The relative stereochemistry of the major stereoisomers of rotaxanes 4 were compared by conversion of 4a, 4c, 4d and 4e to alcohol rotaxane 4b for analysis by 1 H NMR. Whereas samples of rotaxane 4b derived from macrocycles 1b-e were found to be contain the same major diastereomer, macrocycle (S)-1a gives rise to opposite major mechanical epimer. Crystals grown from a sample of 4b (88% de) produced from macrocycle rac-1b were analyzed by single crystal x-ray diffraction (SCXRD) and found to contain rac-(S,Smp)-4b (Figure ). Similarly, crystals grown from a sample of 4a (>96% de) derived from (S)-1e were found by SCXRD to contain the same relative orientation of the macrocycle and axle (Figure ), albeit the absolute stereochemistry is assigned (S,Rmp)-4a (see ESI section S9 for a detailed discussion on the assignment of mechanical stereogenic units). Based on these corroborating data, macrocycles 1b-e produce rotaxanes 4b-e with the same relative orientation of axle and macrocycle, corresponding to the (S,Smp) diastereomer of rotaxane 4b, whereas (S)-1a produces 4a with the opposite relative orientation of axle and macrocycle.