Anion recognition is at the basis of processes such as sensing or transmembrane transport. The recognition of phosphate species is of particular interest in the biological field. Various phosphate species play important roles in a wide range of biological processes, including encoding of genes, energy storage, and as a crucial building block for bones and teeth. Furthermore, phosphate species can also be linked to diseases such as hyperphosphatemia, characterized by increased levels of phosphate in the blood, or pseudogout, a form of arthritis caused by calcium pyrophosphate dihydrate (CPPD) deposits. Pyrophosphate in serum is formed primarily by the hydrolysis of ATP, while it is in turn hydrolyzed to inorganic phosphate. Pyrophosphate is essential to control mineralization, avoiding undesired calcium phosphate (hydroxyapatite) formation, while growing CPPD crystals at elevated pyrophosphate levels. As a result of the importance of phosphate species, receptors capable of effectively binding, sensing, or transporting these species are of great interest. One of the most common approaches for the development of phosphate and pyrophosphate receptors has been the use of NH-or OH-based H-bond donors, mimicking the way that natural receptors achieve phosphate recognition. It is well established that the proper preorganization of the molecular structure contributes to the affinity. A macrocyclic approach is therefore often used. Size complementarity between receptor and substrate was shown early on by Dietrich et al. to have a significant effect on the stability of cryptand anion complexes, and numerous other contributions have ensued this seminal work. Focusing on neutral tetralactam-based macrocycles as anion receptors, Chmielewski and Jurczak have shown that the size of macrocyclic receptors plays an essential role in both the selectivity and strength of receptor-anion interactions. Their work also showed that pyridyl N-atoms in dipicolinic bisamides contribute to the preorganization of the amides for anion binding, resulting in significant increases in binding affinities compared to isophthalamides. The comparison of the 18membered tetralactam receptor with ethylene spacers of Szumna and Jurczak with its aromatic and rigid counterpart prepared by Smith and co-workers seems to support the notion that more rigid macrocycles lead to higher anion affinities. Similarly, Flood and co-workers obtained higher binding affinities with more rigid triazole based anion receptors compared to their more flexible counterparts. On the other hand, examples can also be found where an increase in flexibility within a macrocyclic structure can be beneficial for binding, such as the work carried out by Glas et al. in which they investigated the effect of conformational flexibility of two similar peptide based macrocyclic receptor on the stability of the corresponding macrocycle-ligand complexes. The most flexible of the two showed the highest affinity. With this notion of the importance of size, flexibility, and preorganization in mind, we set out to develop a series of macrocyclic receptors for dihydrogen pyrophosphate (H2P2O7 2-), exhibiting identical macrocyclic ring sizes and possessing in all cases four NH-based hydrogen bond donors. Here, we describe the design, synthesis, and characterization of macrocycles 1, 2, and 3 (Figure ) and their ability to bind H2P2O7 2-, as well as the smaller anions H2PO4 -and Cl -in solution and in the solid state.