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Electronic supplementary material



High Levels of Acrylate in the Great Barrier Reef Coral Acropora millepora

D. M. Tapiolas, C. A. Motti, P. Holloway, S. G. Boyle



Australian Institute of Marine Science, PMB No 3, Townsville MC, QLD, 4810, Australia
D. M. Tapiolas (Corresponding author); C. A. Motti; S. G. Boyle

Australian Institute of Marine Science, PMB 3,

Townsville, QLD, 4810, Australia

e-mail: d.tapiolas@aims.gov.au


P. Holloway

On leave from Department of Biology,

University of Winnipeg,

Manitoba, R3B 2E9, Canada



Table of Contents


Additional experimental details

NMR data in CD3OD and D2O:-


Figure S1. 1H NMR spectrum of MeOH extract of Acropora millepora with 1H NMR spectrum of commercially obtained Na acrylate inserted for comparison

Figure S2. 13C NMR spectrum of acrylate from Acropora millepora

Figure S3. COSY spectrum of acrylate from Acropora millepora

Figure S4. HSQC spectrum of acrylate from Acropora millepora

Figure S5. HMBC spectrum of acrylate from Acropora millepora

Figure S6. 1H NMR spectrum of the methanol extract of Acropora spathulata



Figure S7. 1H NMR spectrum of the methanol extract of Acropora tenuis

Experimental

Chemicals


Compressed gases were supplied by BOC Gases (Townsville, Australia) and were at least 99.99% pure. Purified water was obtained from a MilliQ water purification system (Millipore, MA, USA). Extraction and chromatography solvents were HPLC grade (Mallinckrodt, MO, USA). Deuterated NMR solvents (Cambridge Isotope Laboratories Inc) were sourced from Novachem Australia (Victoria, Australia), DMSP was supplied by Research Plus Inc., (Barnegat, NJ, USA), bleach (NaHClO) was purchased from Kwikmaster Products (WA, Australia) and reversed phase-C18 silica gel was from Phenomenex Australia (Lane Cove, NSW, Australia). All other chemicals were sourced from Sigma Aldrich (Australia).

NMR and MS Analyses


NMR spectra were recorded on either a Bruker Avance 600 NMR spectrometer with cryoprobe or Bruker Avance 300 MHz NMR spectrometer using standard Bruker pulse sequences. Deuterated MeOH (CD3OD), deuterated chloroform (CDCl3) or deuterated water (D2O) was used as solvent and the spectra were referenced to the residual 1H and 13C resonances. Mass spectra measurements were performed on an unmodified Bruker BioAPEX 47e FT-ICR mass spectrometer (FTMS) equipped with an Analytica of Branford model 103426 (Branford, CT) electrospray ionisation (ESI) source in positive mode (cylinder = -1.9 kV, source = -4.0 kV and end cap = -3.5 kV, capillary exit = 140 V and skimmer potential = 10 V). Sodium acrylate was dissolved in MeOH (2 mg ml-1) and diluted 1:9 in MeOH (0.2 mg ml-1). The crude extract was dissolved in H2O (20 mg ml-1) and diluted 1:9 into MeOH (2 mg ml-1). Direct infusion was carried out using a Cole Palmer 74900 syringe pump at a rate of 100 l h-1. The instrument was calibrated using a methanolic solution of CF3COONa (0.1 mg ml-1 MeOH).

qNMR Analyses


For all qNMR experiments, CD3OD/D2O (1:1) containing 0.025% benzene as internal standard was used as solvent. An acrylate standard curve was generated using sodium acrylate. A stock solution of 40 mM sodium acrylate was prepared in CD3OD/D2O (1:1) containing 0.025% benzene, and serial dilutions were performed into CD3OD/D2O (1:1) containing 0.025% benzene to give eight concentrations; (20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156 mM) of sodium acrylate in CD3OD/D2O (1:1) containing 0.025% benzene as external standard. Spectra were acquired non-spinning, with a sweep width of 13ppm, a 90° pulse to maximize sensitivity, a relaxation delay of 43 sec, 2 dummy scans and 16 acquisition scans. The data were Fourier transformed after line broadening of 0.3 Hz and the resulting FID was zero filled to twice the number of points acquired. The spectra were phased manually and the baseline corrected with a fifth-order polynomial.

1D and 2D NMR data

F

Figure S1. 1H NMR spectrum of MeOH extract of Acropora millepora with 1H NMR spectrum of commercially obtained Na acrylate inserted.



Figure S2. 13C NMR spectrum of acrylate from Acropora millepora


Figure S3. COSY spectrum of acrylate from Acropora millepora


Figure S4. HSQC spectrum of acrylate from Acropora millepora



Figure S5. HMBC spectrum of acrylate from Acropora millepora



Figure S6. 1H NMR spectrum of the methanol extract of Acropora spathulata



Figure S7. 1H NMR spectrum of the methanol extract of Acropora tenuis





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