Solid fungal extraction and C8 reversed phase chromatography

Subculture fungus onto ~40 Petri dishes of media of choice and seal the plates with parafilm. Grow at the appropriate growth temperature until the fungus reaches the required age or coverage.

Once grown, freeze plates overnight (-20°C)

Freeze dry over two days (-80°C) until plates are dry and crisp.

Weigh a clean 500ml beaker and break up dry fungal tissue into small pieces. Reweigh the beaker to obtain the dry weight of the fungus.

Submerge dry fungal tissue in methanol (MeOH) for 4 hours.

Filter into a 500ml round bottom flask.

Dry at reduced pressure on a rotary evaporator at 130 mbar.

Re-submerge fungal tissue in dichloromethane (CH₂Cl₂), cover with tinfoil and leave to soak overnight.

The following day filter into the same round bottom flask and dry at 500 mbar.

Remove some crude product from the flask for downstream biological testing.

Dissolve the remaining crude product in 10%M MeOH before loading onto C₈ reversed-phase silica gel. Elute with a gradient of H₂0:MeOH (25% increments of 100ml volume) to afford five fractions (F1-F5). Apply pump pressure and collect the fractions in 250ml round bottom flasks.

Dry the 5 fractions at reduced pressure on the rotary evaporator at 130mbar (MeOH) and 40mbar (Water).

When flasks are dry add 0.35-0.4ml of D4 MeOH to each flask (Fraction 1 isn't necessary as it will contain sugars and salts) and mix. Using a 1ml syringe and needle draw up the methanol-fungi mixture into the syringe and add it to a clean, labelled NMR tube.

Cap samples gently and load them on to the AvanceIII-400mhz instrument.

Name each of the samples and use these settings:

• Proton H+
• 64 scans
• Solvent: d4 Methanol

Label and weigh 5 empty biotesting vials for further testing. Add the contents of the NMR tubes to each of the tubes and allow to dry. Reweigh the dry tubes and label them with the mass (in milligrams). Parafilm the lids and send back for extract testing.