Exploring the Effect of Aliphatic Substituents on Aryl Cyano Amides on Enhancement of Fluorescence upon Binding to Amyloid-β Aggregates

Abstract

The self-assembly of amyloid-β (Aβ) peptides into amyloid aggregates is a pathological hallmark of Alzheimer’s Disease. We previously reported a fluorescent Aryl Cyano Amide (ARCAM) probe that exhibits an increase in fluorescence emission upon binding to Aβ aggregates in solution and in neuronal tissue. Here, we investigate the effect of introducing small aliphatic substituents on the spectroscopic properties of ARCAM both free in solution and when bound to aggregated Aβ. We found that introducing substituents designed to hinder the rotation of bonds between the electron donor and acceptor on these fluorophores can affect the overall brightness of fluorescence emission of the probes in amyloid-free solutions, but the relative fluorescence enhancement of these probes in amyloid-containing solutions is dependent on the location of the substituents on the ARCAM scaffold. We also observed the capability to tune the excitation or emission wavelength of these probes by introducing electron-donating or -withdrawing substituents that putatively affect either the energy required for photoexcitation or the stability of the photoexcited state. These studies reveal new design principles for developing ARCAM-based fluorescent Aβ-binding probes with an enhanced fluorescence signal compared to background and tunable spectroscopic properties, which may lead to improved chemical tools for aiding in the diagnosis of amyloid-associated neurodegenerative diseases.

Graphical Abstract

INTRODUCTION

Alzheimer’s Disease (AD) is a neurodegenerative disease associated with cognitive decline and dementia and is the sixth leading cause of death in the United States.^1,2 Pathologically, AD is characterized by the accumulation and deposition of aggregated amyloid-β (Aβ) peptides that purportedly cause damage to healthy neurons in the brain.^3,4 Current methods for the detection of amyloids comprised of Aβ or other amyloidogenic proteins include the use of small-molecule probes that target these aggregates.^5,6 For instance, on the one hand, Positron Emission Tomography (PET) agents are currently used to aid in a clinical diagnosis of AD, but their utility outside of clinical trials has been limited by the short half-life of the radioligands and the high cost associated with PET scans.^7,8 Fluorescent amyloid-binding agents, on the other hand, have emerged as a viable alternative approach to aiding in the diagnosis of amyloid-associated neurodegenerative diseases^9–11 that have many advantages over PET agents, including (1) high spatial resolution, (2) low cost, and (3) the ability to spectroscopically discriminate between amyloid species of different disease origin.^6,12–14

Historically, fluorescent probes such as Thioflavin T (ThT) and Congo Red (CR) have been used for the detection of Aβ plaques in post-mortem brain samples,¹⁵ but these probes have not been used for in vivo ante-mortem detection of amyloidosis, presumably due, in part, to their poor biocompatibility properties.¹⁶ ThT and CR are part of a large class of fluorescent compounds called molecular rotors, which consist of electron-rich donor (D) units in conjugation through a π-scaffold to electron-poor acceptors (A) (otherwise known as the D–π–A motif).^15–17 Upon photoexcitation, molecular rotors either form a fluorescent locally excited (LE) state or a twisted intramolecular charge-transfer (TICT) complex.¹⁷ When in the LE state, photoexcited molecular rotors can relax to the ground state and release energy through the emission of a photon. However, while in the TICT state, molecular rotors typically release energy through internal nonradiative modes.¹⁸

We previously showed that the fluorescence properties of molecular rotors can be significantly influenced by the surrounding microenvironment.^14,19–22 For instance, ARyl Cyano AMide 1 (ARCAM, Figure 1A) exhibits an enhanced fluorescence intensity upon binding to Aβ aggregates in solution and in tissue compared to the free probe in solution.^21–23 We hypothesize that the binding of ARCAM to an amyloid binding pocket can restrict the rotation of specific bonds (e.g., bonds a and b in Figure 1B) between the D and A groups, leading to a higher fraction of molecules in the LE state versus the TICT state and resulting in a higher overall emission intensity compared to unbound molecules. This enhanced fluorescence property of ARCAM when bound to amyloids has recently been shown to enable and facilitate the ante-mortem detection of Aβ-containing deposits in the retinas of a mouse model for AD.²³

Figure 1.

Examples of amyloid-binding molecular rotor fluorophores. (A) Structure of ARCAM (1). (B) 3D rendering of ARCAM highlighting potential rotatable bonds (a and b) between the electron donor (blue) and acceptor (yellow). WSG = water solubilizing group. (C) Structures of ThT, CR, and new fluorescent ARCAM analogues 2–5.

