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. 2024 Mar 7;29(6):1196.
doi: 10.3390/molecules29061196.

Fluorimetric Detection of Insulin Misfolding by Probes Derived from Functionalized Fluorene Frameworks

Álvaro Sarabia-Vallejo  1 , Ana Molina  2 , Mónica Martínez-Orts  2 , Alice D'Onofrio  1 , Matteo Staderini  1 , Maria Laura Bolognesi  3 , M Antonia Martín  2 , Ana I Olives  2 , J Carlos Menéndez  1
Affiliations

Fluorimetric Detection of Insulin Misfolding by Probes Derived from Functionalized Fluorene Frameworks

Álvaro Sarabia-Vallejo et al. Molecules. .

Abstract

A group of functionalized fluorene derivatives that are structurally similar to the cellular prion protein ligand N,N'-(methylenedi-4,1-phenylene)bis [2-(1-pyrrolidinyl)acetamide] (GN8) have been synthesized. These compounds show remarkable native fluorescence due to the fluorene ring. The substituents introduced at positions 2 and 7 of the fluorene moiety are sufficiently flexible to accommodate the beta-conformational folding that develops in amyloidogenic proteins. Changes in the native fluorescence of these fluorene derivatives provide evidence of transformations in the amyloidogenic aggregation processes of insulin. The increase observed in the fluorescence intensity of the sensors in the presence of native insulin or amyloid aggregates suggest their potential use as fluorescence probes for detecting abnormal conformations; therefore, the compounds can be proposed for use as "turn-on" fluorescence sensors. Protein-sensor dissociation constants are in the 5-10 μM range and an intermolecular charge transfer process between the protein and the sensors can be successfully exploited for the sensitive detection of abnormal insulin conformations. The values obtained for the Stern-Volmer quenching constant for compound 4 as a consequence of the sensor-protein interaction are comparable to those obtained for the reference compound GN8. Fluorene derivatives showed good performance in scavenging reactive oxygen species (ROS), and they show antioxidant capacity according to the FRAP and DPPH assays.

Keywords: antioxidants; fluorescence probes; insulin aggregates detection; insulin amyloid conformation; turn-on fluorescence.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Rationale for the design of compounds 1–4.
Scheme 1
Scheme 1
Synthesis of fluorene derivatives 1–4.
Figure 2
Figure 2
Effect of solvent polarity on the fluorescence emission spectra of compound 1 (A) and compound 2 (B) at maximum excitation wavelength corresponding to each solvent (Table S3). The inset shows the expanded view of the spectra in order to compare the magnitude of the fluorescence emission intensity in different solvents.
Figure 3
Figure 3
Effect of the addition of increasing volumes of native insulin on the fluorescence emission spectra of compound 1 at λex = 289 nm (A) and compound 4 at λex = 304 nm (B). The protein:sensor molar ratio is shown in the inset.
Figure 4
Figure 4
Effect of the additions of increasing volumes of insulin fibrils/aggregates on the fluorescence emission spectra of compound 3: (A) at λex = 304 nm; (B) at λex = 327 nm, employing method A for protein aggregation. Same experiments for compound 4: (C) at λex = 304 nm; (D) at λex = 327 nm, employing method A for protein aggregation. The protein:sensor molar ratios are in the inset. The peaks at 340 (A,C) and 370 nm (B,D) correspond to Raman dispersion.
Figure 5
Figure 5
(A) Comparison of the increase in the fluorescence intensity of compound 3 (λex = 327; λem = 435 nm) when employing method A (GLY) and method B (HCL) for inducing insulin β-amyloid protein aggregation. (B) Same experiments for compound 4.
Figure 6
Figure 6
Protein fibrils–sensor binding assay. Fluorescence plots for determination of the dissociation constant of compound 4 and fibrils of insulin produced by method A. Dots correspond to average experimental data and the line corresponds to the mathematical fitting.
Figure 7
Figure 7
(A) Fluorescence emission spectra (λex = 275 nm) of amyloid insulin in the presence of increasing concentrations of compound 4. (B) Stern–Volmer plot for the quenching emission (λem = 305 nm) of amyloid insulin induced by compound 4. (C) Double logarithmic plot for describing the fluorescence quenching (λem = 305 nm) of amyloid insulin induced by compound 4. Amyloid insulin fibrils obtained according to method A. Concentration of insulin fibrils 5 × 10−6 M. The concentration of compound 4 varies from 0 to 12.5 × 10−6 M.
Figure 8
Figure 8
Total antioxidant capacity (TAC) of compounds 1–4 as compared to Trolox and curcumin.
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