Synthesis and Characterization of FAM-InsP5

Synthesis of FAM-InsP₅General chemistry experimental: All chemicals and solvents were supplied by Sigma–Aldrich and Alfa–Aesar. Unless otherwise stated, HPLC-grade solvents were used, and commercial reagents were used without further purification. Thin-layer chromatography (TLC) was performed on precoated plates (Merck TLC aluminium sheets silica 60 F₂₅₄) with detection by UV light or with phosphomolybdic acid in methanol or alkaline aqueous KMnO₄, followed by heating. Flash chromatography was performed on an ISCO CombiFlash R_f automated flash chromatography system with RediSep R_f disposable flash columns. Ion-exchange chromatography was carried out on Q Sepharose Fast Flow with a Pharmacia Biotech Gradifrac system and a P-1 pump, with elution at 5 mL min⁻¹ with gradients of aqueous triethylammonium bicarbonate (TEAB) buffer. Low-pressure reversed-phase chromatography was performed on Lichroprep RP-18 (Merck) with use of the Gradifrac system and elution at 5 mL min⁻¹ with gradients of acetonitrile in TEAB buffer (0.05 m). All water used in the purification of water-soluble polyphosphates was of MilliQ quality. During the manipulation of fluorescent compounds, light was excluded by covering reaction vessels, columns etc. with aluminium foil. RP-HPLC analyses of FAM-InsP₅ (5) were performed with a Waters 2695 Alliance module fitted with a Waters 2996 photodiode array detector (210–600 nm). The chromatographic system consisted of a Phenomenex Security Guard cartridge system for HPLC and a Phenomenex Gemini 5 μm C₁₈ 10 Å column (150×4.60 mm), with elution at 1 mL min⁻¹ with a gradient (5 % to 70 %) of acetonitrile in aqueous triethylammonium acetate (0.1 m) over 10 min, with detection at 254 nm. Proton ¹H NMR spectra were recorded with a Bruker Avance III (400 MHz) spectrometer. Proton chemical shifts are reported in ppm (δ) relative to internal tetramethylsilane (TMS, 0.0 ppm) or with the solvent reference relative to TMS employed as the internal standard ([D₆]DMSO: 2.50 ppm; D₂O: 4.79 ppm). ¹³C and DEPT spectra were recorded with a Bruker Avance III (100 MHz) spectrometer with complete proton decoupling. Carbon chemical shifts are reported in ppm (δ) relative to internal tetramethylsilane (TMS: 0.0 ppm) or with the solvent resonance relative to TMS employed as the internal standard ([D₆]DMSO: 39.51 ppm). ³¹P NMR spectra were recorded with Bruker Avance III (109 MHz and 162 MHz) spectrometers with complete proton decoupling. Phosphorus chemical shifts are reported in ppm (δ) relative to an H₃PO₄ (85 %) external standard (H₃PO₄: 0.0 ppm). Melting points were determined with a Reichert–Jung Thermo Galen Kofler block or a Stanford Research Systems Optimelt MPA100 automated melting point system and are uncorrected. Microanalysis was carried out at the University of Bath microanalysis service. Mass spectra were recorded at the SERC Mass Spectrometry Service Centre, Swansea, and at the University of Bath on VG Autospec or MicroTOF instruments.
