β -KETOENOLE DYES: SYNTHESIS AND STUDY AS FLUORESCENT SENSORS FOR PROTEIN AMYLOID AGGREGATES

This work supported in the framework of Marie Skłodowska-Curie Research and Innovation (RISE) ABSTRACT The series of the new β-ketoenole dyes ((2E,5Z,7E,9E)-6-hydroxy-2-(alkylamino)-10-phenyldeca-2,5,7,9-tetraen-4-ones) with variation of alkylamino tail groups was synthesized and studied as potential probes for the sensing of protein aggregates amyloid fibrils. The dyes are low fluorescent when free but able to increase their emission intensity in dozens of times in the presence of fibrillar insulin. The fluorescent response of the dye on fibrillar insulin strongly depends on the nature of the alkylamino tail group. For compounds with propylamino (dye 13 ) and 2-hydroxyethylamino (dye 14 ) fragments the fluorescence intensity in the presence of fibrillar insulin exceeds that for the native one in 22 and 66 times correspondingly. However dyes demonstrate from low or moderate exceed of the fluorescence intensity in the presence of aggregated lysozyme compared to native one (up to 8.7 times for the dye 53 bearing methyl ester tail group), due to their pronounced sensitivity to native lysozyme. The dyes in complexes with insulin have rather height quantum yield up to 0.15, the large Stokes shifts values (about 100 nm and more), their binding constant values are about 10 5 M -1 . The dye 14 allows fluorescent detection of the insulin amyloid fibrils in the concentration range 1-50 μg/ml. This causes an interest in the future study of the β-ketoenole as prospective fluorescent amyloid-sensitive molecules.


INTRODUCTION
One of the most convenient methods for the analysis of biomolecules is the use of extrinsic fluorescent probes that noncovalently bind to them by electrostatic, van der Waals and hydrophobic interactions. The wide range of fluorescent molecules is developed for high efficient sensing, quantification and visualization of proteins and nucleic acids in in vitro and in vivo assays [1][2][3][4].
The spontaneous aggregation of proteins leading to formation of insoluble betapleated aggregates (amyloid fibrils) is among the actual targets in the biomedical researches, since these aggregates are connected with the range of harmful human diseases including neurodegenerative ones. This causes an interest in the development of new appropriate analytical tools to be used in the study of this process.
The extrinsic fluorescent probes are used for the detection and quantification of amyloid fibrils, monitoring of the kinetics of their formation and study of the factors and agents affecting these processes. For these purposes, amyloid sensitive fluorescent probes Thioflavin T and it`s derivatives are commonly applied. The histological dyes Congo Red and Chrysamine G are used for the staining and study of amyloid formations on tissues [5][6][7]. Recently we discovered and developed mono and polymethine cyanines dyes as efficient fluorescent probes for the detection of protein β-pleated aggregates [8,9]. Cyanines with high sensitivity to the amyloid fibrils and wide detection range (1.5-120 µg/ml for trimethine cyanine 7519 [10]) surpassing that of Thioflavine T were proposed. On the base of these cyanine dyes the inhibitory assay for the search of the compounds with anti-fibrillogenic activity was developed and applied. At the same time further search for the amyloidsensitive probes with high fluorescent response to the fibrillar protein presence is still urgent.
One of the necessary requirements for the molecule to be applicable as the fluorescent probe for the amyloid formations detection is high affinity of the complex formation between this molecule and the amyloid fibril. As the most probable mode of such complex formation, the insertion of the dye molecule into the groove of the amyloid fibril is suggested [11]. As a result of such binding, the fluorescent response is observed due to the quite rigid fixation of the dye molecule, and the polarization of the absorbed light is caused by the same orientation the bound dye molecules. Thus, in order to obtain an efficient response to the presence of amyloid fibril, the molecule should have shape complimentary to that of the fibril groove and the size fitting the fibril groove (about 6,5-7 Å) [12]. (Fig 1)   Figure 1. Scheme of the layout of the dye molecule in the fibrilar groove (left) [11] and AFM image of the insulin amyloid fibrils (right) [13].
In the present work we firstly studied the β-ketoenole dyes as potential fluorescent probes for the sensing of amyloid aggregates of proteins (Table 1). The β-ketoenoles are the molecules of the elongated shape that is suggested as preferable for fitting to the groove of the amyloid fibril; besides they have rather flexible aliphatic chromophore chain providing the low intrinsic fluorescence intensity to the unbound dye. Unlikely to the majority of the amyloid-sensitive dyes bearing either positive (Thioflavin T, cyanine dyes) or negative (Congo Red) charge, the molecules of β-ketoenoles are uncharged.
With this aim the series of ((2E,5Z,7E,9E)-6-hydroxy-2-(alkylamino)-10phenyldeca-2,5,7,9-tetraen-4-ones) dyes with variation of alkylamyno substituents was synthesized and the fluorescent properties were characterized for the free dyes as well as in the presence of amyloidogenic proteins lysozyme and insulin in the native and aggregated form. The range of detection of the amyloid aggregates with the most efficient dye was determined. Besides, the fluorescent sensitivity of βketoenole dye to the serum albumin able to bind the variety of the small molecules was studied for the comparison.

