Engineering a Green Fluorescent Protein-Core-Inspired NIR-Photocage: Exploring meso-GFP-PRPG toward Alzheimer's Disease Therapeutics

Abstract

NIR light-activated photocage with inherent protein tagging ability is unprecedented in contemporary photochemistry. Herein, we introduce a series of protein-taggable NIR-photocages derived from green fluorescent protein (GFP) chromophore analogs with spatiotemporal control for releasing the caged bioactive molecules. Through molecular engineering of the GFP chromophoric scaffold, a series of meso-substituted oxazolone-photocages (meso-GFP-PRPG) were judiciously designed and synthesized. These photocages, anchored with electron-donating groups (EDG) and electron-withdrawing groups (EWG), accommodate diverse payloads, including aliphatic carboxylic acids, expanding the possibilities for tailoring their properties and applications. Notably, under anaerobic conditions, irradiation of meso-GFP-PRPG leads to fast and efficient release of caged molecules. Insightful experimental and theoretical investigations revealed that photorelease is predominantly driven by the triplet state photochemistry in anaerobic conditions. The concept's theranostic potential was demonstrated by the conditional release of valproic acid, a neuroprotective agent for Alzheimer's disease (AD) treatment. meso-GFP-PRPG (15E) showed enhanced NIR emission with Aβ oligomers and fibrils (30-37 fold vs ThT) and effectively degraded amyloid fibrils under 640 nm light, offering a promising targeted treatment approach for neurodegenerative disorders.

1

(A) Examples of meso-methyl positions for photorelease; (B) photocleavage of coral fluorescent protein: a prior idea for photocleavable and photostable protein; (C) rational design of GFP-core for photocleavage and protein tagging; (D) donor–acceptor complex to achieve red-shifted photocages for early sensing and prevention of amyloid fibrillogenesis.

1. (A) General Synthesis of Meso-Substituted GFP-Photocages, (B) Examples of Meso-Substituted GFP-Photocages, and (C) Synthetic Procedure for Amphiphilic Modification on GFP-Photocages and Examples

2

(a) Normalized UV–vis of photocages 3A, 3B, 3C, 15C, 15E and 15F in chloroform (1 × 10^–5 M); normalized emission spectra of (b) 3A, 3B, and 3C (c) 15C, 15E, and 15F in chloroform (1 × 10^–5 M); (d) solvent-dependent emission spectra of photocage 15E (1 × 10^–5 M); (e) change in the emission spectra of photocage 15E in different aromatic solvents; (f) change in the singlet state lifetime of photocage 3A in the presence and absence of oxygen.

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(a) Pictorial diagram of the experimental setup in degassed condition; (b) RP-HPLC diagram of the course of the photolysis of photocage 3C; (c) ¹H NMR study for monitoring the course of the photolysis [photocage 3C (1 mg) in 0.4 mL DMSO-d ₆ + 0.1 mL D₂O, degassed]; (d) photorelease of photocage 3C with dose-dependent triplet state quencher, potassium sorbate (1 mg of photocage 3C in 7:3 ACN: H₂O, degassed, ≥410 nm); (e) photorelease of photocage 3C in the presence of singlet oxygen scavenger, furfuryl alcohol (3C + furfuryl alcohol 1:10, aerated ethanol 10 mL); (f) change in emission spectra of SM-94 after sensing of singlet oxygen (3C + fluorescence reporter SM-94 1:1); (g) photolysis of photocage 3C with dose-dependent radical scavenger, TEMPO (1 mg of photocage 3C in 7:3 ACN: H₂O, degassed, ≥410 nm); (h) probable photorelease mechanism of GFP-photocage 3C (cis-trans).

4

(a) Change in the emission spectra of photocage 15E in the presence of Aβ₄₀ oligomer and fibril; (b) day to day emission spectra of 15E along with fibrillogenesis; (c) monitoring of Aβ₄₀ protein aggregation kinetics with 15E and thioflavin-T; (d) emission response of photocage 15E in the presence of different analytes; a: Gly; b: Arg; c: Lys; d: Leu; e: Gln; f: Thr; g: Cys; h: Tyr; i: insulin; j: Na⁺; k: K⁺; l: Ca²⁺; m: NO⁺; n: H₂O₂; o: ClO^–; p: TBHP; q: PBS; r: Aβ40; s: BSA.

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(a) Wavelength-specific photorelease of 15E and percentage of photoconversions (7:3 ACN: H₂O, degassed, 1× 10^–5 M); (b) wavelength-specific photorelease of 15E and percentage of photoconversions in different pH solutions (1.5 mL ACN + 1.5 mL pH solutions, degassed, 1× 10^–5 M); (c) change in the CD spectra of Aβ₄₀ fibril after photoirradiation with green light (525 ± 10 nm); (d) change in the CD spectra of Aβ₄₀ fibril in different concentrations of valproic acid; (e) TEM image of Aβ₄₀ fibril before and after the photoirradiation (525 ± 10 nm); (f) photolysis of 15E (10 μM) by 595 ± 10 nm light after sensing the amyloid oligomers.