1,6:3,4-Bis-[O-(2,3-dimethoxybutane-2,3-diyl)]-myo-inositol (6): Trimethyl orthoformate (100 mL), butanedione (25 mL, 285 mmol) and (±)-10-camphorsulphonic acid (0.5 g) were added to a stirred suspension of myo-inositol (25.0 g, 139 mmol) in MeOH (250 mL). The mixture was heated under N₂ at reflux for 96 h and then allowed to cool, giving a cherry-red suspension. The precipitate was filtered off, washed with MeOH (200 mL) and allowed to dry, giving crude diol 6 as a white solid (22.4 g, 85 % pure by ¹H NMR). This material was crystallised from boiling CHCl₃/MeOH (1:1, v/v, 650 mL) and dried under vacuum at 60 °C to give 6 as colourless crystals (15.4 g, 37.7 mmol, 27 %); m.p. >300 °C with sublimation and decomposition; R_f=0.24 (EtOAc); R_f=0.29 (CHCl₃/acetone 2:1); ¹H NMR (400 MHz, [D₆]DMSO): δ=1.17 (s, 6 H; CH₃), 1.18 (s, 6 H; CH₃), 3.13 (s, 6 H; OCH₃), 3.15 (s, 6 H; OCH₃), 3.25 (dt, ³J=5.6, 9.3 Hz, 1 H; H-5), 3.35 (dd, ³J=10.2, 2.4 Hz, 2 H; H-1, H-3), 3.67 (dd, ³J=9.8, 9.8 Hz, 2 H; H-4, H-6), 3.76 (dt, ³J=4.6, 2.4 Hz, 1 H; H-2), 4.99 (d, ³J=4.8 Hz, 1 H; OH-2), 5.05 ppm (d, ³J=5.6 Hz, 1 H; OH-5); ¹³C NMR (100 MHz, [D₆]DMSO): δ=17.64 (CH₃), 47.12 (OCH₃), 47.42 (OCH₃), 67.57 (C-2), 68.37 (C-1, C-3), 69.13 (C-5), 69.33 (C-4, C-6), 98.41 (BDA quaternary C), 98.97 ppm (BDA quaternary C); HRMS: m/z calcd for C₁₈H₃₂O₁₀: 431.1888 [M+Na]⁺; found: 431.1880; elemental analysis calcd (%) for C₁₈H₃₂O₁₀: C 52.93, H 7.90; found: C 52.6, H 7.92.
5-O-Benzoyl-1,6:3,4-bis-[O-(2,3-dimethoxybutane-2,3-diyl)]-myo-inositol (7): A solution of diol 6 (2.04 g, 5.00 mmol) in anhydrous pyridine (20 mL) was stirred at 0 °C under N₂. Benzoic anhydride (1.24 g, 5.5 mmol) was added, followed by a catalytic amount of DMAP (60 mg, 0.50 mmol). The cooling bath was removed, and the solution was allowed to reach room temperature. After 18 h, the solution was diluted with CH₂Cl₂ (200 mL), washed well with HCl (1.0 m, 2×250 mL) and saturated NaHCO₃ solution (100 mL) and then dried (MgSO₄) and concentrated under reduced pressure. The residue was purified by flash chromatography on silica (EtOAc in CH₂Cl₂, 0 to 40 %) to give the 5-O-benzoate ester 7 as a white solid (2.15 g, 4.19 mmol, 84 %); crystals from boiling EtOH, m.p. 292.5–295.5 °C; R_f=0.24 (CH₂Cl₂/EtOAc 5:1); ¹H NMR (400 MHz, CDCl₃): δ=1.19 (s, 6 H; CH₃), 1.32 (s, 6 H; CH₃), 2.44 (br s, 1 H; 5-OH), 3.13 (s, 6 H; OCH₃), 3.25 (s, 6 H; OCH₃), 3.72 (dd, ³J=10.2, 2.6 Hz, 2 H; H-1, H-3), 4.10 (t, ³J=2.6 Hz, 1 H; H-2), 4.26 (dd, ³J=10.1, 9.9 Hz, 2 H; H-4, H-6), 5.39 (t, ³J=9.9 Hz, 1 H; H-5), 7.41–7.45 (m, 2 H; meta-H of Bz), 7.55 (tt, ³J=7.4, 1.3 Hz, 1 H; para-H of Bz), 8.05–8.08 ppm (m, 2 H, ortho-H of Bz); ¹³C NMR (100 MHz, CDCl₃): δ=17.61 (CH₃), 17.64 (CH₃), 47.58 (OCH₃), 48.00 (OCH₃), 67.28 (C-4, C-6), 68.63 (C-1, C-3), 68.95 (C-2), 70.96 (C-5), 99.33 (BDA quaternary C), 100.13 (BDA quaternary C), 128.37 (Bz meta-C), 129.59 (Bz ortho-C), 130.40 (Bz ipso-C), 132.76 (Bz para-C), 165.16 ppm (Bz C=O); HRMS: m/z calcd for C₂₅H₃₆O₁₁: 535.2150 [M+Na]⁺; found: 535.2153; elemental analysis calcd for C₂₅H₃₆O₁₁: C 58.58, H 7.08; found: C 58.5, H 7.14.
