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4 protocols using absolute ethanol

1

Epothilone B and D Synthesis Protocol

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Epothilone B was purchased from Toronto Research Chemical (Toronto, Ontario, Canada) and epothilone D was purchased from Austin Chemical (Buffalo Grove, Illinois). Dichloromethane, ferric chloride, phenylmethylsulfonyl fluoride (PMSF), cremaphor, dithiothreitol (DTT), anhydrous Dichloromethane (DCM), anhydrous N,N-dimethylformamide (DMF), N-methylmorpholine (NMM), 4-dimethylaminopyridine (DMAP), and lithium hydroxide monohydrate (LiOH·H2O) were purchased from Sigma-Aldrich (St. Louis, Missouri). Trehalose was purchased from Carbosynth (Berkshire, United Kingdom). Dialysis tubing with 3500 molecular weight cut-off (MWC) and tangential flow filtration filters with 10 kDa pore size were purchased from Spectrum Labs (Rancho Dominguez, California). Pass-through sterile 0.22 micron filters were purchased from Sartorious (Goettingen, Germany). Thermo Syncronis C18 HPLC column (97105-254630), HPLC grade methanol, HPLC grade acetonitrile, n-butyl chloride, absolute ethanol, K2EDTA tubes, black wall, clear bottom 96-well plates, tetrahydrofuran (THF), isopropyl alcohol, methyl tert-butyl ether (MTBE), acetic anhydride (Ac2O), acetone, and acetic acid were purchased from Fisher Scientific (Waltham, Massachusetts). absolute ethanol was purchased from Decon Labs. Hydroxylamine (50% aqueous solution) was obtained from Alfa Aesar (Haverhill, Massachusetts).
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2

Synthesis and Characterization of Calcium Compounds

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Calcium oxide (99.9%) and nanosized calcium
oxide (<160 nm, 98%) were purchased from Sigma-Aldrich Inc. and
used as received. Calcium carbonate (99.999%) was used as received
from Mallinckrodt. Calcium hydroxide (99%) and hydrochloric acid (36
wt %) were obtained from Fischer Chemicals, and absolute ethanol (200
proof) was acquired from Decon Labs Inc. Dissolved acetylene and industrial
nitrogen were sourced from Airgas. For conductivity measurements,
gold foil (99.9%) was obtained from Aesar and aluminum foil from Reynolds.
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3

Synthesis and Characterization of Ace-DEX

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Chemicals were purchased from MilliporeSigma (St. Louis, MO) unless otherwise indicated. Absolute ethanol was purchased from Decon Labs (King of Prussia, PA). 2-ethoxypropene was purchased from Matrix Scientific (Elgin, SC). Full length wild type (WT) influenza A (H1N1) HA protein amino acid sequences from 3,078 human H1N1 viruses collected from May 1, 2014 – September 30, 2016 were downloaded from the GISAID Epiflu database. These sequences were organized by their collection date, and put into the COBRA algorithm to generate the sequence of Y2 COBRA HA, which was produced and purified as previously described (Huang et al., 2021b (link)).
Ace-DEX was synthesized from dextran from Leuconostoc mesenteroides with an average molecular weight of 60,000–76,000 Da as previously described (Broaders et al., 2009 (link); Chen et al., 2016 (link); Kauffman et al., 2012 (link)). To achieve 20 and 60 CAC, the reaction was carried out for four minutes or two hours, respectively. The polymer CAC was determined by degradation in 10% v/v deuterium chloride in deuterium oxide and quantification of the evolved acetone and ethanol using 1H NMR spectroscopy (Inova 400 MHz spectrometer) (Broaders et al., 2009 (link)).
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4

COBRA Influenza A Hemagglutinin Protein

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70 kDa dextran, anhydrous dimethylsulfoxide, triethylamine, pyridinium p-toluenesulfonate, and sulfuric acid were purchased from MilliporeSigma (St. Louis, MO). Absolute ethanol was purchased from Decon Labs (King of Prussia, PA). 2-ethoxypropene was purchased from Matrix Scientific (Elgin, SC). cGAMP was purchased from Invivogen (San Diego, CA). Full length wild type (WT) influenza A (H1N1) HA protein amino acid sequences from 3,078 human H1N1 viruses collected from May 1, 2014 – September 30, 2016 were downloaded from the GISAID Epiflu database. These sequences were organized by their collection date, and put into the COBRA algorithm to generate the sequence of Y2 COBRA HA, which was produced and purified as previously described.(Huang et al., 2021 (link))
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