Catenane and rotaxane stereochemistry -conclusions. Our simple, first principles approach has allowed us to identify all the possible conditional stereogenic units of rotaxanes and catenanes, and confirm that, now that a previously overlooked MGI-2 rotaxane stereochemistry has been found, the pantheon of unique stereogenic units is complete. Based on this analysis, methods exist to stereoselectively synthesise all conditional mechanical stereogenic units of [2]catenanes and [2]rotaxanes apart from MGI-2 rotaxanes; although until 2014, 19 chiral stationary phase HPLC was required to produce enantioenriched samples of mechanically chiral molecules, 20 since this time methodologies for the stereoselective synthesis of MPC and MAC 5 catenanes and rotaxanes have been disclosed. Similarly, the first stereoselective synthesis of MGI-1 rotaxanes was reported in 2005 24 using calixarene rings and since then many examples based on cone-shaped macrocycles, 25 and more recently simple prochiral 26 rings, 5b,27 have been reported. The corresponding MGI catenanes are less well studied but yield to similar strategies to the corresponding rotaxanes. Retrosynthetic analysis of the "new" MGI-2 stereogenic unit. Having identified the MGI-2 stereogenic unit, we considered what strategies could be used for its selective synthesis. Notionally, the challenge in the synthesis of MGI-2 rotaxanes is the same as that of MPC rotaxanes -how to thread an oriented ring onto an axle with control over their relative orientation (Figure ). We previously achieved this for MPC rotaxanes 22a,e using an active template Cu-mediated alkyne-azide cycloaddition (AT-CuAAC 30,31 ) approach in which the intermediates leading to the different enantiomers are diastereomeric due to a covalent chiral auxiliary. Although it may seem counterintuitive to synthesise the achiral MGI-2 stereogenic unit using chiral starting materials it should be noted that, as with the synthesis of MAC rotaxanes, almost regardless of where the prochiral axle is subdivided, 32 a chiral starting material is produced, which is symmetrised during mechanical bond formation and so no additional auxiliary removal step is required (Figure ). Furthermore, a racemic mixture of starting materials would lead to the same MGI-2 product mixture using this direct approach. Attempted direct synthesis of MGI-2 rotaxanes 5. Thus, we initially attempted a synthesis of a rotaxane expressing the MGI-2 stereogenic unit using a stepwise AT-CuAAC approach. Reaction of oriented macrocycle 1, 33 serine-based azide (S)-2 and alkyne 3 under our AT-CuAAC conditions in CH2Cl2 gave rotaxane 4 as a mixture of diastereomers (17% de, 34 Scheme 1, entry 1) that differ in their MGI-2 configuration but have the same co-conformational covalent configuration. The same reaction in THF (entry 2) or EtOH (entry 3) gave lower selectivity (3% and 16% de respectively), whereas lower temperatures (entries 4 and 5) gave increased selectivity at the expense of reduced conversion. Unfortunately the (Zm,Sco-c)-4 and (Em,Sco-c)-4 diastereomers proved hard to separate; the best we could achieve was a 59% de sample starting from a 17% de sample after several rounds of chromatography. We were also unable to separate rotaxanes 5, which only express the MGI-2 stereogenic unit, obtained by removal of the Boc group from the mixture of rotaxanes 4.