Analysis of the different stereoisomers of rotaxanes 20 by 1 H NMR (Figure ) and CD (Figure ) revealed the expected features. The structures assigned as enantiomers (Rco-mp,Rco-mp)-20 and (Sco-mp,Sco-mp)-20 produced the same 1 H NMR spectra (e.g., Figure ) and mirror image CD spectra whereas the sample assigned as meso diastereomer (Rco-mp,Sco-mp)-20 was distinct by 1 H NMR (Figure ) and produced no significant CD signal. A mixture of rotaxane 20 stereoisomers produced by direct reaction of bisalkyne 17 with two equivalents of azide 3 and the analogous amine macrocycle lacking the chiral auxiliary contained both diastereomers in a 1 : 1 ratio, as judged by 1 H NMR (Figure ), produced no CD signal and contained three species with different retention times by CSP-HPLC in a ~ 1 : 1 : 2 ratio, consistent with a statistical mixture of (Rco-mp,Rco-mp)-20, (Sco-mp,Sco-mp)-20 and (Rco-mp,Sco-mp)-20. HPLC analysis of the stereoenriched samples of rotaxanes 20 revealed peaks with the expected retention times, albeit their absolute stereopurity could not be determined by CSP-HPLC due to the broad peaks obtained and the overlap of the peaks associated with (Sco-mp,Sco-mp)-20 and (Rco-mp,Sco-mp)-20. However, the high diastereopurity of the products at all steps of the synthesis, combined with the strong CD response of the chiral diastereomers and the qualitative appearance of the CSP-HPLC chromatogram confirms they are highly stereoenriched.