[Urbanium]

I'm using biotin (in the form of methyl ester) and DL-cysteine as starting materials in one total synthesis sequence. Since the commercial samples I have are slightly older, I took a proton NMR of a small amount in CDCl₃ for the former and D₂O for the latter.

In both cases the "urea"-like protons in biotin and amino and SH protons in cysteine are completely absent from the spectra. Why is that so? I expected to see broad singlets somewhere downfield.

Just out of pure curiosity, is there some more sophisticated method to see or make these protons visible?

[discodermolide]

How about exchange with D?

[Urbanium]

How about exchange with D?

By that you consider the explanation or a method to find out what is happening?

I'm just curious why I can't see these protons for both molecules. When I go to some empirical spectral database, these peaks (at least for cysteine) are completely absent. Why?

[MOTOBALL]

For aged cysteine at least, I that your NMR solution is detecting the oxidation product---the disulfide, therefore no --SH protons.

Perhaps you can check the structure of biotin to determine if an oxidation/cross-linking may be responsible for the absent protons.

[discodermolide]

Exchange of the protons with deuterium from the D2O. This is a standard method for detection of exchangeable protons.

[Babcock_Hall]

It would be easy to check for oxidation of cysteine using a DTNB assay (see Riddles and coworkers, 1979, in Methods in Enzymology, A reassessment of Ellman's reagent).  I agree with Discodermolide that exchange is the most likely explanation.  If the biotin were soluble in some other (dry) solvent where exchange with residual water is slow, it might be possible to observe the urea-like protons.

[OrgXemProf]

"Shake the sample with a few drops of D2O" is the oft-suggested strategy to reveal the presence of an exchangeable proton in a 1H NMR spectrum. However, in the present example the potentially-exchangeable proton may be buried under other proton absorption signals and, thus, hidden from view.

So, the question now becomes: How do you know whether or not there is a squirrel clinging to the wrong side of a tree?
 
The presence or absence of an exchangeable proton (the "squirrel") can be detected by integrating the NMR spectrum before and after D2O exchange.

In the case of exchangeable H's attached to nitrogen, quadrupole broadening of the proton NMR signal due to the attached 14N nucleus can be so extensive that the absorption signal becomes barely detectable as a gentle "swelling" above baseline. Nevertheless, spectral integration will reveal the presence of this squirrel, too.