Propargyl Chloroformate Synthesis Essay

General methods

Chemicals and solvents were purchased from Fisher Scientific, Sigma-Aldrich or VWR International Ltd. Nuclear magnetic resonance (NMR) spectra were recorded at ambient temperature on a 500-MHz Bruker Avance III spectrometer (Bruker). Chemical shifts are reported in parts per million (p.p.m.) relative to the solvent peak. Rf values were determined on Merck TLC Silica gel 60 F254 plates (Merck) under a 254-nm ultraviolet source. Purification of compounds was carried out via manual flash column chromatography using commercially available silica gel (220–440 mesh, Sigma-Aldrich). Cell lines were grown in culture media supplemented with serum (10% fetal bovine serum) and L-glutamine (2 mM) and incubated in a Heracell 240i tissue culture incubator at 37 °C and 5% CO2. Human colorectal carcinoma HCT116 cells (a kind gift from Dr Van Schaeybroeck) were cultured in Dulbecco's Modified Eagle Media (DMEM). Human pancreas adenocarcinoma BxPC-3 cells (a kind gift from Dr Mark Duxbury) were cultured in Roswell Park Memorial Institute media. Human hepatocyte carcinoma HepG2 cells were obtained from Sigma-Aldrich and cultured in DMEM.

Synthesis and characterization of Pd0-resins

NovaSyn TG amino resin HL (1,000 mg, 0.39 mmol NH2 per g) and palladium acetate (263 mg, 1.17 mmol) were added into a 25 ml Biotage microwave vial (Biotage) with toluene (10 ml) and heated to 80 °C under stirring for 10 min. The mixture was then stirred at room temperature for 2 h and the resins subsequently filtered and washed with dichloromethane (5 × 20 ml) and methanol (5 × 20 ml). Resins were dispersed in 10% hydrazine monohydrate in methanol (10 ml) and stirred at room temperature for 25 min. The resins were then filtered and washed with methanol (5 × 20 ml) and dichloromethane (5 × 20 ml). Resins were added to a solution of Fmoc-Glu(OH)-OH (216 mg, 0.59 mmol), Oxyma (166 mg, 1.17 mmol), N,N′-diisopropylcarbodiimide (DIC) (148 mg, 1.17 mmol) and DCM/DMF (2:1, 9 ml) and stirred for 2 h at room temperature. The resins were filtered and washed with dichloromethane (5 × 20 ml), methanol (5 × 20 ml) and H2O (5 × 20 ml) and dried in an oven at 40 °C for 3 days. Complete coupling was verified by the ninhydrin test after the last methanol wash. Scanning electron microscope/transmission electron microscope images and Pd0 quantification (by inductively coupled plasma-optical emission spectrometry) were carried out as previously described8. Resins were characterized using X-ray difractometry (Bruker D2 PHASER with LYNXEYE detector (Bruker), Cu radiation source at 30 kV, 10 mA and a Ni filtre) before and after Pd0-functionalization. Without any extra preparation or post-processing, samples were mounted on a 1 inch diameter standard holder. The phase constituents were identified as per Supplementary Fig. 1c.

Synthesis and characterization of 5FU prodrugs

5FU (200 mg, 1.54 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (269 μl, 1.80 mmol) were dissolved in dry DMF (2 ml) under N2 atmosphere and cooled to 4 °C. Either allyl, propargyl or benzyl bromide (1.54 mmol) were dissolved in dry DMF (0.5 ml). The solution was added dropwise to the mixture and the resulting mixture stirred at room temperature overnight. Solvents were then removed under reduced pressure and the crude purified via flash chromatography (3% MeOH in DCM).

1-Allyl-5FU (All-5FU): colourless solid, 80 mg (31% yield); Rf=0.5 (6% MeOH in DCM); 1H NMR (500 MHz, dimethylsulphoxide (DMSO)-d6) δ=11.80 (br s, 1H, NH), 8.01 (d, J(HH)=6.7 Hz, 1H, ArH), 5.88 (ddt, J(HH)=17.0, J(HH)=10.5 Hz, J(HH)=5.5 Hz, 1H, N–CH2–CH), 5.19 (m, 2H, CH2–CH=CH2), 4.24 (d, J(HH)=5.3 Hz, 2H, N–CH2–CH); 13C NMR (126 MHz, DMSO) δ=157.45 (d, J(CF)=25.5 Hz, C), 149.46 (C), 139.68 (d, J(CF)=229.5 Hz, C), 132.63 (CH), 129.79 (d, J(CF)=33.0 Hz, CH), 117.64 (CH2), 49.34 (CH2); high-resolution mass spectrometry (HRMS) fast atom bombardment (FAB) (m/z): [M]+ calcd for C7H7O2N2F, 170.0486; found, 170.0489.

