Phosphine

The American Type Culture Collection (ATCC) provided the genomic DNA used to clone Acel_2062 (ATCC Number: ATCC 43068). Protein production and crystallization of the Acel_2062 protein was carried out by standard JCSG protocols
[8 (link)]. Clones were generated using the Polymerase Incomplete Primer Extension (PIPE) cloning method
[9 (link)]. The gene encoding Acel_2062 (GenBank: YP_873820[GenBank:YP_873820]; UniProtKB: A0LWM4[UniProtKB:A0LWM4]) was synthesized with codons optimized for Escherichia coli expression (Codon Devices, Cambridge, MA) and cloned into plasmid pSpeedET, which encodes an expression and purification tag followed by a tobacco etch virus (TEV) protease cleavage site (MGSDKIHHHHHHENLYFQ/G) at the amino terminus of the full-length protein. Escherichia coli GeneHogs (Invitrogen) competent cells were transformed and dispensed on selective LB-agar plates. The cloning junctions were confirmed by DNA sequencing. Expression was performed in a selenomethionine-containing medium at 37°C. Selenomethionine was incorporated via inhibition of methionine biosynthesis
[10 (link)], which does not require a methionine auxotrophic strain. At the end of fermentation, lysozyme was added to the culture to a final concentration of 250 μg/ml, and the cells were harvested and frozen. After one freeze/thaw cycle the cells were homogenized in lysis buffer [50 mM HEPES, 50 mM NaCl, 10 mM imidazole, 1 mM Tris(2-carboxyethyl)phosphine-HCl (TCEP), pH 8.0] and passed through a Microfluidizer (Microfluidics). The lysate was clarified by centrifugation at 32,500 x g for 30 minutes and loaded onto a nickel-chelating resin (GE Healthcare) pre-equilibrated with lysis buffer, the resin was washed with wash buffer [50 mM HEPES, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP, pH 8.0], and the protein was eluted with elution buffer [20 mM HEPES, 300 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP, pH 8.0]. The eluate was buffer exchanged with TEV buffer [20 mM HEPES, 200 mM NaCl, 40 mM imidazole, 1 mM TCEP, pH 8.0] using a PD-10 column (GE Healthcare), and incubated with 1 mg of TEV protease per 15 mg of eluted protein for 2 hours at 20°–25°C followed by overnight at 4°C. The protease-treated eluate was passed over nickel-chelating resin (GE Healthcare) pre-equilibrated with HEPES crystallization buffer [20 mM HEPES, 200 mM NaCl, 40 mM imidazole, 1 mM TCEP, pH 8.0] and the resin was washed with the same buffer. The flow-through and wash fractions were combined and concentrated to 15.6 mg/ml by centrifugal ultrafiltration (Millipore) for crystallization trials.

tert-Butyl(phenyl)phosphine chloride (1.003 g, 10.00 mmol) and 20 mL dichloromethane were added to a dry round bottom flask, which was filled with nitrogen. And then pure water (0.1802 g, 10.00 mmol) was slowly added dropwise under ice bath. When all the pure water had been added, the resulting solution was allowed to warm to room temperature and stirred for 5 h. After the hydrolysis reaction was completed, the mixture was directly rotary evaporated to obtain a white solid compound 2a (0.8864 g, 97% yield). ¹H NMR (400 MHz, CDCl₃): δ = 7.70–7.48 (m, 5H), 1.16 (dd, J = 17.0, 1.3, 9H). ¹³C NMR (101 MHz, CDCl₃): δ = 132.88 (d, J = 2.8), 131.09 (d, J = 10.3), 128.70 (d, J = 11.9), 31.97 (d, J = 68.8), 23.42 (d, J = 2.2). ³¹P NMR (162 MHz, CDCl₃): δ 49.24. MS (ESI⁺): Calcd for C₁₀H₁₆OP [M + H]⁺ requires 183.1, found 183.1. (NMR spectra were shown in Additional file 1: Figure S20–S22, and MS spectrum was shown in Additional file 1: Figure S54.)

A dry round-bottomed flask, equipped with a magnetic stirring bar, sealed with a septum, and protected with an Ar balloon, was charged with 4- or 3-(trifluoromethyl)phenyl boronic acid (190 mg, 1.0 mmol or 570 mg, 3.0 mmol) in 1.0 mL of THF. A solution of 1.0 M diisobutyl aluminium hydride (DIBAL) in toluene (3.0 or 5.0 mmol) was added to the mixture, and the corresponding phosphine was added at 0 °C. The mixture was stirred at room temperature and monitored using TLC. Once completed, the reaction was quenched by the addition of a saturated aqueous potassium sodium tartrate solution and extracted with AcOEt. The organic layer was washed with brine and dried over Na₂SO₄. The solvent was evaporated, and the residue was purified using silica gel column chromatography (eluent = hexane/AcOEt) to generate the desired phosphine–borane derivatives. For compounds 20–22 and 32–34, the corresponding phosphines were prepared immediately before addition, without purification. The details of the characterization and spectra of the compounds are presented in the Supporting Information.

AC (401.4 mg) was placed into an oven-dried Schlenk flask,
dried in vacuo for 30 min at 90 °C, and placed under a dry nitrogen
atmosphere.⁸⁹ In a second oven-dried Schlenk
flask, PⁿBu₃ (1) (29.4 mg, 0.145 mmol, 28% surface coverage) was dissolved in dry
and oxygen-free THF⁹⁰ (10 mL) under a dry
nitrogen atmosphere. The phosphine solution was then transferred to
the AC and stirred for 20 min under a dry nitrogen atmosphere. Subsequently,
THF⁹⁰ was removed in vacuo at 30 °C
until the AC was dry, which took 25 min. Meanwhile, a plastic desiccator
was filled with a mixture of drierite and molecular sieves (3 Å).
Both desiccants had been dried in a drying oven at 250 °C for
3 h and dried at RT in vacuo for an additional h. For the oxidation
of the adsorbed phosphine, a gentle stream of air, monitored by a
bubbler, was blown through the desiccator and into the Schlenk flask
containing 1 adsorbed on AC for 10 min. After the air
flow was stopped, the flask was sealed to allow for an additional
50 min of oxidation time. Then, the AC with adsorbed 1_ox was again placed under a dried nitrogen
atmosphere, 4 mL of dry, degassed THF⁹⁰ was added, and the mixture was stirred for 5 min. A portion of this
suspension was then added to a dried NMR tube under a nitrogen atmosphere,
and a ³¹P NMR spectrum was recorded (Figure S22).