5-O-Benzoyl-2-O-cyanomethyl-1,6:3,4-bis-[O-(2,3-dimethoxybutane-2,3-diyl)]-myo-inositol (8): Sodium hydride (468 mg of a 60 % suspension in mineral oil, 11.7 mmol) was added under N₂ to a suspension of 7 (2.00 g, 3.90 mmol) in dry acetonitrile (20 mL). The suspension was stirred at room temperature for 30 min. The suspension first became almost clear, and then thickened as evolution of gas ceased. The suspension was then cooled to −30 °C (acetonitrile/solid CO₂ bath), and bromoacetonitrile (1.5 mL, 23 mmol) was added dropwise over 2 min. The suspension was stirred at −20 °C for 3.5 h and then allowed to reach room temperature overnight. The resulting brown suspension was concentrated under reduced pressure to give a solid residue, which was dispersed in dichloromethane (100 mL in portions with use of an ultrasound bath) and filtered through Celite, leaving a pale yellow solution. The solution was concentrated, and the residue was purified by flash chromatography (EtOAc in CH₂Cl₂, 0 to 50 %) to give 8 as a white solid (1.83 g, 3.32 mmol, 85 %); m.p. 264–266 °C (from EtOH); R_f=0.54 (CH₂Cl₂/EtOAc 5:1); ¹H NMR (400 MHz, CDCl₃): δ=1.17 (s, 6 H; CH₃), 1.30 (s, 6 H; CH₃), 3.13 (s, 6 H; OCH₃), 3.25 (s, 6 H; OCH₃), 3.76 (dd, ³J=10.3, 2.4 Hz, 2 H; H-1, H-3), 4.01 (t, ³J=2.4 Hz, 1 H; H-2), 4.14 (dd, ³J=10.2, 10.0 Hz, 2 H; H-4, H-6), 4.69 (s, 2 H; CH₂CN), 5.37 (t, ³J=9.8 Hz, 1 H; H-5), 7.42–7.45 (m, 2 H; meta-H of Bz), 7.55 (tt, ³J=7.2, 1.2 Hz, 1 H; para-H of Bz), 8.05–8.08 ppm (m, 2 H; ortho-H of Bz); ¹³C NMR (100 MHz, CDCl₃): δ=17.57 (CH₃), 17.58 (CH₃), 47.64 (OCH₃), 48.09 (OCH₃), 56.77 (OCH₂CN), 67.43 (C-4, C-6), 68.68 (C-1, C-3), 71.03 (C-5), 76.22 (C-2), 99.27 (BDA quaternary C), 99.97 (BDA quaternary C), 116.28 (OCH₂CN), 128.39 (Bz meta-C), 129.62 (Bz ortho-C), 130.29 (Bz ipso-C), 132.83 (Bz para-C), 165.16 ppm (Bz C=O); HRMS: m/z calcd for C₂₇H₃₇NO₁₁: 574.2259 [M+Na]⁺; found: 574.2269; elemental analysis calcd for C₂₇H₃₇NO₁₁: C 58.79, H 6.76, N 2.54; found: C 58.4, H 6.85, N 2.48.
