Having established the (4-fluorophenyl) acetamido group as the preferred substituent at the Nterminus, we sought to optimize the C-terminal fragment while fixing the N-terminus of the ligand (Figure , 1G). Close inspection of our substrate bound co-crystal structures revealed a SOCS2-SH2 groove between Ile110 and Thr93 (named the "IT" channel) comprising of hydrophobic residues Leu95, Leu106, Leu150 and EF loop residues (Ile109, Ile110, Val112 and Leu116) , that is utilised by both EpoR and GHR peptides to form hydrophobic interactions (Figure and). We hypothesized that SOCS2 ligands could be optimally grown out of the C-terminus, by fitting snugly within the narrow groove of the IT channel, with a goal to access this largely hydrophobic region. Moreover, the flexible nature of the flanking EF loop (residues 107-116) can help accommodate a wide range of chemical groups in the groove. Towards this aim, we designed and synthesized a next library of SOCS2 ligands using commercial available Fmoc-Tyr(PO(NMe2)2)-OH as a starting material. Fmoc-Tyr(PO(NMe2)2)-OH was coupled with various benzylamines followed by Fmocdeprotection and subsequent coupling with 4-fluorophenylacetic acid. Deprotection of the N,Ndimethyldiamide phosphate protecting group with TFA afforded the desired phosphotyrosines (Supplementary Scheme 2). The binding of all obtained molecules was quantified using ITC and SPR (Table ). This second round of structure-activity relationship (SAR) exploration identified several ligands with improved binding affinity. We found that 4-fluorobenzylamino 9 and 3-methylbenzylamino 10 improved binding affinity by 10-fold compared to 5, showing Kd (SPR) = 5 μM and 3 μM, respectively (Figure , Table ). ITC titrations yielded Kd of 2.6 μM for 9 and 1.1 μM for 10, comparable to SPR, and evidenced exothermic binding ΔH = -4.1 and -5.8 kcal/mol (Table , and Supplementary Figure ). To elucidate the ligand binding mode, we solved the crystal structures of SBC in complex with 9, via ligand soaking (Figure ). Many of the key interactions formed by the pY residue are conserved between the ligand and the substrate peptides . These include: 1) hydrogen bonds between the phosphate group of the ligand and SOCS2-SH2 pocket residues Arg73, Ser75, Ser76 and Arg96; 2) cation-π interaction between the pY aromatic ring and Arg96; and 3) hydrogen bonds between the amide bonds surrounding pY and the backbone amides and side chain groups of Thr93 and Asn94. Pleasingly, the C-terminal 4-fluorobenzylamino group of 9 successfully occupied and engaged the hydrophobic patch residues Thr93, Leu95, Ile110, and Leu150 of the IT channel, as designed (Figure ).