Catalysis driven by an amyloid-substrate complex

Abstract

Amine modification through nucleophilic attack of the amine functionality is a very common chemical transformation. Under biorelevant conditions using acidic-to-neutral pH buffer, however, the nucleophilic reaction of alkyl amines (pKa ≈ 10) is not facile due to the generation of ammonium ions lacking nucleophilicity. Here, we disclose a unique molecular transformation system, catalysis driven by amyloid-substrate complex (CASL), that promotes amine modifications in acidic buffer. Ammonium ions attached to molecules with amyloid-binding capability were activated through deprotonation due to the close proximity to the amyloid catalyst formed by Ac-Asn-Phe-Gly-Ala-Ile-Leu-NH₂ (NL6), derived from islet amyloid polypeptide (IAPP). Under the CASL conditions, alkyl amines underwent various modifications, i.e., acylation, arylation, cyclization, and alkylation, in acidic buffer. Crystallographic analysis and chemical modification studies of the amyloid catalysts suggested that the carbonyl oxygen of the Phe-Gly amide bond of NL6 plays a key role in activating the substrate amine by forming a hydrogen bond. Using CASL, selective conversion of substrates possessing equivalently reactive amine functionalities was achieved in catalytic reactions using amyloids. CASL provides a unique method for applying nucleophilic conversion reactions of amines in diverse fields of chemistry and biology.

Keywords: amyloid; amyloid catalyst; catalysis; nucleophilic reaction.

Fig. 1.

CASL approach. (A) Illustration to explain the structural match between the enzyme and substrate to catalyze the reaction. The activation of a substrate amine by the carbonyl oxygen of the backbone amide of enzyme is shown as an example. (B) Conceptual illustration of CASL to explain that the amine functionality attached to the amyloid-binding molecule (regarded as substrate) is activated due to close proximity to the amyloid catalyst.

Fig. 2.

Optimization of the amyloid catalyst. (A) Screening of amyloid catalysts for N-benzoylation CASL with substrate 1, in which the amino group is attached to the amyloid-binding azobenzene motif. Reaction conditions: 25 μM 1, 100 μM Bz donor, 200 μM amyloid catalyst, acetate buffer (pH 6.0), 37 °C, 6 h. *Fibrils were clearly observed in amyloid catalysts exhibiting a sufficient increase in ThT fluorescence intensity (entries 2, 3, 4, and 6 as representative examples) by microscopic analyses (SI Appendix, Fig. S6). **Yields of 1-Bz were determined by the HPLC peak area (at 475 nm) using the standard curve (SI Appendix). (B) Correlation diagram of API vs. reaction yield (%). Circle: initial screening of amyloid catalysts; square: derived from NL6; see text for explanations of red fill and blue fill.

Fig. 3.

Substrate and reaction scope. (A) Substrate scope of N-benzoylation CASL. Reaction conditions: 25 μM substrate, 100 μM Bz donor, 200 μM NL6, acetate buffer (pH 6.0), 37 °C, 6 h. (B) Correlation diagram of affinity for NL6 amyloid (IC₅₀) vs. reaction yield (%). (C) Reaction scope of amine modification CASL. Reaction conditions: (i) 25 μM 1, 500 μM 2,4-dinitrofluorobenzene (DNFB), 200 μM NL6, phosphate buffer (pH 6.4), 37 °C, 24 h; (ii) 25 μM 1, 500 μM 2-ethynylbenzaldehyde, 200 μM NL6, acetate buffer (pH 6.0), 37 °C, 24 h; (iii) 25 μM 1, 2.5 mM benzaldehyde, 2.5 mM sodium cyanoborohydride, 200 μM NL6, acetate buffer (pH 6.0), 37 °C, 24 h.

Fig. 4.

Insights into the reaction mechanism. (A–C) Crystal structures of pNL and the docking study with 1. (B) Side view. (C) Cross-section view. These structures are shown by sticks with oxygen, nitrogen, carbon, and hydrogen colored red, blue, wheat (except for 1 colored in green), and white, respectively. (D) Chemical modification studies of NL6.

Fig. 5.

Substrate-selective conversion. (A) N-Benzoylation of 1 vs. 2b. Common reaction conditions: 12.5 μM 1, 12.5 μM 2b, 50 μM Bz donor, 37 °C, 6 h. Differences in conditions: (condition 1) 200 μM NL6 amyloid, acetate buffer (pH 6.0); (2) phosphate buffer (pH 7.4); (3) phosphate buffer (pH 8.0); (4) water, 25 μM potassium carbonate; (5) acetonitrile, 125 μM triethylamine. (B) N-Arylation of 1 vs. lysozyme. Common reaction conditions: 25 μM 1, 25 μM lysozyme, 25 μM DNFB, 37 °C, 14 h. Differences in conditions: (condition 1) 200 μM NL6 amyloid, phosphate buffer (pH 6.8); (2) phosphate buffer (pH 8.0). (Inset) MALDI-MS spectra around m/z corresponding to the molecular weight of lysozyme.