PHAL 241 - LECTURE 5

What is fractionation

• it is the method for seperating metabolites within an extract

what is fractionation dependant on

• dependant on the structural and chemical differences in the metabolites - eg polarity, pH and size

what is chromatography

• a method of fractionation - seperates metabolites with similar features - metabolites are seperated according to the strength of their interactions with different phases

what are the two main types of chromatography

• liquid chromatography - column chromatography - size exclsuion chromatography

explain liquid-liquid chromatography

• the key idea is that 'like dissolves like' - extracts are mixed into solvents - one thats polar and one thats less polar - when the extracts are added, the polar metabolites will be attracted to the polar solvents whereas the nonpolar metabolites will be attracted to the nonpolar solvents and so two distinct layers will be formed

what is column chromatography

• similar to liquid chromatography but allows us to seperate a larger range of fractions - Columns contain a polar gel which is stationary and a less polar solvent which is moble - the less polar metabolites move with the solvent through the column faster than the polar metabolites which will stay with the gell - metabolites with similar polarity are collected as they leave the column

what is size exclusion chromatography

• extracted seperated into metabolites by size

explain how size exclusion chromatography works

• the extract and solvent are added to a column with porous beads (with holes in them) - large metabolites move with the solvent because they cant fit into the holes of the beads - the smaller metabolites enter the beads and so they go through the column slower - metabolites of similar size are collected as they leave the column

what happens once a bioactive extract or fraction has been found

• bioactivity screening (antioxidant screening, toxicity screening etc)

how does chromatography aid in finding the metabolite responsible for bioactivity

• it is used to purfiy and increase the metabolite abundance - it is used in combination with bioactivity screening - once the metabolite of interest is determined, chromatography is used to isolate the single active metabolite

how is the metabolite structure found (once the one responsible for the bioactivity is found)

• spectroscopy

what is spectroscopy

• technique used to identify metabolites based on their specific pattern of light absorption or radiation

what are 2 examples of spectroscopy

• infrared spectroscopy - nuclear magnetic resonance spectroscopy

how does infrared spectroscopy work

• metabolites absorb infrared light at different wavelenghts - light is passed through them, and the wavelengths of light that arent absorbed produce a spectra - functional groups in the metabolites appear as downward peaks (because they have been absorbed and so fall below the baseline) - the spectra are compared to standards (known compounds) or used to figure out the structure of unknown metabolites

how does NMR work

• when specific nuclei are put in a mag field, they change in spin - if we put radio waves through it, the nuclei absorbs the energy until it reaches resonance state - the energy then gets released - we detect the change in energy back down to normal which is the emission frequency - depending on what the nuclei had around them (ie what functional group they were a part of), they will release a different energy which we can detect and compare

what do we need to turn a metabolite to a drug

• we need to be able to produce that metabolite at scale - large amounts of it - this requires commercial production and harvest of biological material - extraction and isolation of the metabolite

what is the best approach to producing large amounts of metabolites for drug production

• chemical synthesis

what is chemical synthesis

• chemical reactions to create copies of natural metabolites

what is the overall flow of natural product drug discovery

collect organism Extract metabolites Fractionate extract (separate compounds) Test fractions for biological activity Isolate active metabolite Determine chemical structure (spectroscopy) Synthesise and optimise compound