1,6:3,4-Bis-[O-(2,3-dimethoxybutane-2,3-diyl)]-2-O-[2-(2,2,2-trifluoroacetylamino)ethyl]-myo-inositol (9): A solution of 8 (1.78 g, 3.23 mmol) in dry THF (25 mL) was added dropwise over 30 min, under N₂ at 0 °C, to a solution of LiAlH₄ in THF (10 mL of a 1.0 m solution, 10 mmol). The mixture was stirred at room temperature for a further 1 h and then quenched by careful addition of a saturated solution of potassium sodium tartrate (75 mL). Ether (75 mL) was then added, and the mixture was stirred vigorously for 30 min until two distinct layers formed. The ether layer was separated, and the aqueous layer was re-extracted with ether (2×100 mL). The combined organic extracts were dried (MgSO₄) and concentrated to give the crude amine (1.8 g, R_f=0.16 in CH₂Cl₂/MeOH/NH₃, 200:20:1) as a foam, which was taken up in dry THF (10 mL) and stirred with ethyl trifluoroacetate (2 mL, 8.4 mmol) at room temperature. After 2 h, the solution was concentrated, and the residue was purified by flash chromatography (ethyl acetate in petroleum ether, 0 to 100 %) to give 9 as a white solid (1.16 g, 2.12 mmol, 66 %); m.p. 206–208 °C (from EtOAc/petroleum ether); R_f=0.25, (CH₂Cl₂/EtOAc 1:1); ¹H NMR (400 MHz, CDCl₃): δ=1.29 (s, 6 H; CH₃), 1.32 (s, 6 H; CH₃), 2.62 (br s, 1 H; 5-OH), 3.24 (s, 6 H; OCH₃), 3.28 (s, 6 H; OCH₃), 3.54 (dd, ³J=10.2, 2.6 Hz, 2 H; H-1, H-3), 3.54–3.57 (m, 2 H; OCH₂CH₂N), 3.62 (t, ³J=2.6 Hz, 1 H; H-2), 3.65 (td, ³J=9.5, 2.1 Hz, 1 H; H-5), 3.81 (dd, ³J=5.1, 4.7 Hz, 2 H; OCH₂CH₂N), 3.94 (dd, ³J=10.0, 9.8 Hz, 2 H; H-4, H-6), 7.32 ppm (br t, 1 H; NHC(O)CF₃); ¹³C NMR (100 MHz, CDCl₃): δ=17.50 (CH₃), 17.69 (CH₃), 39.52 (OCH₂CH₂N), 47.96 (OCH₃), 48.06 (OCH₃), 68.47 (C-1, C-3), 69.26 (C-4, C-6), 70.11 (C-5), 70.80 (OCH₂CH₂N), 77.46 (C-2), 99.27 (BDA quaternary C), 99.89 (BDA quaternary C), 115.99 (q, ²J_H,F=288 Hz; CF₃), 157.24 ppm (q, ³J_H,F=37 Hz; C(O)CF₃); HRMS: m/z calcd for C₂₂H₃₆F₃NO₁₁: 570.2133 [M+Na]⁺; found: 570.2151; elemental analysis calcd for C₂₂H₃₆F₃NO₁₁: C 48.26, H 6.63, N 2.56; found: C 48.1, H 6.52, N 2.41.
2-O-[2-(2,2,2-Trifluoroacetylamino)ethyl]-myo-inositol 1,3,4,5,6-pentakis(dibenzylphosphate) (10): Compound 9 (450 mg, 0.822 mmol) was dissolved in aqueous TFA (95 %, 10 mL). The solution was stirred at room temperature for 20 min and then concentrated by evaporation under reduced pressure. Ethanol was added and evaporated several times to remove traces of TFA. The solid pentaol product (R_f=0.20, CH₂Cl₂/MeOH 3:1) was dried under vacuum for 16 h and then suspended in dry dichloromethane (10 mL). The suspension was stirred at room temperature under N₂, and 5-phenyltetrazole (900 mg, 6.16 mmol) was added, followed by bis(benzyloxy)diisopropylaminophosphine (1.7 mL, 4.9 mmol). The mixture was stirred at room temperature for 2 h, after which time a clear solution remained. The solution was cooled to −78 °C, and 3-chloroperoxybenzoic acid (57 %, 2.5 g, 8.2 mmol) was added in portions over 1 min. A precipitate formed during the oxidation reaction. The resulting suspension was allowed to warm to room temperature and diluted with EtOAc (50 mL), giving a clear solution. The solution was washed with aqueous sodium sulfite solution (10 %, 2×50 mL), dried over MgSO₄ and concentrated, leaving an oily residue, which was purified by flash chromatography with elution with acetone in dichloromethane (0 to 50 %) to give 10 (1.17 g, 0.722 mmol, 88 %) as a colourless oil; R_f=0.20 (CH₂Cl₂/acetone 5:1); ¹H NMR (400 MHz, CDCl₃): δ=3.31–3.34 (m, 2 H; OCH₂CH₂N), 3.69 (t, ³J=4.6 Hz, 2 H; OCH₂CH₂N), 4.25 (ddd, ³J_H,H=9.5, 2.2 Hz, ³J_H,P=9.5 Hz, 2 H; H-1, H-3), 4.40–4.49 (m, 2 H; H-2, H-5), 4.92–5.03 (m, 22 H; CH₂Ph, H-4, H-6), 7.15–7.29 (m, 50 H; Ph), 8.06 ppm (broad, 1 H; amide NH); ¹³C NMR (100 MHz, CDCl₃): δ=40.09 (OCH₂CH₂N), 69.59–69.96 (with ³J_C,P couplings; OPOCH₂Ph), 71.04 (OCH₂CH₂N), 74.67, 75.24, 75.49 and 75.86 (broad signals with J_C,P couplings; inositol ring CH), 115.95 (¹J_C,F=288 Hz; CF₃), 127.96–128.71 (CH of Ph), 135.21–135.80 (ipso-C of POCH₂Ph), 157.62 ppm (²J_C,F=36.8 Hz; C(O)CF₃); ¹⁹F NMR (376 MHz, CDCl₃): δ=−75.14 ppm; ³¹P NMR (162 MHz, CDCl₃): δ=−2.05 (2 P), −1.45 (1 P), −1.43 ppm (2 P); HRMS: m/z calcd for C₈₀H₈₁F₃NO₂₂P₅: 1618.3818 [M]⁻; found: 1618.3788.
