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200 180 160 140 120 100 80 60 40 20 ppm

200 180 160 140 120 100 80 60 40 20 ppm

!H ^ 13C NOE (up to 3x enhanced signal) observed in the 13C spectrum reduces the difference in sensitivity somewhat (without NOE it would take 16 times longer than the DEPT to acquire a 13C spectrum), but as we need to see the slowly relaxing quaternary carbons in the 13 C spectrum, most of the time is allotted to this experiment. The 13 C spectrum (bottom) includes the intense CD3OD solvent peak at 49.15 ppm (seven peaks, 1:3:6:7:6:3:1 ratio), which goes off-scale at the top of the spectrum. The solvent peak is intense because we are comparing it to the very weak 13C peaks of the dilute sample. The DEPT-90 spectrum (top) clearly shows six strong peaks (*), but a number of weak peaks result from incorrect calibration of the final 90o 1H pulse. Because all of these are positive, including those that show up negative in the DEPT-135, it is clear that the final 1H pulse was less than 90o, so that we have some DEPT-45 mixed in with the DEPT-90. It is impossible to calibrate the pulse on such a dilute sample, but it would have been possible to calibrate on a concentrated sample of menthol or cholesterol dissolved in CD3OD before starting the experiment. This was not done, however, so this is what we have to work with. It is easy to see that the weak peaks in the DEPT-90 are a combination of CH2 peaks (negative in the DEPT-135) and CH3 peaks (positive in the DEPT-135), so we can ignore them. We can see two quaternary carbons (@) at 202.59 and 175.61 ppm (ketone C=O and 5 carbon of a,5 unsaturated C=O, respectively), and six CH (*) carbons (olefinic at 124.24, two alcohols at 81.97 and 69.58, two "crowded" aliphatic and one "normal" aliphatic). We count seven CH2 carbon peaks (O) in the DEPT-135 (negative peaks) and two CH3 (#) peaks (positive in DEPT-135 and weak in DEPT-90) at the upfield end of the spectrum. Two more quaternary (@) carbons, the bridgehead C10 and C13 carbons, are seen in an expanded plot (Fig. 7.40) at 43.47 and 40.38 ppm. From this data alone we can infer that one of the eight CH2 groups of testosterone was converted into a CH group bearing an alcohol (OH). Testosterone has only one alcohol, and there are two "alcohol" carbons in the metabolite. To find out which position in the steroid bears the "new" alcohol functionality would require 2D experiments.

Testosterone Metabolite, 0.19 mg in CD3OD

Testosterone Metabolite, 0.19 mg in CD3OD

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