Zinc powder

PceA reductive dehalogenase activity was assayed in anaerobic conditions at room temperature (inside a Coy Laboratory glove-box with a 97%/3% (v/v) N₂/H₂ atmosphere). After protein separation by CN-PAGE, the gel piece (corresponding to up to three lanes) was vertically placed in a 500-mL anaerobic serum flask with 10 mL oxygen-free 50 mM Tris-HCl buffer (pH 7.5). The flask was removed from the glove-box, gas-exchanged for N₂ and placed back into the glove-box. The buffer was removed and replaced by 0.5 mL of a 250 mM methyl viologen (MV) solution, that was previously reduced with 1 g of zinc powder (Sigma-Aldrich). The gel was incubated for approximately for 15–30 min, until a homogeneous blue staining was obtained. After removing the excess MV solution, 1 mL of an anaerobic 10 mM PCE solution in ethanol was added to the gel and the assay was incubated at room temperature until colourless bands appeared on the gel due to the MV oxidation by the reductive dehalogenase activity. When bands were clearly visible, 5 mL of a 300 mM 2,3,5-triphenyltetrazolium chloride solution was added producing homogeneous and oxygen-resistant red staining of the MV-reduced portions of the gel, leaving the bands with enzymatic activity colourless.

AAI was purchased from Sigma‐Aldrich. The LLC‐PK1 porcine cell line (ATCC® CL‐101™) was obtained from the American Type Culture Collection (ATCC). Dulbecco's modified Eagle medium (DMEM), phosphate‐buffered saline and trypsin‐EDTA were purchased from Gibco and fetal calf serum from Lonza BioWhittaker. dA, deoxyguanosine (dG), N,N‐dimethylformamide, zinc powder, phosphodiesterase I from Crotalus adamanteus (venom phosphodiesterase), phosphodiesterase II from bovine spleen (spleen phosphodiesterase), nucleus PI and alkaline phosphatase were purchased from Sigma‐Aldrich. Dimethyl sulfoxide (>99.9%) was obtained from Acros Organics. Acetonitrile (ACN; ULC/MS grade) was obtained from Biosolve BV. Formic acid and ethanol were obtained from VWR Merck.

Anhydrous magnesium sulfate (MgSO₄, 99.5%), pyrene (Py), benzophenone (99%), PdCl₂(PPh₃)₂, bromine solution (Br₂), copper(I) iodide (CuI, 99%), dichloromethane (DCM), glacial acetic acid (AcOH), hydrogen peroxide (H₂O₂), potassium carbonate (K₂CO₃, 99.9%), Pd(PPh₃)₄, thianthrene (THT, 99%), titanium tetrachloride (TiCl₄, 99.9%), trimethylsilylacetylene (TMSA, 98%), triphenylphosine (PPh₃, 99%), zinc powder (Zn, 98%) were purchased from Sigma-Aldrich (Gillingham, UK) and Acros Organics (Geel, Belgium).

Materials used for the fabrication of DSSCs such as Fluorine doped tin oxide (FTO), absolute ethanol, zinc powder, hydrochloric acid (HCl), titanium (IV) isopropoxide (TTIP), platinum (Pt) paste and Di-tetrabutylammonium cis-bis(isothiocyanato)bis(2,2′-bipyridyl-4,4′-dicarboxylato)ruthenium(II) (N719) dye with analytical grade quality were purchased from Sigma Aldrich. Titanium dioxide (TiO₂) paste and redox electrolyte (iodide-tri iodide ${\mathrm{I}}^{-}/{\mathrm{I}}_3^{-}$ in 3-methoxypropionitrile (EL-HSE)) were procured from Dyesol, Australia.