2-O-(2-Aminoethyl)-myo-inositol 1,3,4,5,6-pentakisphosphate (4): Palladium hydroxide on activated charcoal (Fluka, 20 %, 50 % water, 100 mg) was added to a solution of 10 (380 mg, 0.235 mmol) in MeOH (30 mL) and deionised water (8 mL). The suspension was stirred vigorously under hydrogen (balloon) for 16 h. The catalyst was removed by filtration through a PTFE syringe filter (0.2 μm) to give a colourless solution, which was neutralised by addition of N,N-diisopropylethylamine (DIPEA, 0.5 mL). The solvents were then removed by evaporation under reduced pressure. A ¹H NMR spectrum of the product in D₂O at this stage showed no residual aromatic signals, thus indicating that hydrogenolysis was complete. The product was redissolved in deionised water (1 mL), excess DIPEA (1 mL) was added, and the solution was heated under N₂ at 60 °C for 20 h. The solution was concentrated, and the residue was redissolved in deionised water and lyophilised to give the diisopropylethylammonium salt of 4 as a fawn solid (325 mg); ¹H NMR (400 MHz, D₂O): δ=1.18–1.22 (m, 75 H; DIPEA CH₃), 3.07 (q, ³J=7.4 Hz,10 H; DIPEA CH₂), 3.13 (t, ³J=5.1 Hz, 2 H; OCH₂CH₂NH₃⁺), 3.58 (heptet, ³J=6.7 Hz,10 H; DIPEA CH), 3.96 (t, ³J=5.1 Hz, 2 H; OCH₂CH₂NH₃⁺), 4.02–4.08 (m, 3 H; H-1, H-3, H-5), 4.11 (t, ³J=2.5 Hz, 1 H; H-2), 4.38 ppm (q, ³J=9.7 Hz, 2 H; H-4, H-6); ¹³C NMR (100 MHz, D₂O): δ=12.11 (DIPEA CH₂CH₃), 16.22 (DIPEA CH(CH₃)₂), 17.69 (DIPEA CH(CH₃)₂), 39.52 (OCH₂CH₂NH₃⁺), 42.51 (DIPEA CH₂CH₃), 54.32 (DIPEA CH(CH₃)₂), 69.37 (OCH₂CH₂NH₃⁺), 73.89 and 76.13 (C-1, C-3, C-4, C-6), 77.27 (C-5), 79.17 ppm (C-2); ³¹P NMR (162 MHz, D₂O): δ=−0.39 (2 P), 0.63 (2 P), 0.80 ppm (1 P; P-5); HRMS: m/z calcd for C₈H₂₂NO₂₁P₅: 621.9300 [M]⁻; found: 621.9311.
This material was used in subsequent conjugation reactions, but for biological evaluations of 4, a portion was purified by ionexchange chromatography on Q Sepharose Fast Flow resin with elution with TEAB (0 to 2.0 m) to give the triethylammonium salt of 4, which was accurately quantified by total phosphate assay.