To develop molecular rotors with an improved fluorescence contrast when bound to amyloid versus free in solution, here we systematically introduced small aliphatic substituents on the vinyl group or on the 2-position of the piperidine of ARCAM (compounds 2–5, Figure 1C) to explore whether these modifications could affect the fluorescence intensity of the probe when bound versus unbound to Aβ aggregates. We hypothesized that these substituents could sterically impede the rotation of bonds a or b between the naphthalene and the donor piperidine or acceptor cyano groups in ARCAM. Introducing these steric interactions could, in turn, disfavor the formation of the LE state (or promote the formation of the TICT state) and reduce the overall background fluorescence intensity of the free molecule in solution. We also hypothesized that the binding of these rotationally restricted analogues of ARCAM (2–5) to amyloid aggregates could help drive a planarization of the molecules and result in an increased ratio of the LE versus TICT photoexcited state, leading to an improved enhancement of the fluorescence intensity of the amyloid-bound compounds compared to the parent ARCAM compound. We compared the effects of aliphatic substituents on the quantum yield (QY) of ARCAM analogues 2–5 as well as the relative fluorescence enhancement of these probes bound to Aβ aggregates in solution. We also investigated whether electronic effects of aliphatic substituents could influence the excitation or emission profile of these ARCAM analogues. We found that only certain aliphatic substituents on the ARCAM scaffold led to an improvement of the fluorescence enhancement when bound versus unbound to amyloid aggregates, which represents an important step toward developing new design principles for generating high-contrast fluorescent probes for detecting amyloids associated with neurodegenerative diseases.

RESULTS AND DISCUSSION

Within the ARCAM scaffold, there are two potential rotatable bonds between the D and A that may affect the fluorescence intensity, one between the piperidine and the 6 position of the naphthalene group (rotatable bond a, Figure 1B) and one between the vinyl and the 1 position of the naphthalene group (rotatable bond b). In order to influence the rotational barrier and planarity of the molecule at rotatable bond a, we designed and synthesized compounds 4 and 5 (Figure 1C), which contained either a methyl or ethyl group on the 2-piperidinyl position of the ARCAM scaffold. To affect the rotation and planarity of rotatable bond b, we designed and synthesized compounds 2 and 3 (Figure 1C), which comprised either a methyl or trifluoromethyl group on the vinylic position of the ARCAM scaffold.

The synthesis of ARCAM (1) was reported previously.²¹ The syntheses of ARCAM analogues 2–5 are shown in Scheme 1. Briefly, commercially available methyl 6-bromo-2-naphthoate (6) was converted to the corresponding bromoaldehyde (7) by a reduction of the ester using diisobutylaluminum hydride (DIBAL-H), followed by an oxidation of the primary alcohol to the aldehyde using pyridinium chlorochromate (PCC). Intermediate 7 was then further reacted in three ways, namely, (1) for the synthesis of piperidinylketone 8 through a nucleophilic attack with methyl Grignard, oxidation to the ketone, and Buchwald-Hartwig coupling with piperidine, (2) for the synthesis of trifluoromethylketone 9 by first a Buchwald-Hartwig coupling with piperidine, followed by a nucleophilic attack with (trifluoromethyl)trimethylsilane and oxidation, or (3) for the synthesis of piperidinylaldehydes 10 and 11 through a Buchwald-Hartwig coupling with 2-methyl or 2-ethylpiperidine, respectively. Intermediates 8–11 were then subjected to a Knoevenagel condensation with previously reported²¹ α-cyanoamide 12 to afford fluorescent ARCAM analogues 2–5. Because of the limitations on the commercial availability of enantiomerically pure 2-substituted piperidines, we generated ARCAM analogues 4 and 5 as racemic mixtures in this initial study. The Supporting Information contains the details of the synthesis and characterization of these probes.

Scheme 1.

