On this basis, we performed a systematic backbone N-methyl scan for all 13 residues of the parent SIM peptide. Peptides were prepared using Fmoc-based solid phase peptide synthesis (see the Supporting Information for procedures and characterization) and their relative binding affinities to SUMO were estimated using a fluorescence anisotropy competition assay. N-methylation at six sites (Me-Asp2705, Me-Asn2706, Me-Glu2707, Me-Ile2708, Me-Ile2712, Me-Lys2717) led to similar or slightly improved inhibitory potency to the parent SIM peptide (Fig. ; Fig. , Table ). By contrast, N-methylation at five sites (Me-Val2710, Me-Ile2711, Me-Ile2713, Me-Trp2714, and Me-Lys2716) led to significantly diminished inhibitory potency, and the remaining two sites (Me-Ile2709, and Me-Glu2715) exhibited moderately reduced potency. Significantly, changes in inhibitory potency were observed both within and outside the consensus hot-spot region.