To a solution of nitroalcohol 11 (5.75 g, 20.7 mmol) in THF (80 mL) was added a saturated solution
of NH₄Cl (80 mL). The resulting biphasic mixture was cooled
to 0 °C, and zinc powder (8.14 g, 124.4 mmol, 6.0 equiv, Sigma-Aldrich,
10 μm) was added in a few portions. Then the reaction mixture
was vigorously stirred at rt for 24 h, and the resulting suspension
was filtered (washing with 1 × 50 mL of EtOAc). Then the aqueous
phase was separated, saturated with solid NaCl, and extracted with
EtOAc (4 × 50 mL). The combined organic extracts were dried over
Na₂SO₄ and evaporated, and the residue was chromatographed
on silica (10–25% EtOAc/hexanes) to give a pure aniline 12 as a light yellow solid (2.62 g). Fractions containing
impurities were collected and crystallized from n-heptane to give additional (0.60 g) white solid (3.37 g, overall
yield of 66%): mp 111–112 °C (DCM/n-heptane); ¹H NMR (400 MHz, CDCl₃) δ 6.81 (br s, 3H),
6.09–5.96 (m, 1H), 5.43–5.35 (m, 1H), 5.31–5.25
(m, 1H), 5.00 (q, J = 7.5 Hz, 1H), 4.50–4.45
(m, 2H), 4.34 (br s, 2H); ¹³C{¹H} NMR (100 MHz,
CDCl₃) δ 153.5, 136.8, 133.4, 125.3 (q, J_CF = 281.2 Hz), 123.5, 122.2, 117.8, 116.7, 116.1, 72.4
(q, J_CF = 32.0 Hz), 69.5; ¹⁹F NMR (376 MHz, CDCl₃) δ −77.6; HRMS (ESI) m/z calcd for C₁₁H₁₃F₃NO₂ [M + H]⁺ 248.0898, found 248.0895.

To a solution of nitroalcohol 14 (2.73 g, 9.37 mmol) in THF (20 mL) was added a saturated solution
of NH₄Cl (20 mL); the mixture was cooled to 0 °C,
and zinc powder (3.67 g, 56.2 mmol, 6.0 equiv, Sigma-Aldrich, 10 μm)
was added in a few portions. Then the reaction mixture was vigorously
stirred at rt for 16 h and filtered through pad of Celite (washing
with EtOAc). The resulting mixture was diluted with brine (30 mL)
and extracted with EtOAc (3 × 20 mL). The combined organic extracts
were washed with brine (2 × 20 mL), dried over Na₂SO₄, and evaporated. The residue was chromatographed on
silica (15–50% EtOAc/hexanes, Combi Flash, 80 g column) to
give a light yellow solid (1.92 g, 78%): ¹H NMR (400 MHz,
CDCl₃) δ 6.81–6.72 (m, 3H), 5.95–5.82
(m, 1H), 5.20–5.07 (m, 2H), 4.97 (q, J = 7.5
Hz, 1H), 4.26 (s, 3H), 3.95 (t, J = 6.7 Hz, 2H),
2.56–2.46 (m, 2H); ¹³C{¹H} NMR (100 MHz,
CDCl₃) δ 153.8, 136.2, 134.4, 125.1 (q, J_CF = 283.2 Hz), 123.6 (q, J_CF = 1.4 Hz), 122.4, 117.0, 116.4, 115.9, 72.4 (q, J_CF = 32.1 Hz), 67.8, 33.6; ¹⁹F NMR (376 MHz,
CDCl₃) δ −77.6; HRMS (ESI) m/z calcd for C₁₂H₁₄F₃NO₂ [M + H]⁺ 262.1055, found 262.1042.

To a solution of nitroalcohol 20 (3.23 g, 11.7 mmol) in THF (50 mL) was added a saturated solution
of NH₄Cl (50 mL), and then zinc powder (4.61 g, 70.4 mmol,
6.0 equiv, Sigma-Aldrich, 10 μm) was added in a few portions
(a slight increase in the temperature of the reaction mixture was
detected). After 18 h, the reaction mixture was filtered through a
pad of Celite, and the aqueous phase was separated, saturated with
solid NaCl, and extracted with EtOAc (4 × 50 mL). The combined
organic extracts were dried over Na₂SO₄ and
evaporated. The residue was filtered through a pad of silica (25%
EtOAc/hexanes) and then crystallized form n-heptane
to give 21 as a light yellow solid (1.29 g, 45%): mp
82–83 °C (n-heptane); ¹H NMR
(400 MHz, CDCl₃) δ 6.89 (br s, 3H), 5.09–5.01
(m, 1H, CHOH), 4.81 (br s, 3H, NH₂ and OH), 4.64 (d, J = 2.4 Hz, 2H, CH₂), 2.51 (t, J = 2.4 Hz, 1H, C≡CH); ¹³C{¹H} NMR (100 MHz, CDCl₃) δ 152.2, 137.5,
125.2 (J_CF = 281.3 Hz), 123.1, 121.8,
117.1, 116.6, 78.7, 75.8, 72.2 (J_CF =
32.1 Hz), 56.7; ¹⁹F NMR (376 MHz, CDCl₃) δ
−77.6; HRMS (ESI) m/z calcd
for C₁₁H₁₀F₃NO₂ [M + H]⁺ 246.0742, found 246.0732.