Synthetic Route for the Preparation of Fluorescent Compounds 2–5

With compounds 1–5 in hand, we first evaluated whether aliphatic substituents near the rotatable single bonds a and b on the ARCAM scaffold (Figure 1B) had an effect on the QY of the free probes in solution compared to the parent ARCAM compound (see the Supporting Information Figures S1–S5). We measured the absorption and emission spectra of compounds 1–5 in an aqueous solution using a similar protocol as previously described for an estimation of the QY of ThT.²⁴ Table 1 summarizes the estimates for QY for compounds 1–5. These solution studies revealed that the QY of ARCAM (1) is an order of magnitude higher than the QY we estimated for ThT (0.0006) (see the Supporting Information Figure S6). Interestingly, in all cases where we introduced aliphatic substituents on the ARCAM scaffold, the QY decreased compared to the parent ARCAM (1), with the substituents on the vinylic position causing a larger decrease in QY than substituents on the piperidine ring (Table 1). This trend of decreased QY for substituted ARCAM analogues was reflected in the relative intensity of the fluorescence emission spectra of the free probes (black lines, Figure 2) in aqueous solution, as compounds 2 and 3 decreased in fluorescence intensity by 2 orders of magnitude compared to ARCAM (1), while the fluorescence intensity of free compounds 4 and 5 in solution were lower but similar in magnitude to that of ARCAM (1). These trends did not change when the emission was normalized to account for the slight differences in absorbance levels of each probe (see the Supporting Information Figure S7).

Table 1.

Spectroscopic and Amyloid-Binding Characteristics of Compounds 1–5

Cmpd no.	Ex (λmax) (Free) (nm)	Ex (λmax) (with Aβ) (nm)	Ex (λmax) (Free) (nm)	Ex (λmax) (with Aβ) (nm)	fold increase with Aβ	KD with Aβ (μM)	QY
1	409 ± 11	421 ± 4	590 ± 4	560 ± 10	2.1 ± 0.7	6.3 ± 0.7	0.0066 ± 0.0023
2	372 ± 14	364 ± 6	601 ± 2	547 ± 4	3.5 ± 0.9	5.0 ± 1.7	0.0016 ± 0.0005
3	419 ± 4	410 ± 5	547 ± 5	514 ± 6	5.4 ± 0.9	3.7 ± 1.1	0.0012 ± 0.0004
4	431 ± 4	439 ± 3	583 ± 1	559 ± 8	1.9 ± 0.5	4.4 ± 2.3	0.0062 ± 0.0003
5	445 ± 1	443 ± 5	575 ± 6	567 ± 5	1.2 ± 0.1	3.2 ± 0.6	0.0055 ± 0.0010

Figure 2.

Fluorescence emission spectra probes 1–5 free in aqueous solution (black) or in the presence of aggregated Aβ (red).

In order to probe whether the aliphatic substituents could introduce any electronic effects on the spectroscopic properties of ARCAM analogues, we compared the excitation (black dotted lines, Supporting Information Figure S8) and emission (black lines, Figure 2 and Supporting Information Figure S7) profiles of compounds 1–5. We found that an introduction of the methyl or ethyl group on the piperidine ring (as in probes 4 and 5, respectively) led to a bathochromic or red-shift in the excitation maximum (Table 1) relative to ARCAM (1). These observations suggest that an introduction of σ-donating groups near the electron-donating nitrogen within the piperidine group lowers the energy required for photoexcitation. The introduction of substituents on the vinylic position of the ARCAM scaffold also exhibited an effect on excitation maxima, where a σ-donating methyl group on the vinylic position (as in probe 2) led to a hypsochromic or blue-shift in the excitation maximum, and a σ-withdrawing trifluoromethyl group on the vinylic position (as in probe 3) led to a bathochromic shift in the excitation maximum compared to the parent ARCAM (1); the effects of substituents at the vinylic position in probes 2 and 3 are consistent with the expectation that σ-donating groups near the electron acceptor of the fluorophore will increase the energy required to generate the photoexcited state and that σ-withdrawing groups will have the opposite effect. For the effects of substituents on emission profiles, we found that the introduction of the methyl group in 2 resulted in a bathochromic shift in the maximum emission wavelength (λ_max) of the free probe in solution compared to the parent ARCAM (1) (Table 1), whereas the introduction of the trifluoromethyl group in 3 resulted in a hypsochromic shift in emission λ_max compared to 1. These observations suggest that, on the one hand, electron-donating groups (e.g., the methyl group in 2) near the electron-acceptor region (e.g., the nitrile group in 1–5) of the molecular rotor can help stabilize the dipolar photoexcited LE state, leading to a lower energy (or higher wavelength) of photon emission, whereas electron-withdrawing groups (e.g., the trifluoromethyl group in 3) at the same position lead to a destabilization of the LE state and a higher energy (or lower wavelength) of photon emission upon relaxation to the ground state. Electron-donating substituents on the 2-piperidinyl position of the ARCAM scaffold (e.g., the methyl or ethyl groups in 4 and 5), on the other hand, apparently increased the energy of fluorescence emission of the free probe compared to the parent ARCAM probe, albeit the effect was relatively small. The excitation and emission λ_max of 1–5 spanned ranges of 73 and 54 nm, respectively, demonstrating that additions of small aliphatic groups on the ARCAM scaffold can lead to very large changes in spectral characteristics of these fluorophores and can help fine-tune the spectroscopic properties of these probes for specialized applications.^14,16,17,25

