Synthesis of substituted terpyridine ligands for use in protein purification Chih-Pei Lin, Pas Florio, Eva M. Campi, Chunfang Zhang, Dale P. Fredericks, Kei Saito, W. Roy Jackson, Milton T.W. Hearn * Centre for Green Chemistry, School of Chemistry, Monash University, Clayton, Victoria 3800, Australia article info Article history: Received 20 July 2014 Received in revised form 12 September 2014 Accepted 22 September 2014 Available online xxx Keywords: Heterocyclic compound synthesis 4 0 -Terpyridones 4 0 -Halo-terpyridines 4,4 00 -Disubstituted-4 0 - aminoethanesulphanylterpyridines Affinity chromatography Protein purification abstract A number of 4,4 00 -disubstituted terpyridines bearing a 4 0 -thioethanamine linker, and regioisomers thereof, have been synthesised from 4-substituted picolinates. The substituted terpyridines were im- mobilised onto epoxy-activated SepharoseÔ FF gel, creating functionalised solid supports for the frac- tionation of proteinsdincluding antibodiesdby mixed mode affinity chromatography. The metal chelating properties of the immobilised ligand render the stationary phase also amenable for use in immobilised metal-ion affinity chromatography (IMAC), and have been demonstrated with the purifi- cation of suitably tagged green fluorescent protein (GFP). Ó 2014 Published by Elsevier Ltd. 1. Introduction Affinity chromatography in its various forms is an important tool for the analytical and preparative purification of proteins and other biomolecules. 1e6 In recent years there has been a consider- able interest in the development of low molecular weight affinity ligands for the functionalisation of support materials for the chromatographic purification of proteins. 7e10 Such efforts typi- cally seek to address limitations in protein purification such as specificity, capacity, ligand toxicity and leaching. 11,12 The de- velopment of synthetic ligands for use in affinity chromatography has utilised traditional ligand discovery approaches ranging from ligand-based design, structure based design and ligand screening. 13 The use of low molecular weight pyridine-based ligands in protein purification is well documented. 7e10 Such ligands present opportunities for protein binding through a number of interactions such as hydrogen bonding, hydrophobic interactions, ionic in- teractions, thiophilic interactions, etc., and are therefore amenable to various forms of chromatography. A recent study has reported the synthesis of an array of pyridine based ligands for use in mixed mode affinity chromatography. 10 The pyridine nucleus in these li- gands contained a thioethanamine unit to create both a thiophilic centre 10 and an easy modality for immobilisation onto suitably activated support materials and solid phase surfaces. Both the na- ture and arrangement of the substituents on the pyridine nucleus of these ligands were demonstrated to affect their binding capacities and specificities for cell culture derived monoclonal antibodies (mAbs). As part of an ongoing research program into the synthesis of hetero-aromatic ligands for use in protein separation, 10,14,15 a range of substituted terpyridines bearing a thioethanamine functionality have now been prepared (Scheme 1). These ligands incorporate the terpyridine skeleton as a structural motif with substituents on the peripheral terpyridine rings to influence the steric and electronic properties of the ligand. Starting from the appropriately 4-substituted alkyl picolinates 1 , the 4,4 00 -di- substituted terpyridines of interest 2 were prepared via a number of alternative synthetic strategies building upon modifications to procedure of Constable et al.. 16 The advantages of these synthetic approaches will be discussed, and applications documented of the target compounds as ligands used to prepare novel chromato- graphic supports for the separation of antibodies and suitably tagged proteins. * Corresponding author. Tel.: þ61 3 9905 4547; fax: þ61 3 9905 8501; e-mail address: milton.hearn@monash.edu (M.T.W. Hearn). Contents lists available at ScienceDirect Tetrahedron journal homepage: www.elsevier.com/locate/tet http://dx.doi.org/10.1016/j.tet.2014.09.074 0040-4020/Ó 2014 Published by Elsevier Ltd. Tetrahedron xxx (2014) 1e12 Please cite this article inpress as: Lin, C.-P.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.09.074