Stereoselective synthesis of chiral catenane 5. Catenane 3 was synthesized (Fig. ) using an active template Cu-mediated alkyne-azide cycloaddition reaction between azide/alkyne pre-macrocycle alkene (Z)-1 and chiral bipyridine macrocycle (S)-2 (98% ee) . H NMR analysis confirmed that catenane 3 was isolated as a single diastereomer (>96% de), consistent with the covalent stereochemistry of (S)-2 efficiently controlling the relative orientation of the bipyridine and alkene containing macrocycles . Catenane 3 also contains a coconformational geometric stereogenic unit , the configuration, Eco-c, of which is controlled by the geometry of the starting pre-macrocycle, (Z)-1, and fixed in the interlocked product due to the steric bulk of the styrene and silyl ether units which prevent shuttling of the bipyridine macrocycle between the two triazole-containing compartments. The pendant covalent chiral auxiliary unit of catenane 3 was removed following a simple chemical sequence (steps iii → v) followed by cleavage of the silyl ether unit (step vi) to give catenane 5 which contains no covalent stereogenic unit. Catenanes 3, 4 and 5 were characterized in full (see ESI for details). Samples of catenanes 3, 4 and 5 were also synthesized starting from (R)-2 (98% ee) and rac-2. The solidstate structure obtained by single crystal x-ray diffraction analysis of intermediate catenane 4 derived from rac-2 (Fig. ) contains the rac-(S,Smp,Eco-c)-4 stereoisomer and thus the absolute stereochemistry of catenanes 3 and 4 derived from (S)-2 can be assigned as (S,Rmp,Eco-c)-3 and (S,Smp,Eco-c)-4. The absolute stereochemistry of the expected major enantiomers of catenane 5 derived from (S)-2 as and (R)-2 can thus be assigned as (Rmp)-5 and (Smp)-5 respectively (see Supplementary Information section 9 for a detailed discussion on stereochemical assignment in such systems). Stereochemical analysis of catenane 5. Care had to be taken throughout the synthesis of catenanes 5 to ensure the double bond geometry, which defines the orientation of the triazole-containing macrocycle, was maintained; the electron rich alkene unit is prone to acid-mediated isomerization. Pleasingly, chiral stationary phase HPLC (CSP-HPLC) analysis confirmed that catenanes 5 were formed in excellent stereoselectivity (98% ee, Fig. ) and that the samples derived from (R)-2 and (S)-2 produce mirror image circular dichroism (CD) spectra, whereas the sample derived from rac-2 did not produce a CD response (Fig. ). H NMR analysis of the isolated samples of catenane 5 revealed that the bipyridine macrocycle is in slow exchange between the triazole stations (Fig. ); although the Eco-c isomer was isolated and characterized, this slowly evolved in solution (CD2Cl2, 303 K) to an equilibrium mixture containing both co-conformations (~80 : 20 Eco-c : Zco-c, Supplementary section 7 for a detailed discussion). This process takes place by shuttling of the bipyridine macrocycle around the triazole-containing macrocycle, past the benzylic alcohol unit, and demonstrates that the double bond of catenane 5 has no defined geometry. Thus, the only fixed stereogenic unit of catenane 5 results from the relative orientation of the two rings, as depicted in its highest symmetry representation 3d). Finally, CSP-HPLC analysis revealed a second stereoisomerization process; alongside the isomerization between Eco-c and Zco-c, a slower racemization process was also observed (Fig. ). Thus, over time in CD2Cl2 solution, the stereopurity of (Rmp)-5 decreased from 98% ee to 84% ee (Fig. ). Based on the apparent protonation of catenane 5 over time and the behavior of an analogous rotaxane model (Supplementary section 8), we tentatively propose that the enantiomerization of 5 may take place by reversible protonation of the electron rich double bond. This process is the chemical equivalent of the topologically allowed isomerization of the catenane stereogenic unit by a double bond isomerization process accompanied by shuttling of the bipyridine ring around the triazole-containing ring combined (Fig. ).