In order to examine the binding and fluorescence properties of ARCAM and its analogues in the presence of Aβ aggregates, we prepared a solution of aggregated Aβ(1–42) peptides using a previously reported protocol (see the Methods section for details of this preparation and the Supporting Information for the characterization).²⁶ Binding measurements²¹ revealed that all of the compounds 1–5 bound with similar low micromolar affinities to aggregated Aβ (Table 1 and Supporting Information Figure S9), demonstrating that small aliphatic substituents on the ARCAM scaffold do not significantly affect the binding to amyloids. Interestingly, all of the probes with aliphatic substituents show a trend of stronger binding to Aβ aggregates, which could be due to increased hydrophobic interactions betwen these ARCAM analogues with the binding pockets on Aβ aggregates compared to the parent ARCAM (1).²⁷ However, we found substantial differences in the fluorescence enhancement properties between these probes in amyloid-containing versus amyloid-free solutions. For the parent compound ARCAM (1), we observed a 2.1-fold increase in the fluorescence intensity in the presence of aggregated Aβ compared to the background fluorescence of the probe in the absence of Aβ (Figure 2 and Table 1). For compounds 2 and 3, containing substituents on the vinylic group, we found a 3.5- and 5.4-fold increase in the fluorescence intensity in the presence of aggregated Aβ compared to the background fluorescence, respectively. ARCAM analogues 4 and 5 with substituents on the piperidine ring, however, exhibited only a 1.9- and 1.2-fold increase in the fluorescence intensity in the presence of aggregated Aβ compared to the background, respectively. While the changes in the maximal excitation wavelength of all probes varied upon binding to aggregated Aβ (Table 1 and Supporting Information Figure S8 (red dotted lines)), all of the probes displayed a hypsochromic shift in emission λ_max in the presence of aggregated Aβ compared to free probes in solution. These observations are consistent with previous studies on ARCAM (1) and suggest that spectroscopic measurements for all of the probes in the presence of aggregated Aβ were dominated by their fluorescence properties in the bound state.^{14,15,17–21} These results also demonstrate that the location and the identity of the substituent on the ARCAM scaffold are important for exhibiting the overall effect on the fluorescence enhancement of these probes in the presence of aggregated Aβ. While aliphatic substituents at the vinylic position on ARCAM significantly lowered the background fluorescence of these molecules, these substituents apparently had a larger effect on increasing the relative fluorescence intensity when bound to an amyloid. Conversely, aliphatic substituents on the 2-position of the piperidine ring of ARCAM appear to only lower the fluorescence enhancement properties of the probes when bound to amyloids, without any substantial effect on the fluorescence of the free probes in solution.

In conclusion, we show that aliphatic substituents near the rotatable bonds of ARCAM can affect the fluorescence properties of probes both free in solution and when bound to aggregated Aβ. When we introduced substituents on the vinylic position of the ARCAM scaffold, the overall brightness of the probes was lowered, but the enhancement upon binding to aggregated Aβ compared to background was increased. When aliphatic substituents are introduced on the 2-piperidinyl position of the ARCAM scaffold, the overall brightness of the free probe in solution was essentially unchanged, but the relative fluorescence intensity of these probes upon binding to aggregated Aβ was decreased compared to the parent ARCAM compound. We also observed that electronic effects of aliphatic substituents on the vinylic position of ARCAM can affect the emission wavelength of the free probe, with a red-shift or blue-shift depending on whether the group is electron-donating or electron-withdrawing, respectively. Such changes in the emission wavelength are typically achieved by varying the extent of conjugation in the π-framework of the D-π-A motif of molecular rotors,¹⁴ rather than structural changes as simple as the introduction of an aliphatic group outside of the π-system. Probes 1–5 all had similar binding affinities to aggregated Aβ, supporting that the changes in spectroscopic properties observed in the presence of aggregated Aβ were not due to differences in the amyloid-binding capabilities of these compounds. This study shows that consideration of the rotational freedom in the design of molecular rotors can lead to fluorescent amyloid-targeting probes with an increased contrast between bound and unbound states, which may serve as new design principles for tuning both their spectroscopic as well as fluorescence enhancement properties for development as chemical tools to aid in the diagnosis of amyloid-associated diseases.

