TIPOS DE VALIDEZ

A.1 Introduction

The majority of the methods described below were published as supplemental data to Lichtenstein et al.1 _{Where appropriate additional methodology has been added and} described. Raw spectra found in the paper’s supplementary data have been removed for brevity. As elsewhere, Naq is represented by the single letter code Ϙ, where appropriate.

A.2 General Methods

All reactions were carried out under a positive pressure of argon or nitrogen unless otherwise indicated. Commercial reagents were from Sigma Aldrich, unless otherwise indicated, and used as received. HPLC grade diethylether, ethyl acetate, hexanes, methylene chloride, and acetonitrile were purchased from Fisher Scientific. HPLC grade carbon tetrachloride was purchased from Sigma Aldrich. Anhydrous dimethylformamide (DMF)

Sulfuric acid, glacial acetic acid and hydrochloric acid were purchased from Fisher Scientific. All other solvents were reagent grade and used as received. Flash chromatography was performed using Fisher 230-400 silica gel 60 unless otherwise indicated. Analytical TLC was carried out on Analtech silica gel HLF pre-coated glass plates with detection by UV illumination unless otherwise indicated. 1_{H and} 13_{C NMR spectroscopy of small molecules} was performed on a Varian Inova-500 (at 500 MHz and 125 MHz, respectively) using CDCl₃ as solvent (unless otherwise noted) and referenced with the residual solvent signal (CDCl3, 7.24 and 77.0 ppm, respectively) or referenced externally. The following abbreviations are used to describe peak patterns where appropriate: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad). Small molecule mass spectrometry data were obtained by the Penn State University Proteomics and Mass Spectrometry Core Facility. Peptide synthesis was carried out either a Pioneer Peptide Synthesis System or CEM Liberty Microwave Synthesiser using standard Fmoc chemistry (protected natural amino acids from Novabiochem) with HATU/HOAt (Genscript Corporation) as the coupling reagents (4-5.0 mol equivalents of activator/Fmoc-amino acid) on PAL-PEG-PS resin (Applied Biosciences, most peptides) or Fmoc-Ser(tBu)-Wang resin (Anaspec, peptides containing C- terminal serine carboxylates) . After cleavage and deprotection of the natural amino acids (Reagent R: 90:5:3:2 TFA/Thioanisole/Ethane Dithiol/Anisole) under argon, the resin was filtered and washed with 10 mL TFA (3x). The TFA was removed in vacuo, and the crude peptide precipitated with cold methyl tert-butyl ether. The identity of all peptides was confirmed with MALDI-TOF-MS using either α-cyano-4-hydroxycinnamic acid or sinapic acid (Sigma) as the peptide matrix. MALDI-TOF-MS determinations were performed on a PerSeptive Biosystem Voyager-DE RP. Kjedahl analysis was performed by Galbraith

Laboratories, Inc. CHN elemental analysis was performed by Midwest Microlab, LLC. UV/vis spectra were acquired on an Agilent 8453 UV-Visible Spectrophotmeter or a Varian Cary-50 Spectrophotometer. CD Spectra were acquired on either an AVIV 405 or 410 CD Spectrophotometer. Automated titrations were performed with a Hamilton Company automated diluter.

A.3 Naq Synthetic Methods

A.3.1 Methyl 1-hydroxy-4-methoxy-2-naphthoate (1)

(For alternative method see Boger and Jacobson2_{) 40.83 g (200 mmol) 1,4-} dihydroxynaphthoic acid was suspended in methanol (200 mL) at room temperature. With stirring, sulfuric acid (30 mL, 2 eq) was added dropwise to the solution such that the reaction mixture was nearly refluxing when the addition was complete. The reaction was refluxed for 24 hr at which point an additional 2 equivalents of sulfuric acid and 50 mL of methanol were added. After an additional 24 hr of refluxing the reaction appeared complete by TLC (eluent toluene). The reaction was diluted into 700 mL of CH2Cl2 and was added to 1 L of saturated aqueous sodium bicarbonate into a 2 L beaker with stirring. Solid sodium bicarbonate (Fisher Scientific) was added until the aqueous layer no longer evolved carbon dioxide at which point the layers were separated. The organic layer was dried over anhydrous magnesium sulfate (Fisher Scientific) and the solvent was removed in vacuo. Silica column

chromatography (eluent toluene) afforded 43.40 g (93.4%) of the methyl 1-hydroxy-4- methoxy-2-naphthoate as a pale green solid. NMR 1_{H (500 MHz) d 3.97 (s, 3H), 4.00 (s,} 3H), 7.02 (s, 1H), 7.57 (t, 1H, J=7.5 Hz), 7.63 (t, 1H, J=7.5 Hz), 8.19 (d, 1H, J=8.2 Hz), 8.39 (d, 1H, J=8.2 Hz), 11.62 (s, 1H); 13_{C (125 MHz) d 52.49, 55.95, 100.71, 104.52, 122.14,} 124.04, 125.78, 126.66, 129.26, 130.14, 147.96, 155.88, 171.57; Expected MS: M-H+ 231.1, Found 231.1.

