Desmodium incanum, DC.

Hao, B., Caulfield, J. C., Hamilton, M. L., Pickett, J. A., Midega, C. A. O., Khan, Z. R., Wang, J. & Hooper, A. M., 2016, Biosynthesis of natural and novel C-glycosylflavones utilising recombinant Oryza sativa C-glycosyltransferase (OsCGT) and Desmodium incanum root proteins, Phytochemistry 125, pp. 73-87 : 76

2.5. Synthesis of 6- and 8-C-glucosylflavones from synthetic 2- hydroxyflavanones by D. incanum soluble root proteins

In our previous work demonstrating the biosynthesis of allelopathic C -glycosylflavonoids found in the root exudates of Desmodium spp ., we demonstrated C -glucosylation in this genus requires a 2-hydroxyflavonone substrate ( Hamilton et al., 2009). We therefore challenged the soluble root proteins of D. incanum with the synthetic 2-hydroxyflavanones to assess whether there are significant differences in the capacity of D. incanum to C -glucosylate 2-hydroxyflavanones compared to the rice recombinant OsCGT protein ( Table 3 ). Where standards were not available from the experiments with OsCGT which allowed NMR characterisation, the two products were analysed by high cone voltage ESIMS experiments to verify the C -glucosyl link to the flavonoid core through the loss of 90 and 120 amus ( Fig. 1 ) ( Cuyckens and Claeys, 2004) (Supplementary data). An example of the mass spectrometric analysis is shown for incubation assays with 3h (3 0 ,4 0 -methylenedioxy) ( Fig. 2 ) and in this case the products are fully characterised as 5h and 6h. The differences between the two systems are small but relevant. OsCGT cannot C -glucosylate the sterically bulky 3n (t -butyl) and 3q (phenyl) while D. incanum can, implying a relaxed steric constraint. However, this is minimal as 3p (hexamethylenoxyl) still cannot be C -glycosylated by D. incanum as is the case with OsCGT. Minor differences include the acceptance by OsCGT of a brominated substrate 3v although at a very low level and 3i (3 0 ,5 0 -hydroxy) which was surprisingly not C -glycosylated by D. incanum proteins. A significant difference was the ability of D. incanum proteins to turn over chlorinated substrates which would allow the potential for generating chlorinated biologically active synthons for cross-coupling. These results demonstrate that crude D. incanum soluble protein has a different, though very similar capacity to accept novel substrates. These differences may be due to a single protein having a less sterically hindered substrate specificity for the substrate B-ring (3n and 3q) along with some different electronic requirements (3i, 3v, 3w, and 3x) or it may be due to different C -glycosyltransferases with a combined greater range of acceptable chemical space for 2-hydroxyflavanones. In any case, the plant has a greater capacity for producing novel C -glucoslyflavones than those possible using the single rice recombinant OsCGT.

Figures

Fig. 1. Fragmentation of C-hexosides and C-pentosides under high cone voltage ESIMS conditions to generate fragments losses of 60, 90 and 120 amus.

Fig. 2. (A) Ion scan for [M—H] — 459 showing two new metabolites found in (B) the LCMS ion trace. (C) Molecular ions of two new metabolites and (D) fragmentation under high cone voltage showing C-link fragments.