METHODS

Synthesis and Characterization of ARCAM Analogues.

The synthesis of ARCAM (1) was reported previously.²¹ The Supporting Information summarizes the synthesis and characterization for compounds 2–5.

Quantum Yield Measurements.

To determine the QY of compounds 1–5, we used a procedure that was similar to a previously reported protocol for estimating the QY for Thioflavin T.²⁴ Briefly, we first measured the absorbance spectrum of each probe (in 5% dimethyl sulfoxide (DMSO)/H₂O), in addition to the absorbance spectrum for the reference standard (Coumarin 30, QY = 0.67 in acetonitrile).²⁸ The wavelength at which the two normalized absorbance curves intersected was used as the excitation wavelength. A serial dilution of each probe and the standard was made with absorbance values at the excitation wavelength in the range from 0 to 0.1 absorbance units. At each concentration, the emission spectra for each probe and the standard were measured. Technical triplicates of these experiments were performed for all probes, and the averages were recorded. From these emission spectra, the area under the curve was calculated and plotted against the absorbance at each concentration (see Figures S1–S6 in the Supporting Information). The data were fitted to eq 1 to obtain estimates for QY

$$\text{Q}{\text{Y}}_{\text{p}}=\text{Q}{\text{Y}}_{\text{r}}\left(\frac{A_{\text{r}}}{A_{\text{p}}}\right)\left(\frac{E_{\text{p}}}{E_{\text{r}}}\right){\left(\frac{{\eta }_{\text{p}}}{{\eta }_{\text{r}}}\right)}^2$$ (1)

where QY represents the quantum yield, A represents the absorbance, E represents the integrated fluorescence emission, η represents the refractive index of the solvent,^29,30 and the “p” and “r” subscripts signify the probe or the reference compound, respectively.

Preparation of Aggregated Aβ (1–42) Peptides.

Synthetic Aβ (1–42) peptide was purchased from Biopeptide, Co., LLC. Aggregated Aβ (1–42) was prepared as previously described.²⁶ Briefly, Aβ (1–42) was dissolved in 100% 1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP) to a concentration of 1 mM and put on a shaker at room temperature (RT) for 24 h. The solution was then diluted in cold nanopure water (2:1 H₂O/HFIP). Aliquoted fractions were lyophilized for 3 d before being dissolved in nanopure water to a concentration of 100 μM. Aggregated Aβ solutions were incubated and shaken at 37 °C for 3 d before use. The formation of soluble aggregates (67% fibrils as a mixture with 13% oligomers and 20% monomer) was confirmed using a standard ThT assay and by Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel analysis (see the Supporting Information, Figures S10 and S11).

Estimation of Binding Constant (K_D) to Aggregated Aβ (1–42) Peptides.

Aggregated Aβ(1–42) at a final concentration of 5 μM (based on the molecular weight of monomer) was mixed with an increasing concentration of each probe in 5% DMSO in nanopure water. K_D values were determined as previously described.²¹ The data shown in Supporting Information Figure S9 and in Table 1 represent average values and standard deviations from measurements of three independent experiments.

Estimation of Fluorescence Enhancement of Probes when Bound Versus Unbound to Aggregated Aβ (1–42) Peptides.

Aggregated Aβ(1–42) at a final concentration of 5 μM (based on the molecular weight of monomer) was mixed with each fluorescent probe at a final concentration of 4 μM in 5% DMSO in nanopure water. The solution (80 μL) was transferred to a black opaque 96-well plate, and the fluorescence was read using a microplate reader. Technical triplicates of these experiments were performed for all probes, and the averages and standard deviations were recorded. For all probes, the relative fluorescence enhancement of the bound versus unbound state was estimated by comparing the intensity at λ_max of the amyloid-bound state.

ABBREVIATIONS

Aβ

Amyloid Beta

AD

Alzheimer’s Disease

ARCAM

Aryl Cyano Amide

PET

Positron Emission Tomography

ThT

Thioflavin-T

CR

Congo Red

D

Donor

A

Acceptor

LE

Locally Excited

TICT

Twisted Intramolecular Charge-Transfer

QY

Quantum Yield

DIBAL-H

Diisobutylaluminum Hydride

PCC

Pyridinium Chlorochromate

DMSO

Dimethyl Sulfoxide

H₂O

Water

HFIP

1,1,1,3,3,3-Hexafluoroisopropanol

RT

Room Temperature

SDS-PAGE

Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis