Anaerobic conditions were attained by sparging with nitrogen gas containing less than 5 ppm O2 (AGA GAS AB, Sundbyberg, Sweden) before inoculation.
Anaerobic conditions were obtained by continuously sparging with 0.2 L min−1 nitrogen gas containing less than 5 ppm O2 (AGA GAS AB, Sundbyberg, Sweden) controlled by a mass flow metre (Bronkhorst Hi-Tech, Ruurlo, the Netherlands).
Anaerobic conditions were obtained by sparging with nitrogen gas containing less than 5 ppm O2 (AGA GAS AB, Sundbyberg, Sweden) at a constant gas flow of 0.2 litre min-1 controlled by mass flow meters (Bronkhorst HI-TEC, Ruurlo, the Netherlands).
The strong downfield shift of HC15 (to 4.38 ppm) as part of an AB system at 4.24/4.38 ppm (J=7.5) was consistent with the presence of a de-shielding substituent at C15. H,H-NOE correlations of HC15 with methyl group singlets at 2.00 ppm (H3C13) and at 1.52 ppm (H3C17) helped to put this assignment on a safe ground.
As anticipated, H NMR analysis of the isolated product, 4, showed the methylene protons of the DPA moiety as an AB quartet (5.03/4.96 ppm) with a downfield shift from their original appearance as a singlet in the spectrum of 3 (4.62 ppm).
The closely spaced signals, at 4.19 and 4.17 ppm, of HC15 and of HC16 generate an AB system, which shows NOE correlations with the singlet at 2.11 ppm of H3C17.
The spectra were manually corrected (phasing, baseline correction, referencing to the DSS peak at 0.0 ppm) in the Chenomx NMR Suite 6.1 software (Chenomx Inc., Edmonton, AB, Canada) [ 23].
For compounds 4 b– e, the P NMR spectra feature AB coupling patterns with typical doublets at chemical shifts of δ=161.5 and 16.0 ppm (JPP=35 Hz) for heterocycle 4 e and approximately 152 and 15 ppm (JPP=28 Hz) for compounds 4 c, d.
Nitric oxide (1,000 ppm; AGA Gas AB, Lidingö, Sweden) was delivered in an air/oxygen mixture from a low-flow air oxygen blender (AGA AB, Sundbyberg, Sweden) into the low-pressure gas-flow inlet of the ventilator.