2-O-[2-(5-Fluoresceinylcarboxy)aminoethyl]-myo-inositol 1,3,4,5,6-pentakisphosphate (5): Dry DIPEA (10 μL) was added to a suspension of 4 (20 mg DIPEA salt, 14 μmol) in dry propan-2-ol. Solid 5-carboxyfluorescein NHS ester[14 ] (13 mg, 28 μmol) was added to the resulting clear solution, followed by further dry DIPEA (60 μL). The flask was covered in foil to exclude light, and the reaction mixture was stirred under N₂ at 60 °C for 24 h and was then allowed to cool and concentrated under reduced pressure. The residue was dissolved in TEAB (0.05 m, pH approx. 7.5, 5 mL) and applied to a column of Q Sepharose Fast Flow resin (bicarbonate form, 70 mm×20 mm). The column was washed well with milliQ water, followed by TEAB (0.8 m, pH approx. 7.8) until the eluent ran colourless. This required approximately 400 mL of buffer. The column was then eluted with a gradient of TEAB (0.8 to 2.0 m, over 300 mL), with collection of 10 mL fractions. A fluorescent product eluted at high buffer concentration (>1.6 m TEAB). Fractions containing this product were combined and concentrated to give an orange solid, which was re-dissolved in TEAB (0.05 m, pH approx. 7.5, 5 mL) and applied to a small column (100 mm×10 mm) of Lichroprep RP-18. The column was eluted with a gradient of acetonitrile (0 to 30 % in 0.05 m TEAB over 300 mL), with collection of 10 mL fractions. Fluorescent fractions were combined and concentrated to leave a solid residue, which was re-dissolved in milliQ water and lyophilised to give the pure triethylammonium salt of 5 (containing 4.5 Et₃NH⁺ per equiv of 5) as a fluffy orange solid (16 mg, 11 μmol, 79 %); ¹H NMR (400 MHz, D₂O): δ=1.12 [t, ³J=7.5 Hz, approx. 40 H; (CH₃CH₂)₃NH⁺], 3.03 [q, ³J=7.5 Hz, approx. 27 H; (CH₃CH₂)₃NH⁺], 3.61 (t, ³J=5.1 Hz, 2 H; OCH₂CH₂NH), 3.97 (t, ³J=5.1 Hz, 2 H; OCH₂CH₂NH), 4.04–4.14 (m, 3 H; H-1, H-3, H-5), 4.19 (br s, 1 H; H-2), 4.41 (dt, ³J_H,P=9.4 Hz, ³J_H,H=9.4 Hz, 2 H; H-4, H-6), 6.65–6.68 (m, 4 H; fluorescein H-2′, H-4′, H-5′, H-7′), 6.99 (d, ³J=9.7 Hz, 2 H; fluorescein H-1′, H-8′), 7.43 (d, ³J=8.2 Hz, 1 H; fluorescein H-7), 8.13 (dd, ³J=8.2 Hz, ⁴J=1.9 Hz, 1 H; fluorescein H-6), 8.31 ppm (d, ⁴J=1.9 Hz, 1 H; fluorescein H-4); ¹³C NMR (100 MHz, D₂O): δ=8.14 [(CH₃CH₂)₃NH⁺], 40.34 (OCH₂CH₂NH), 46.53 [(CH₃CH₂)₃NH⁺], 71.67 (OCH₂CH₂NH), 74.10 and 76.29 (C-1, C-3, C-4, C-6), 77.49 (C-5), 78.62 (C-2), 102.64, 112.66, 116.55, 126.60, 127.77, 130.97, 131.45, 132.48, 135.97, 144.62, 155.23, 165.24, 169.21 (C=O), 170.98 ppm (C=O); ³¹P NMR (109 MHz, CD₃OD): δ=0.97 (2 P), 1.89 (2 P), 2.21 ppm (1 P; P-5); HRMS: m/z calcd for C₂₉H₃₁NO₂₇P₅⁻: 979.9777 [M]⁻; found: 979.9741; analytical RP HPLC: t_R=4.40 min (see General Chemistry Experimental).
