5. Chemistry of the Fungiflexes
We have to confess here at the outset that our research was prematurely terminated by events outside our control. So, although we were tantalisingly close to our goals, we did not achieve purification to homogeneity, nor complete the chemical characterisation. Nevertheless, we did identify a string of facts about the active agents in our bioassays that narrow the chemical focus onto a small family of molecules which are candidates to be Fungiflex 1 and Fungiflex 2.
The function of the Fungiflexes is to co-ordinate and orchestrate the behaviour of multihyphal systems. Our bioassay identified two separate activities with contrasting behaviours and different timings:
- Fungiflex 1 activity was demonstrated when mushroom stipes bent towards the side on which a drop of the extract was applied within 1 h of its application;
- the stipe grew back to the vertical in about 6 to 9 h, but then started bending away from the point of application of the drop. By 18 h from application of the drop, the stipe had bent to an angle of 90° from the vertical away from the site of application. This was Fungiflex 2 activity.
These two activities are determined by two different chemicals, sufficiently different in their basic chemistries to be separated quite readily.
Both Fungiflex 1 and Fungiflex 2 were stable to boiling for 10 min. Both were small molecules of low molecular mass, certainly less than 12000 MW. Both Fungiflex 1 and Fungiflex 2 were soluble in water and methyl alcohol but virtually insoluble in ethyl alcohol, acetone, ethyl acetate and less polar organic solvents.
- Fungiflex 1 was preferentially solubilised in methyl alcohol and was also more negatively charged than Fungiflex 2 (as determined with the positively charged resin diethylaminoethyl cellulose (DEAE) in ion-exchange column chromatography), suggesting that Fungiflex 1 is more polar than Fungiflex 2.
- The possibility exists that Fungiflex 1 may be a breakdown product or the oxidised form of Fungiflex 2.
Biological activity of both Fungiflex 1 and Fungiflex 2 was unaffected by digestion with alpha-amylase or a variety of proteinases. These observations suggest, respectively, that neither substance is likely to depend for activity on 1,4-glycosidic bonds, or peptide bonds.
Similarly, activities of both Fungiflexes survived treatment with tyrosinase + catecholase (= neither substance is a polyphenol or contains tyrosine), and digestion with nuclease or RNase (= neither substance is likely to be a single stranded nucleot(s)ide sequence).
Both Fungiflex 1 and Fungiflex 2 bioassay activities were stable to basic conditions, but Fungiflex 2 was labile in acidic conditions, suggesting that it may contain a carboxylic or activated amine group (a feature also suggested by Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) data).
Both were also stable to treatment with sodium dodecyl sulfate (cold or boiled for 5 minutes) suggesting that their activities do not depend on disulfide bonding.
Fungiflex 1 activity was stable to reducing conditions (treatment with 5 mM dithiothreitol (DTT)), but Fungiflex 2 was unstable in these conditions although it regained activity when the DTT solution was boiled for 5 min and then cooled prior to bioassaying. These results with DTT indicate, again, that Fungiflex 1 and 2 are not proteins or peptides but that Fungiflex 2 may have required an oxidative environment for activity.
Extensive spectral analyses, particularly FTIR spectroscopy and NMR spectroscopy, have given useful results.
- There is evidence for hydrogen-bonded hydroxyl groups (-OH), amide (-CONH), and possibly amine (-NH1-3) functionalities in the Fungiflexes.
- There are at least 4 different types of carbonyl (C=O) groups in Fungiflex, possibly including a carboxylic acid, a cyclic lactam (5-membered ring including N), a secondary amide or a salicylate.
- The presence of individual amino acids is also possible although not as part of a peptide chain.
- There are many hydroxyl groups and acidic protons suggesting the presence of sugar rings.
- There is no evidence for the presence of aromatic rings but there is evidence of the presence of double or triple bonds since the compound fluoresces at 415 nm when exposed to 260 nm UV.
Elemental microanalysis of crude Fungiflex suggested it contains phosphorus; there was spectral evidence for the presence of diphosphate groups. An interesting point is that P=O groups are hygroscopic and Fungiflex was also a very hygroscopic compound.
1H-NMR confirmed the presence of an amide and sugar groups, with signals characteristic of sugar skeleton protons, including both α-anomeric and β-anomeric protons, so the preparations could have contained both isomeric forms (α and β) of one sugar molecule.
In particular, signals in the 1H-NMR spectra were very characteristic of the C5 and C6 protons in the methyl pentoses, fucose (6-deoxy-L-galactose [view Wikipedia entry]) and rhamnose (6-deoxy-L-mannose [view Wikipedia entry]).
Signals in the 13C-NMR spectra were also typical for sugar skeleton carbon atoms, with peaks typical of the carbon of a deoxysugar, and possibly those characteristic of N-acetate groups.
Copyright © David Moore & Lily Novak-Frazer 2016