Anhydrous pyridine

Twenty-five milliliters of YNB containing 1% DMSO and the indicated concentration of diethylstilbestrol or hexestrol was inoculated with SC5314 at a concentration of 1 × 10³ cells/ml. Cultures were incubated at 37°C for 18 h. Nonsaponifiable lipids were extracted using alcoholic KOH. Samples were dried in a vacuum centrifuge (Heto) and were derivatized by the addition of 100 µl 90% N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)–10% trimethylsilyl (TMS [Sigma]) with 200 µl anhydrous pyridine (Sigma) and heating for 2 h at 80°C. TMS-derivatized sterols were analyzed and identified by gas chromatography-mass spectrometry (GC-MS) (Thermo 1300 gas chromatograph coupled to a Thermo ISQ mass spectrometer; Thermo Scientific) with reference to retention times and fragmentation spectra for known standards. GC-MS data files were analyzed using Xcalibur software (Thermo Scientific) to determine sterol profiles for all isolates and for integrated peak areas (40 (link)).

Anhydrous dimethyl acetamide (DMAc, 99%), anhydrous pyridine (Py), dimethyl amino pyridine (DMAp), trimethylsilylchloride (TMSC, >98%), N, N dimethyl formamide (DMF), 3-methoxycarbonyl-phenylboronic acid and tetrahydrofuran (THF, 99%) were purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA) and used as received. NaOH (sodium hydroxide), sulfuric acid (H₂SO₄), Tetrakis(triphenylphosphine) palladium (0) (Pd(PPh₃)₄), potassium carbonate (K₂CO₃), hydrochloric acid and thionyl chloride (SOCl₂) were obtained from Scharlau (Scharlab, Barcelona, Spain). The diacid 5′-tert-butyl-m-terphenyl-3,3″-dicarboxylic acid (tBTmDA) was synthesized following the procedure previously reported [27 (link)].
o-Hydroxydiamine 2,2-bis(3-amino-4-hydroxy phenyl)-hexafluoropropane (APAF) was purchased from Apollo Scientific (Apollo Scientific, Stockport, Cheshire, UK) and purified through sublimation at 220–225 °C before use. 4,4′-(hexafluoroisopropylidene) dianiline (6FpDA, Apollo Scientific, Stockport, Cheshire, UK) purified through sublimation at 220 °C and 5′-terbutil-m-terphenyl-3,3″-dichloride (tBTmCl) were employed as monomers to create the polyamide without o-hydroxy groups (PA). The synthesis of the corresponding HPA was carried out as described elsewhere [28 (link)].
The dichloride monomer (tBTmCl) was prepared from the corresponding diacid, as described below.

All chemicals used throughout this study were of analytical reagent grade. D,L-HTL, D,L-Met, CysGly, symmetrical disulfides of D,L-Hcy, D,L-Cys, and L-GSH, MSTFA, TMCS, TCEP, DTT, 2-ME, THP, HSA, sodium chloride, and anhydrous pyridine were from Sigma-Aldrich, (St. Louis, MO, USA). PCA, hydrochloric acid, acetic acid, sodium hydroxide, HPLC-gradient grade MeCN, ethanol, chloroform, methanol, sodium hydrogen phosphate heptahydrate, sodium dihydrogen phosphate dihydrate were from J.T. (Baker, Deventer), the Netherlands. CMLT was prepared as previously described [24 (link)]. Deionized water was produced in our laboratory.

Butylamine (99 +%), pentylamine (99%), and hexylamine (99%) were purchased from Acros Organics (Morris Plains, NJ, USA). 3,5-dinitrobenzoyl chloride (DNBC) (96.5%), propylamine (98%), tert-Butylamine (99.5%), 4,4′-(hexafluoroisopropylidene) diphthalic anhydride (6FDA) (99%), anhydrous N,N-dimethylacetamide (DMAc) (99.8%), triethylamine (TEA) (99.5%), hydrazine monohydrate (80%), anhydrous pyridine (99.8%), and Pd/C (10%) were purchased from Sigma-Aldrich (Milwaukee, WI, USA). All other reagents and solvents were purchased commercially as analytical grade and used without further purification.

Fluconazole-evolved strains and the parental clinical isolate were grown to the exponential-growth phase at 30°C in RPMI liquid media with or without fluconazole supplemented at 16 mg/liter. Alcoholic KOH was used to extract nonsaponifiable lipids. A vacuum centrifuge (Heto) was used to dry samples, which were then derivatized by adding 100 μl 90% N,O-bis(trimethylsilyl)-trifluoroacetamide–10% tetramethylsilane (TMS) (Sigma) and 200 μl anhydrous pyridine (Sigma) while heating at 80°C for 2 h as previously described (22 (link), 34 (link)). gas chromatography-mass spectroscopy (GC-MS) (with a Thermo 1300 gas chromatography system coupled to a Thermo ISQ mass spectrometer; Thermo Scientific) was used to analyze and identify TMS-derivatized sterols through comparison of the retention times and fragmentation spectra for known standards. Sterol profiles for each sample were determined by analyzing the integrated peak areas from GC-MS data files using Xcalibur software (Thermo Scientific). All sterol analysis was performed in biological triplicate. Error bars for each data point represent the standard deviations of results from three independent measurements of technical replicates.