Cell lines and cell culture: NCI-H1299 (H1299), HCT-116, CaCo-2, MDA-MDB-231, MCF-7 and Mevo cells were purchased from ATCC. Skin fibroblasts cells were a gift from Ulla Kasten-Pisula (Hamburg, Germany), and PT4323 (primary tumour) and LN2343 (the corresponding lymph node metastasis) were both freshly isolated from primary lung adenocarcinomas (for details see ref. [8 (link)]). The well-established cell line H1299 derives from a lymph node metastasis of lung adenocarcinoma cells. HCT-116 and CaCo-2 are colon cancer cells, MDA-MDB-231 and MCF-7 are breast cancer cells, and Mevo are melanoma cells. The cell lines MDA-MDB-231, NCI-H1299 and HCT-116 were cultured in Dulbecco's modified Eagle's medium (DMEM), Mevo and fibroblasts were grown in RPMI, PT4323 and LN2343 in RPMI with supplements (see ref. [8 (link)]) and CaCo-2 in MEM alpha medium. All media were supplemented with foetal calf serum (FCS, 10 %, v/v), l-glutamine (4 mm), streptomycin (100 μg mL⁻¹) and penicillin (100 U mL⁻¹) and were purchased from Invitrogen.
Analysis of cellular uptake of FAM-InsP₅ (5) in intact cells by fluorescence microscopy: Cells were grown on poly-l-lysine-covered cover slips to 20, 50 or 80 % confluence, and 5 or fluorescein (TEA⁺ salt in each case, 20 μm) as control (both diluted in deionised water) was added. After incubation for different times, the cells were washed three times with phosphate saline (PBS), fixed with paraformaldehyde (3 %), washed again three times with PBS and finally embedded in Fluoromount-G (Southern Biotech, Birmingham, Alabama. USA). Uptake of 5 was analysed by performing z-stacks (about 50 stacks per image of 1 to 1.5 μm in size) of bright and fluorescence light with a BZ-9000 E microscope from Keyence (Neu Isenburg, Germany). After overlay of these z-stacks, a 3D analysis was performed (software: BZ-H1RE), and the position of 5 was determined in xz- and in xy-layers of single stacks. Positioning of 5 in the middle cell layers was defined as cellular uptake, whereas accumulation in the first layers was defined as cell surface localisation of 5. The micrographs shown here represent focus stackings of bright and fluorescence light overlays by using software (BZ-H1RE) that selects the sharpest areas from multiple frames. Every experiment was performed in triplicate, and at least 100 cells were analysed per experiment.
Analysis of cellular uptake of FAM-InsP₅ (5) in lysed cells by fluorescence photometry: H1299 cells grown to about 80 % confluence in 10 cm dishes were treated with 5 (25 μm) for 3 h. The cells were then washed five times with PBS, MPER buffer (1.1 mL) was added, the cells were scraped, and the suspension was frozen in N₂ and thawed twice. The resulting suspension was diluted 1:1 with PBS, and the fluorescence was analysed with a Tecan Infinitive M100 fluorescence reader at excitation 490 nm and emission 535 nm. To prepare a standard curve, different concentrations of 5 were added to untreated lysed cells and, after cautious mixing, the fluorescence of the standards was measured in parallel to the samples as well as to the washing fractions. To analyse bleaching, a solution of 5 was added to cell suspension or to PBS, and fluorescence was analysed as above over a time period of 3 h. Under both sets of conditions (cell suspension and PBS) we measured 20 % loss by bleaching.
Immunofluorescence: Cells preincubated with 5 for 16 h in chamber slides were washed twice with PBS, fixed with paraformaldehyde (3 %) for 10 min, washed three times with PBS and treated with Triton-100 (0.3 %) for 5 min at RT. After washing of the cells three times with PBS they were blocked in BSA/PBS (Sigma—Aldrich, 2.5 %) for 20 min, incubated with antibodies against early endosome antigen 1 (EEA1; Abcam, ab2900, Cambridge, UK) or lysosome-associated membrane protein-2 (LAMP-2; Santa Cruz #sc-5571), respectively, at a dilution of 1:200 in BSA/PBS (0.7 %, w/v) for 16 h at 8 °C. After having been washed again three times with PBS, the cells were finally treated with anti-rabbit secondary antibodies coupled to Alexa Fluor 568 at a dilution of 1:1000 for 1 h at 22 °C. After the cells had been washed with PBS, images were captured on a fluorescence microscope (Keyence BZ-9000).