First, we decided to optimize the N-terminal region of 1 while maintaining the N-methylamide group at the C-terminus. To generate analogues rapidly, we employed a solid phase synthesis using methyl indole AM resin. After Fmoc deprotection, the resin was loaded with Fmoc-Tyr (PO(OBzl)2)-OH . Subsequent coupling with various commercially available carboxylic acids and cleavage with TFA allowed the rapid synthesis of N-methylcarboxamide phosphotyrosines (Supplementary Scheme 1). We introduced different substituents at the N-terminus to cover a range of physicochemical properties (e.g. alicyclic, heterocyclic, aromatic with electron-withdrawing and electron-donating groups) to maximize potential interactions (see Table for representative examples). Binding affinities of the synthesized compounds for SOCS2 were measured via two orthogonal biophysical binding assays: 1) surface plasmon resonance (SPR) with SBC immobilized on the chip (Supplementary Figure ); and 2) 19 F ligand-observed displacement NMR assays, providing two orthogonal biophysical measurements of dissociation constants (KD) . We found excellent correlation between the KD values measured via the two techniques (Table ). Para-substituted benzyl moieties yielded the greatest contributions to binding affinity and there were only slight differences between electron withdrawing groups such as a methyl ester and electron-donating such as methoxy group. At the same time, the 4-fluorobenzyl substituted ligand 5 was found to be the strongest binder in the series with comparable KD = 32 μM by F NMR and 49 μM by SPR, and good LE = 0.21 kcal/mol/NHA. ). LE values for each compound are reported in kcal/mol/NHA and calculated as LE = -RT•ln(Kd)/NHA, there NHA is the number of non-hydrogen atoms 32 .