Although many different approaches to catenanes and rotaxanes have been introduced,[1] few strategies have been successfully developed for the synthesis of molecular knots.[2] Trefoil knots, the simplest prime knot other than the topologically trivial unknot (ie, any ring or simple macrocycle),[3] have been found in DNA,[4] proteins,[5] and in synthetic polymers.[6] Sauvage and co-workers prepared the first synthetic molecular knot by using the preorganization of two ligand strands around two tetrahedral CuI centers as the key template interaction to generate the three crossing points required for a trefoil knot.[7] Subsequently, donor–acceptor interactions,[8] Watson–Crick base pairing,[9] amide hydrogen bonding,[10] and ligand folding around an octahedral metal ion [11] have all been used to template the formation of molecular trefoil knots.[12] A few years ago a strategy for the synthesis of rotaxanes and catenanes was introduced in which metal ions play a dual role, acting as a template to entwine or thread the building blocks while also actively catalyzing the bond-forming reaction that covalently traps the interlocked structure.[13] This “active-template” approach has proven to be an effective route to various types of mechanically interlocked molecules and can be applied by using an increasing number of different transition-metal-catalyzed reactions.[13j] Herein we report an active-template reaction that occurs through a loop generated through classical “passive-template” coordination to synthesize the smallest trefoil knot reported to date. The trefoil knot was characterized by 1H and 13C NMR spectroscopy, mass spectrometry, and by drift tube ion mobility mass spectrometry (DT IM-MS) experiments that show that the molecular knot has a significantly smaller cross-sectional area (with a narrower distribution) than the corresponding open-chain and unknot-macrocycle isomers. To apply active-template synthesis to a trefoil knot architecture, we envisaged a system (Figure 1) in which a single molecular strand with reactive functional groups at each terminus (X and Y) could be geometrically manipulated and knotted through multiple interactions with metal ions(M). First, a loop in the strand would be formed by coordination of two bidentate binding sites in the strand to a tetrahedral metal ion (Figure 1, step 1). A second metal ion, bound endotopically within the loop by a monodentate ligating site, would then perform the twofold tasks of 1) gathering both functional end groups in a specific orientation that is dictated by the metal s preferred coordination geometry and places them on opposite sides of the loop (Figure 1, step 2), and 2) catalyzing a covalent-bond-forming reaction between the end groups to generate the molecular trefoil knot (Figure 1, step 3). Ligand 1 (Scheme 1) was synthesized in nine steps from commercially available starting materials (for experimental