Pro-5FU: colourless solid, 104 mg (40% yield); Rf=0.35 (6% MeOH in DCM); 1H NMR (500 MHz, DMSO-d6) δ=11.91 (br s, 1H, NH), 8.13 (d, J(HH)=5.0 Hz, 1H, ArH), 4.46 (d, J(HH)=2.5 Hz, 2H, N–CH2–C), 3.44 (t, J(HH)=2.5 Hz, 1H, C≡CH); 13C NMR (126 MHz, DMSO) δ=157.35 (d, J(CF)=26.0 Hz, C), 149.11 (C), 139.80 (d, J(CF)=230.5 Hz, C), 128.95 (d, J(CF)=34.0 Hz, CH), 78.40 (C), 76.15 (CH), 37.00 (CH2); HRMS (FAB) (m/z): [M]+ calcd for C7H5O2N2F, 168.0332; found, 168.0330.

1-Benzyl-5FU (Bn-5FU): pale yellow solid, 133 mg (38% yield); Rf=0.44 (6% MeOH in DCM); 1H NMR (500 MHz, DMSO-d6) δ=11.86 (br s, 1H, NH), 8.22 (d, J(HH)=6.7 Hz, 1H, ArH), 7.39–7.28 (m, 5H, ArH), 4.83 (s, 2H, N–CH2–Ph); 13C NMR (126 MHz, DMSO-d6) δ=157.42 (d, J(CF)=25.5 Hz, C), 149.68 (C), 139.62 (d, J(CF)=228.0 Hz, C), 136.52 (C), 130.08 (d, J(CF)=33.5 Hz, CH), 128.67 (CH), 127.75 (CH), 127.49 (CH), 50.63 (CH2); HRMS (FAB) (m/z): [M]+ calcd for C11H9O2N2F, 220.0643; found, 220.0643.

Synthesis and characterization of probe 3

Rhodamine 110 chloride (250 mg, 0.68 mmol) was dissolved in dry DMF (4 ml) under nitrogen atmosphere. Separately, propargyl chloroformate (273 μl, 2.80 mmol) and triethylamine (488 μl, 3.50 mmol) were added dropwise to the mixture. The reaction mixture was stirred at room temperature for 48 h, solvents removed in vacuo and the resulting crude re-suspended in 25% isopropanol in DCM (20 ml) and washed with H2O. The aqueous layer was then washed five times with 25% isopropanol in DCM (20 ml). The combined organic layers were dried over anhydrous MgSO4, the solids filtered off and concentrated in vacuo. Crude was purified via flash chromatography (hexane/ethyl acetate 2:1) and yielded bis-N,N′-propargyloxycarbonyl-rhodamine 110 (3) as a white solid (67 mg, 20%). Rf=0.13 (hexane/ethyl acetate 2:1); 1H NMR (500 MHz, CD3OD) δ=8.02 (d, J(HH)=7.5 Hz, 1H, ArH), 7.77 (td, J(HH)=7.5 Hz, 1.1, 1H, ArH), 7.71 (td, J(HH)=7.5 Hz, 0.9, 1H, ArH), 7.61 (d, J(HH)=2.0 Hz, 2H, ArH), 7.21 (d, J(HH)=7.6, 1H, ArH), 7.08 (dd, J(HH)=8.5 Hz, 2.2, 2H, ArH), 6.68 (d, J(HH)=8.5 Hz, 2H, ArH), 4.78 (d, J(HH)=2.4 Hz, 4H, CH2–C≡CH), 2.94 (t, J(HH)=2.4 Hz, 2H, C≡CH); 13C NMR (126 MHz, MeOD) δ=171.35 (C), 154.59 (C), 154.43 (C), 153.18 (C), 142.65 (C), 136.74 (CH), 131.27 (CH), 129.47 (CH), 127.76 (C), 125.89 (CH), 125.19 (CH), 115.60 (CH), 114.42 (C), 107.04 (CH), 84.48 (C), 79.08 (C), 76.22 (C), 53.40 (CH2); mass spectrometry (electrospray ionization) (m/z): 495.2 [M+H]+.

