I do not believe in the diastereotopicity of the CH2 protons at low pH. Rather, the amide groups prefers to be planar.

The reason for the splitting of the CH2 signal at low pH and of its singlet nature at high pH is slow chemical exchange of NH and H2O in the first case and fast exchange in the latter case. When the exchange rate becomes fast with respect to the coupling constant (3JHH), the CH2 protons "see" only an averaged orientation of the amide proton spin and, hence, appear as a singlet. A comparable (and often observed) case is the appearance of the CH3 signal of the methanol sample used to calibrate the probehead temperature. Low temperature = slow exchange = methyl doublet; high temperature = fast exchange = methyl singlet.

Thanks for your answer John, I should have added more details sorry. The D2O I add to the NMR tube is inside an insert so there's no possible deuteration of N-acetylglycine.

Maybe the pH changes the chemical environment of the methylene protons enough so that they magnetically affect each other? Definitely the carboxyl will get protonated at low pH; maybe this causes the bonds to rotate slower, and that causes the chemical environment felt by each proton to be different? Note that the 3 protons from the terminal methyl protons maintain their singlet at 2 ppm, so that's why the 'different rotation speed' explanation comes to me. Don't know how plausible this sounds though.

At pH=12 you are deprotonating the carboxylic acid. I think that is the only big change you get in your molecule. Is it possible that at low pH the carboxylic acid is lining up with the amide to get an internal hydrogen bond and the protons of the CH2 are diastereotopic, while at higher pH you have two almost flat systems (the delocalized carboxylate and the amide) that make the 2 protons enantiotopic?

I think the question is very simple. If you see doublet at ~3,7 and this is doublet with very small splittings like 1-5 Hz, then this is clear. At pH Nac-Gly has non equivalent CH2 protons they are almost A2 system, but with slightly different chemical shifts. In this case (if J>delta chem. shifts) you should see strong doublet and very tine forbidden transition at offset = J. When you change pH Gly may change electronic structure and happens to have identical CH2 protons. So they will collapse to one singlet. Look here how AX system transforms to AB and then to A2 https://www.chem.wisc.edu/areas/reich/nmr/05-hmr-10-ax-ab.htm

I do not believe in the diastereotopicity of the CH2 protons at low pH. Rather, the amide groups prefers to be planar.

The reason for the splitting of the CH2 signal at low pH and of its singlet nature at high pH is slow chemical exchange of NH and H2O in the first case and fast exchange in the latter case. When the exchange rate becomes fast with respect to the coupling constant (3JHH), the CH2 protons "see" only an averaged orientation of the amide proton spin and, hence, appear as a singlet. A comparable (and often observed) case is the appearance of the CH3 signal of the methanol sample used to calibrate the probehead temperature. Low temperature = slow exchange = methyl doublet; high temperature = fast exchange = methyl singlet.

Thanks to all for your replies. Ludger, I do agree that it most problably has to do with the rate of exchange of the NH proton, which is indeed pH-dependant, what causes the methylene splitting at low pH. Thank you.