g, 1.38 mmol, 87% yield.

lH NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 7.75 (d, J = 7.6 Hz, 2H), 7.58 (d, J = 7.3 Hz, 2H), 7.39 (t, J = 7.32 Hz, 2H), 7.26-7.34 (m, 2H), 6.22 (d, J = 7.1Hz, 1H), 4.59 (m, 1H), 4.37 (d, J = 6.9 Hz, 2H), 4.19 (t, J = 6.9 Hz, 1H), 3.84 (t, J = 6.9 Hz, 2H), 3.75 (s, 3H), 3.09-3.19 (m, 2H), 1.72-1.51 (m, 7H), 1.02-1.31 (m, 6H) 0.68-0.90 (m, 2H).

13C NMR (100 MHz, CDCl3) δ 171.3, 155.9, 143.7, 143.6, 141.3, 136.8, 132.3, 127.8, 127.1, 126.9, 125.3, 125.1, 120.0, 119.96, 119.0, 67.4, 52.8, 50.6, 47.0, 45.7, 37.2, 34.1, 33.1, 28.1, 26.6, 26.5, 26.2.

MS (MALDI-TOF) m/z 516.2 [M+H]+.

Synthesis of N (α)-[(9H-Fluoren-9-ylmethoxy)carbonyl]- N(π)-(3-cyclohexylpropyl)-L- histidine (Compound D2)

The title compound was synthesized in a manner identical to compound D1, absent of C1; instead C2 (0.711 g, 1.38 mmol). Materials: 2 N HCl : 1,4-Dioxane (30 ml: 30 ml). The compound is purified by column chromatography on silica gel, eluted with 5-20% MeOH-DCM.0.297 g, 0.59 mmol, 43% yield. [α]D26.5ºC 31.38 (c 0.5, CHCl3).

lH NMR (400 MHz, CDCl3) δ 8.29 (s, 1H), 7.77 (d, J =7.26Hz, 2H), 7.47-7.68 (m, 2H), 7.33-7.47 (m, 2H), 7.22-7.33 (m, 2 H), 7.14 (s, 1H), 6.20-6.45 (m, 1H), 4.42-4.58 (m, 1H), 4.26-4.42 (m, 2H), 4.11-4.26 (m, 1H), 3.83-4.11 (m, 2H), 3.29 (s, 2H), 1.45-1.83 (m, 7H), 0.97-1.25 (m, 6H), 0.59-0.96 (m, 2H).

13C NMR (100 MHz, CDCl3) δ 171.7, 155.8, 143.7, 143.3, 141.4, 141.3, 133.7, 129.6, 127.8, 127.1, 124.8, 120.0, 118.9, 66.8, 52.9, 47.3, 47.1, 36.9, 33.8, 33.0, 27.4, 26.3, 26.0.

MS (MALDI-TOF) m/z 502.26[M+H] +.

AMP: (WR)3-NH2

Purity of crude product 95% (C18 RP-HPLC). White powder; RP-HPLC Rt = 18.53 min (gradient B: 10-90% / 25 min); MS (MALDI-TOF) m/z 1044.39 [M + H]+.

AMP: (WR)2-NH2

Purity of crude product 95% (C18 RP-HPLC). White powder; RP-HPLC Rt = 17.27 min (gradient B: 10-90% / 25 min); MS (MALDI-TOF) m/z 702.29 [M + H]+.

AMP: RWR-NH2

Purity of crude product 95% (C18 RP-HPLC). White powder; RP-HPLC Rt = 13.62 min (gradient B: 10-90% / 25 min); MS (MALDI-TOF) m/z 516.298 [M + H]+.

SAMP-1, RDR-Amide (where D = 1, Histidine derivative)

Purity of crude product 95% (C18 RP-HPLC).White powder; RP-HPLC Rt = 17.6 min (gradient B: 10-90% / 25 min); MS (MALDI-TOF) m/z 715.47 [M].

SAMP-2, RDR-Amide (where D = 2, Histidine derivative)

Purity of crude product 95% (C18 RP-HPLC). White powder; RP-HPLC Rt = 12.7 min (gradient B: 10-90% / 25 min); MS (MALDI-TOF) 591.42 [M + H]+.

SAMP-3, DR-Amide (where D = 1, Histidine derivative)

Purity of crude product 95% (C18 RP-HPLC). White powder; RP-HPLC Rt = 18.4 min (gradient B: 10-90% / 25 min); MS (MALDI-TOF) m/z 559.48 [M].

SAMP-4, DRR-Amide (where D = 1, Histidine derivative)

Purity of crude product 95% (C18 RP-HPLC). White powder; RP-HPLC Rt = 15.6 min (gradient B: 10-90% / 25 min); MS (MALDI-TOF) m/z 715.58 [M].

SAMP-5, RRD-Amide (where D = 1, Histidine derivative)

Purity of crude product 95% (C18 RP-HPLC). White powder; RP-HPLC Rt = 16.27 min (gradient B: 10-90% / 25 min); MS (MALDI-TOF) m/z 715.58 [M].

Table S1. RP-HPLC retention time of the indicated SAMPs

|Peptides |SAMP-1 |SAMP-2 |SAMP-3 |SAMP-4 |SAMP-5 |

|RP-HPLC retention time (Rt): min |17.6 |12.7 |18.4 |15.6 |16.27 |

RP-HPLC retention time (Rt) was measured using a C18 reverse-phase analytical column (5 (m; 4.6 mm ( 250 mm; Vydac). Peptides were eluted for 25 min, using a linear gradient of 10–90% (v/v) acetonitrile in water containing 0.05% (v/v) trifluoroacetic acid.