Pd0-mediated prodrug dealkylation in biocompatible environ

All-5FU, Pro-5FU and Bn-5FU (100 μM) were dissolved in PBS (1 ml) with 1 mg of Pd0-resins and shaken at 1,400 r.p.m. and 37 °C in a Thermomixer. Reaction crudes were monitored at 0, 6, 24 and 48 h by analytical HPLC (Agilent) using the ultraviolet detector at 280 nm to avoid the detection of PBS salts. Eluent A: water and formic acid (0.1%); eluent B: acetonitrile, formic acid (0.1%); A/B=95:5 to 5:95 in 3 min, isocratic 1 min, 5:95 to 95:5 in 1 min, isocratic 1 min. Catalytic conversion assay: Pro-5FU in 0.1% (v/v) DMSO was dissolved in PBS (1 ml) with Pd0-resins (1 mg ml−1, [Pd0]~266 μM) to reach a final Pro-5FU concentration 300 μM and incubated for 24, 48, 72 h at 37 °C. Pro-5FU presence was monitored by HPLC. For the heterogeneous catalysis assay, Pd0-resins (2 mg) were dispersed into a PBS solution (2 ml) and incubated for 24 h at 37 °C. The mixture was filtrated using a Millipore microfiltre (0.22 μm, Millipore) and, subsequently, Pro-5FU added to the mixture (final concentration=100 μM). The reaction mixture was incubated for additional 24 h and analysed by HPLC (ultraviolet detector at 280 nm).

Cell viability studies of 5FU versus Pro-5FU

Cells were seeded in a 96-well plate format at the appropriate cell concentration (3,000 cells per well for HCT116 and BxPC-3 cells, and 4,000 cells per well for HepG2 cells) and incubated for 48 h before treatment. Each well was then replaced with fresh media containing 5FU (0.01 μM to 1 mM) with DMSO (0.1% v/v); or Pro-5FU (0.01 μM to 1 mM) with DMSO (0.1% v/v) and incubated for 5 days. Untreated cells were incubated with DMSO (0.1% v/v). PrestoBlue cell viability reagent (10% v/v) was added to each well and the plate incubated for 45 min. Fluorescence emission was detected using a PerkinElmer EnVision 2101 multilabel reader (Perkin Elmer; excitation filtre at 540 nm and emissions filtre at 590 nm). All conditions were normalized to the untreated cells (100%) and curves fitted using GraphPad Prism using a sigmoidal variable slope curve.

Pd0-mediated dealkylation of Pro-5FU in cell culture

HCT116 and BxPC-3 cells were seeded in a 96-well plate format with a density of 3,000 cells per well and incubated for 48 h before treatment. Each well was then replaced with fresh media containing: Pd0-resins (0.67 or 1 mg ml−1 for BxPC-3 or HCT116, respectively) with DMSO (0.1% v/v); Pro-5FU (0.01–100 μM) with DMSO (0.1%, v/v); 5FU (0.01 μM to 1 mM) with DMSO (0.1%, v/v) or combination of Pd0-resin+Pro-5FU (0.01–100 μM) with DMSO (0.1%, v/v). Untreated cells were incubated with DMSO (0.1% v/v). For the cell viability assay, cells were incubated with drugs for 5 days. PrestoBlue cell viability reagent (10% v/v) was added to each well and the plate incubated for 45 min. Fluorescence emission was detected and results normalized as described above. For the time lapse imaging study, cell growth studies were carried out at a single dose of 5FU or Pro-5FU (100 μM for HCT116 and 30 μM for BxPC-3). Each well was imaged every 2 h over 5 days under standard incubation conditions using an IncuCyte ZOOM microscope (placed inside the incubator). Imaged-based analysis of cell confluence and Supplementary Movies 1 and 2 were carried out using the IncuCyte software.

