To stereoselectively synthesise a co-conformationally chiral catenane we must: i) incorporate the oriented ring at a defined position around the bilaterally symmetric macrocycle, and ii) ensure that the oriented ring is installed stereoselectively. The first requirement can be met by forming the mechanical bond such that the oriented ring is trapped between bulky groups that prevent co-conformational exchange. The second is more challenging and is essentially the same problem as encountered in the synthesis of any topologically chiral [2]catenane. Although the majority of topologically chiral catenanes in which the mechanical bond is the sole source of stereochemistry have been synthesized as racemates and then separated by chiral stationary phase HPLC (CSP-HPLC), recently we developed an auxiliary approach in which a covalent stereogenic unit directs the stereoselective formation of the mechanical bond. Separation of the mechanical epimers before removal of the auxiliary gave the enantiopure product. However, in this proof-of-concept synthesis the stereoselectivity of the mechanical bond formation was low (dr ~ 2 : 1); unless the mechanical epimers can be separated, which is far from assured, this auxiliary has limited utility for synthesis of structurally more complicated chiral catenanes. Thus, we investigated whether a phenylalanine-based auxiliary developed for the stereoselective synthesis of mechanical planar chiral rotaxanes 18,19 could be applied in the synthesis of topologically chiral [2]catenanes. Tyrosine-derived pre-macrocycle (S)-1a was synthesized (96% ee, Figure ) and reacted under previously reported pseudo high-dilution active template 20 Cu-mediated alkyne-azide cycloaddition (AT-CuAAC) conditions with oriented bipyridine macrocycle 2. Catenane 3a was produced with reasonable stereoselectivity (Table , entry 1), based on 1 H NMR analysis of the crude reaction product (Figure ); proton Ha of the diastereomers of 3a resonate at 8.98 (major) and 9.07 (minor) ppm, respectively. 1 H NMR analysis of the crude reaction product also suggested the presence of several other interlocked species, characterized by higher ppm (9.51 -9.61) triazole resonances. LCMS analysis indicated that these signals were due to [3]catenane 4, which can be formed as 3 diastereomers, and the corresponding [2]catenane (not shown, 2 diastereomers) containing a single bipyridine ring (ESI section S10). Unfortunately, we were unable to obtain pure samples of these intriguing chiral structures. Longer addition times (entry 2) resulted in diminished diastereoselectivity, perhaps due to epimerization of the covalent stereogenic center, and lower conversion of macrocycle 2. Lowering the reaction temperature resulted in enhanced diastereoselectivity (76% de) and reduced quantities of oligomeric species, allowing catenane 3b to be isolated in 39% yield (entry 4).