3. Antimicrobial activity (MIC)

The antimicrobial activity of the peptides against two Gram-positive bacterial strains and two Gram-negative bacterial strains was examined by using the broth microdilution method in sterile 96-well plates. Aliquots (100 (l) of a bacterial suspension at 2 ( 106 colony-forming units (CFU)/ml in 1% peptone were added to 100 (l of the peptide solution (serial 2-fold dilutions in 1% peptone). After incubation for 18(20 h at 37 (C, bacterial growth inhibition was determined by measuring the absorbance at 600 nm with a Microplate Autoreader EL 800 (Bio-Tek Instruments, VT). The minimal inhibitory concentration (MIC) was defined as the minimum peptide concentration inhibited bacteria growth. Two types of Gram-positive bacteria (Staphylococcus epidermidis [KCTC 1917] and Staphylococcus aureus [KCTC 1621]) and two types of Gram-negative bacteria (Escherichia coli) [KCTC 1682] and Pseudomonas aeruginosa [KCTC 1637]) were procured from the Korean Collection for Type Cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Methicillin-resistant Staphylococcus aureus (MRSA) (CCARM 3089, CCARM 3090 and CCARM 3095) were obtained from the Culture Collection of Antibiotic-Resistant Microbes (CCARM) at Seoul Women’s University (Seoul, Korea).

4. Hemolytic activity

Hemolytic activity of peptides was tested against human red blood cells (hRBCs). Fresh hRBCs were washed three times with phosphate buffered saline (PBS; 35 mM phosphate buffer containing 150 mM NaCl, pH 7.4) by centrifugation for 10 min at 1000×g and then resuspended in PBS. The peptide solutions (serial 2-fold dilutions in PBS) were then added to 100 μl hRBCs in PBS to give a final volume of 200 μl and a final erythrocyte concentration of 4% (v/v). The resulting suspension was incubated with agitation for 1 h at 37 °C. The samples were then centrifuged at 1000×g for 5 min, and release of hemoglobin was monitored by measuring the absorbance of the supernatant at 405 nm. No hemolysis (blank) and 100% hemolysis controls consisted of hRBCs suspended in PBS and 0.1% Triton X-100, respectively. Percent hemolysis was calculated using the following equation:

Hemolysis (%) = [(OD405nm sample − OD405nm zero lysis)

/ (OD405nm100 % lysis – OD405nm zero lysis)] × 100

[pic]

Figure S2. Concentration–response curves of percent hemolysis of the peptides against human red blood cells. Peptides are indicated as follows: (WR)3-NH2 (●), (WR)2-NH2 (○), RWR-NH2 (▼), SMAP-1 (▽), SMAP-2 (□), SMAP-3 (■), SMAP-4 (◆), SMAP-5 (◇), LL-37 (▲).

5. Proteolytic stability

Escherichia coli (KCTC 1682) and Staphylococcus aureus (KCTC 1621) were grown overnight for 18 h at 37 ℃ in 10ml of LB broth and then 10μl of this culture was inoculated into 10ml of fresh LB and incubated for an additional 3 h at 37℃ to obtain mid-logarithmic phase organisms. Digestion of each peptide by trypsin was carried out using 50 (g/ml peptide and 0.2 (g/ml trypsin in 50 mM Tris-HCl buffer, pH 7.5 at 37 °C for 1h. The reaction solution (50 (l) was added to 150 (l of a bacterial suspension (2×106 CFU/ml in 1% peptone). After incubation at 37 °C for 18–20 h, the bacterial growth inhibition was determined by measuring absorbance at 600 nm with a Microplate autoreader EL 800 (Bio-Tek Instruments).

[pic]

Figure S3. Proteolytic stability profiles of the designed SAMPs and melittin by trypsin. Relative peptide concentrations were determined by integration of the molecular peak from analytical RP-HPLC chromatograms.

6. Morphological changes of bacteria upon SAMPs addition

Morphological changes of a Gram-negative bacterial strain (Escherichia coli [KCTC 1682]) and a Gram-positive bacterial strain (Staphylococcus aureus [KCTC 1621]) upon the addition of SAMPs (SAMP-1 and SAMP-4) and LL-37 were analyzed using transmission electron microscopy (TEM). Bacterial culture at 2 ( 106 colony-forming units (CFU)/ml in LB media was washed 3 times in phosphate buffered saline (PBS) via a series of centrifugation at 10,000 (g, for 5 minutes, and re-suspension. 100 μl of SAMPs in PBS was added to an equal volume of bacterial suspension to a final concentration at (10 MIC. Following the addition of SAMPs, the samples were incubated for 1 hour at 37 (C. Bacterial cell pellet after centrifugation was re-suspended in 20 μl PBS for TEM specimen preparation. 5 μl of sample solution was loaded onto a carbon film-coated TEM grid that was rendered hydrophilic by glow discharge. After 90 seconds, excess sample solution was washed off with distilled water. 5 μl of 1 % uranyl acetate was loaded onto the grid for negative staining for 1 minute, and excess stain solution was blotted using a piece of filter paper. Samples were imaged using a Tecnai G2 Spirit electron microscope (FEI) equipped with lanthanum hexaboride (Lab6) gun, operating at 120 kV. Images were recorded using Ultrascan 4000 charge-coupled device (CCD) camera (Gatan).

REFERENCES

1. Himes, R.A.; Park, G.Y.; Barry, A.N.; Blackburn, N.J.; Karlin, K.D. J. Am. Chem. Soc., 2007, 129, 5352.

2. Qian, W.; Liu, F.; Burke, T.R. Jr. J. Org. Chem., 2011, 76, 8885-8890.

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