Reverse phase protein array analysis

Cells were plated in a 6-well plate at a density of 480,000 cells per well and incubated for 48 h. Before adding the prodrug to the HCT116 cells, Pro-5FU (100 μM) and Pd0-resins (1 mg ml−1) were incubated in media at 37 °C for 24 h to overcome the time delay required to convert Pro-5FU into 5FU, which was seen to have a relevant effect in this cell type. This pre-incubation procedure was not carried out for the study with BxPC-3 cells (30 μM). Pd0 resin/Pro-5FU combination and the controls (as before, untreated cells; Pd0-resins; Pro-5FU and 5FU) were incubated with each cell line for 6 and 24 h. Afterwards, the cells were washed with PBS (2 × 3 ml) and lysed with Zeptosens CLB1 lysis buffer (90 μl, Zeptosens). Samples were analysed by Zeptosens RPPA (Zeptosens) as previously described31 using the following specific conditions. Tumour cell lysates were normalized to a uniform protein concentration with spotting buffer CSBL1 (Zeptosens-Bayer) before preparing a final fourfold concentration series of 0.2, 0.15, 0.1 and 0.05 mg ml−1

Citation data is made available by participants in Crossref's Cited-by Linking service. For a more comprehensive list of citations to this article, users are encouraged to perform a search inSciFinder.

  • A Carbomethoxylated Polyvalerolactone from Malic Acid: Synthesis and Divergent Chemical Recycling

    Grant W.FahnhorstThomas R.Hoye

    ACS Macro Letters20187 (2), 143-147

    Abstract | Full Text HTML | PDF | PDF w/ Links

  • Self-Associating Poly(ethylene oxide)-block-poly(α-carboxyl-ε-caprolactone) Drug Conjugates for the Delivery of STAT3 Inhibitor JSI-124: Potential Application in Cancer Immunotherapy

    Shyam M.GargMohammad RezaVakiliOmmoleilaMolaviAfsanehLavasanifar

    Molecular Pharmaceutics201714 (8), 2570-2584

    Abstract | Full Text HTML | PDF | PDF w/ Links

  • Synthesis of Functional Polycaprolactones via Passerini Multicomponent Polymerization of 6-Oxohexanoic Acid and Isocyanides


    Macromolecules201649 (7), 2592-2600

    Abstract | Full Text HTML | PDF | PDF w/ Links

  • A Combined Experimental and Computational Study of the Substituent Effect on Micellar Behavior of γ-Substituted Thermoresponsive Amphiphilic Poly(ε-caprolactone)s

    JingHaoYixingChengR. J. K. UdayanaRanatungaSuchithraSenevirathneMichael C.BiewerSteven O.NielsenQianWangMihaela C.Stefan

    Macromolecules201346 (12), 4829-4838

    Abstract | Full Text HTML | PDF | PDF w/ Links

  • In Vitro Efficacy of Paclitaxel-Loaded Dual-Responsive Shell Cross-Linked Polymer Nanoparticles Having Orthogonally Degradable Disulfide Cross-Linked Corona and Polyester Core Domains

    SandaniSamarajeewaRituShresthaMahmoudElsabahyAmolkumarKarwaAngLiRyan P.ZentayJames G.KostelcRichard B.DorshowKaren L.Wooley

    Molecular Pharmaceutics201310 (3), 1092-1099

    Abstract | Full Text HTML | PDF | PDF w/ Links

  • Polymer Micelle with pH-Triggered Hydrophobic–Hydrophilic Transition and De-Cross-Linking Process in the Core and Its Application for Targeted Anticancer Drug Delivery


    Biomacromolecules201213 (12), 4126-4137

    Abstract | Full Text HTML | PDF | PDF w/ Links

  • Functional Polylactide-g-Paclitaxel–Poly(ethylene glycol) by Azide–Alkyne Click Chemistry


    Macromolecules201144 (12), 4793-4800

    Abstract | Full Text HTML | PDF | PDF w/ Links

  • Traceable PEO-poly(ester) micelles for breast cancer targeting: The effect of core structure and targeting peptide on micellar tumor accumulation

    Shyam M.GargIgor M.PaivaMohammad R.VakiliRaniaSoudyKateAgopsowiczAmir H.SoleimaniMaryHitt

  • 0 Replies to “Propargyl Chloroformate Synthesis Essay”

    Lascia un Commento

    L'indirizzo email non verrà pubblicato. I campi obbligatori sono contrassegnati *