A.3.2 Methyl 1,4-dimethoxy-2-naphthoate (2)

(For alternative method see Newman and Choudhary3_{) 35.5 g (153 mmol) of methyl} hydroxynaphthoate 1 was dissolved in 155 mL of anhydrous DMF. To the solution was added 35.5 g (257 mmol, 1.67 eq) of K2CO3 (Fisher Scientific) with stirring. Iodomethane (58 mL, 929 mmol, 6 eq) was added and the reaction was refluxed for 48 hours at which point TLC analysis (eluent toluene) showed that the reaction was complete. The reaction was diluted into 1L ethyl acetate and extracted three times with 1L water, and once with 1L brine. The organics were collected and the solvent removed in vacuo. After chromatography (eluent toluene) the trimethyl naphotoate 2 was obtained in 90% yield (33.9 g) as a beige solid. NMR 1_{H (500 MHz) d 3.99 (s, 3H), 4.00 (s, 3H), 4.01 (s, 3H), 7.16 (s, 1H), 7.58 (m,} 2H), 8.23 (m, 2H); 13_{C (125 MHz) d 52.48, 55.93, 63.51, 103.74, 118.88, 122.50, 123.67,} 127.25, 127.94, 128.99, 129.40, 151.57, 152.25, 167.01; Expected MS: MH+ 247.1, Found 247.1.

A.3.3 1,4-Dimethoxy-2-hdroxymethylnaphthalene

(For alternative method see Bulbule et al.4_{) Methyl naphthoate 2 (33.9 g, 138 mmol)} in 200 mL anhydrous THF was added dropwise to a stirred suspension of LiAlH4 (15.7 g, 414 mmol, 3 eq) in 100 mL anhydrous THF at 0°C. The reaction was allowed to warm to room temperature and was stirred for 2 hours. The reaction was quenched by the dropwise addition of 700 mL of saturated sodium potassium tartrate and stirred overnight. The resulting mixture was diluted with 2L ethyl acetate and was extracted twice with 1L water and once with brine. The ethyl acetate layer was collected and the solvent removed in vacuo

affording the product hydroxymethyl naphthalene (29.7 g, 99%) as a beige solid. NMR 1_H (500 MHz) d 3.90 (s, 3H), 3.96 (s, 3H), 4.87 (s, 2H), 6.79 (s, 1H), 7.47 (t, 1H, J=7.6), 7.53 (t, 1H, J= 7.3 Hz), 8.02 (d, 1H, J=8.3 Hz), 8.22 (d, 1H, J=8.3 Hz); 13_{C (125 MHz) d 55.93,} 61.20, 62.87, 104.05, 122.02, 122.68, 125.75, 126.54, 126.94, 128.70, 128.78, 147.20, 152.43; Expected MS: MH+-H2O 201.1, Found 201.1.

A.3.4 1,4-Dimethoxy-2-bromomethylnaphthalene (3)

(For alternative method see Bulbule et al.4_{) Hydroxymethyl naphthalene (34.6 g, 159} mmol) was dissolved in 200 mL CCl4 and cooled to 0°C. PBr3 (22.4 mL, 238 mmol, 1.5 eq) in 70 mL CCl₄ was added dropwise to this solution. The reaction was stirred for 30 minutes at 0°C and for 1.5 hours at RT. The reaction was quenched by the dropwise addition of saturated aqueous sodium bicarbonate until evolution of CO₂ ceased. The mixture was diluted with 2L CH2Cl2 and extracted three times with 1L saturated aqueous sodium bicarbonate, and then with brine. Collection of the organic layer and removal of the solvent

in vacuo yielded the product aryl bromide 3 (44.6 g, 95.8%) as a tan solid. This product was

unstable and was used immediately in the next reaction.

A.3.5 N-(Diphenylmethylene)-L-1,4-dimethoxy-2-naphthalanine tertbutyl ester (4)

N-(Diphenylmethylene)-glycine tert-butyl ester (36.0 g, 122 mmol), O-allyl-N-(9- anthracenylmethyl)cinchonidinium bromide (7.38 g, 12.2 mmol, 0.1 eq), CsOH•H2O (204.54