After identifying NL6 as a promising amyloid catalyst, we attempted to extract the structural factors necessary for the catalytic activity. Heptapeptides extended by one residue each at the N-terminus and C-terminus of NL6, respectively according to IAPP sequence, gave lower yields than NL6 (entries 7 and 8), suggesting that a six-residue peptide is sufficient to demonstrate the catalytic activity. We next investigated the contribution of each side chain of NL6 by comprehensively substituting the amino acids with an alanine residue (entries 9-13). The structure-API-yield relationship studies revealed that the amyloidogenic ability correlated well with the reaction yield. Thus, the yield of 1-Bz was 2% to 17% in entries 10, 12, and 13 with a low API, 54% in entry 9 with a medium API, and 81% in entry 11 with a high API. On the other hand, the low yield produced despite the high API of Ac-Asn-Phe-Gly-Ala-Phe-NH2 (entry 2), a homologous sequence to NL6, indicates that the amyloidogenic potential alone is not sufficient for the reaction progress and that the side chains of Ile and Leu of NL6 are also important for promoting the reaction. Similarly, the yield of Ac-Leu-Ila-Ala-Gly-Phe-Asn-NH2, in which the amino acid sequence of NL6 was reversed, was half of that of NL6 although the amyloidogenicity was completely maintained (entry 14), suggesting that a specific arrangement of the side chains of NL6 is involved in the reaction catalysis. Finally, replacement of the primary amide of the Asn side chain with carboxylic acid to give Asp abolished both the amyloidogenic ability and catalytic activity (entry 15), presumably due to intermolecular anionic repulsion preventing the self-assembly. Subsequently, we explored the substrate scope of the N-benzoylation CASL using the NL6 catalyst (Fig. ). Throughout the study, we quantified the affinity of the substrates for NL6derived amyloid using a ThT displacement assay according to a previous report. Briefly, the substrate was added to the preformed NL6 amyloid-ThT complex and the substrate concentration at which the fluorescence intensity of ThT was halved was defined as the IC50. The IC50 value of 1, in which N-benzoylation with NL6 amyloid proceeded at 78% yield, was 0.36 µM (Figs. and). Deletion of the 4'-dimethylamino group from the parent substrate (1a) decreased the affinity for NL6 amyloid (IC50 = 3.5 µM) as well as the yield (40%). Conversion of the linkage bond between the azobenzene and alkyl amine from ester to anilide (1b) slightly decreased both the affinity (IC50 = 0.50 µM) and yield (64%) compared with 1. Replacement of the ester with amide (1c) moderately reduced both the affinity (IC50 = 3.0 µM) and yield (33%), while replacement with ether (1d) strengthened both parameters (IC50 = 0.24 µM and 93% yield). These results are consistent with the mechanism of CASL by which the reaction is promoted via the binding of substrates with amyloid catalysts. As a control, N-Fmoc-1,4-butanediamine, which has no binding ability to NL6 amyloid (IC50 > 150 µM), gave only a trace amount of the N-benzoylated product (Fig. ). Interestingly, changing the ester-alkylamine moiety from the p-position of 1 to the m-and o-positions relative to the azo group (2a and 2b) significantly reduced the yields to 35% and 24%, respectively, although the decrease in binding affinities for NL6 amyloid relative to 1 remained small (IC50 = 0.53 µM and 0.75 µM). These results imply that the direction of the amine extension from the amyloid-binding site influences the activation efficiency of the amyloid catalyst. There was little change in the affinity and yield compared to 1 when the alkylamine was moved to the reverse wing of the azobenzene (2c). In addition, the 4'-dimethylaminoazobenzene moiety, which exhibited the strongest binding affinity to NL6 amyloid, was fixed (designated as a binding group, BG), and the effects of the spacer structure attached to the p-position of BG were investigated. Changing the alkyl chain to an ether chain (3a) and introducing a shortened spacer (3b) had little effect on the outcomes compared to 1. The introduction of an ester functionality to the spacer was tolerated (1c vs. 3d), although the addition of an amide moderately reduced the affinity for NL6 amyloid (1b vs. 3c). Comparison of 3c and 3e showed that extending the spacer to approximately 23 Å in the linear distance between BG and reactive amine was tolerated. The benzoylation hardly proceeded with any of the substrates in the absence of the NL6 catalyst.