Figure 3.

The progress of the enzymatic cascade outlined in Scheme 2 was followed by ³¹P NMR spectroscopy to confirm the generation of a) [2-¹³C₁]-PnAA from [2-¹³C]-PEP by the successive actions of RhiH and PnPy decarboxylase, b) 2-[2-¹³C₁, 2-²H₁]-HEP via the reduction of [2-¹³C₁]-PnAA by DhpG in the presence of (4R)-NADP²H, and c) HMP by the action of HEPD. ³¹P chemical shifts of phosphonates are very sensitive to small changes in pH. Therefore, the identity of 2-HEP and HMP was confirmed by spiking with an authentic synthetic standard. The additional resonance in b) and c) is residual [2-¹³C₁]-PnAA. The additional resonance at ~19 ppm in a) is an unknown byproduct of PnAA generation.