Spectral measurements
Fluorescence excitation and emission spectra were registered using the fluorescent spectrophotometer Cary Eclipse (Varian, Austria). Fluorescence emission was excited at the maximum wavelength of excitation spectrum of corresponding dye solution. The quantum yield value of the dyes 14 and 50 (2 μM) in the presence of fibrillar insulin (13.6 μM) was determined using Rhodamine 6G solution in ethanol as the reference (quantum yield value 0.95) [15]. All the spectralluminescent characteristics of unbound dyes in aqueous buffer were studied at room temperature.

Estimation of equilibrium constants of the dye-to-fibril binding
To estimate the equilibrium constant of the dyes 14 and 50 binding to fibrillar insulin, fluorescent titration of the dyes (2 μM) upon addition of 0-45 μM of fibrillar insulin was performed. Taking into account only the points for the concentrations 2 M of protein molecules and higher, we could consider the concentration of the binding sites to be much higher than this of the dyes and thus the concentration of the free protein to be roughly equal to its total concentration. Under this assumption, the equation for the equilibrium constant K of dye-fibril binding could be written as: where C d , C bd and C F are total dye, fibril-bound dye and fibrillar protein concentrations respectively.
Further, let us consider the totally unbound and totally bound with fibrils dye solution to have the fluorescence intensity I 0 and I max respectively. In this case the measured dye fluorescence intensity I at the fibrillar protein concentration C F could be written as I = I 0 ×(C d -C bd )/C d + I max ×C bd /C d , that can be transformed into: C d /C bd = (I max -I 0 )/(I -I 0 ) Together with (1), (2) gives A being the denotation for (I max -I 0 ) difference. Thus the experimental dependence of I -I 0 on C F was approximated with the equation (3), A and K being obtained as approximation parameters. Accounting for several assumptions made, the obtained K value could be regarded as a rough estimation of the binding constant value rather than its precise value. Besides it should be reminded that the estimated binding constant is only an apparent value calculated with respect to protein globule concentration and not this of the binding sites that is unknown; actually the estimation of binding constant with respect to protein globule concentration is common for the ligand-fibril binding studies.

Computer simulations of the dye 14 dimensions
To estimate the dimensions of the dye 14, geometry optimization of the dye structure was first performed using the PM3 method from the HyperChem 6.03 program package. Further the isosurface with the total charge density 0.002 that characterizes the molecular dimensions was built; linear dimensions i.e. length, height and width of the obtained isosurface were then estimated.

Synthesis of the β-ketoenole dyes
In present work we obtained series of new compounds by the pyran ring opening reaction of 4-hydroxy-6-methyl-3 -((2E, 4E) -5-phenylpenta-2,4-dienoyl) -2H-pyran-2-one with primary aliphatic amines. The mechanism of this reaction was studied in [14,16], it occurs through the nucleophilic attack of the 6-carbon atom of the pyran cycle leading to its next opening and decarboxilation. We have found that on the first step of this reaction the interaction of 4-hydroxy-6-methyl-3 -((2E, 4E) -5-phenylpenta-2,4-dienoyl) -2H-pyran-2-one with amines (Scheme 1) leads to the formation of the corresponding salt. When heated, it is dissociated and at the same time the amino group attacks a carbon atom of the methyl group in the pyran ring, causing its opening and subsequent decarboxylation [14,16]. In the case of n-alkyl amines, formation of the corresponding salts, ring opening and decarboxylation occurs quite easily, but iso-amines react considerably worse and require higher reaction temperature.

Scheme. 1. Synthesis of the studied β-ketoenole dyes.
In the case of the tert-butylamine, the reaction stopped at the stage of the salt formation, opening of the pyran ring does not occur (Fig. 1), the behavior of phenyl ethyl amine is similar. This observation indirectly confirms the reaction mechanism proposed in [14,16].
Such molecules could be divided into two parts: hydrophobic ones containing phenyl moiety and polymethine chain, and hydrophilic molecules containing ketoenol fragment and tail alkylamino group. For related compounds the existence of the set of tautomeric forms of the ketoenol fragment was established by NMR [16]. The compounds described here are also able to the formation of the tautomers. In their NMR spectra in addition to the main groups of signals there are the minor signals with similar morphology corresponding to the presence of 3-6% of admixtures.
According to data of LC/MS the molecular ions of these admixtures have the same molecular weight as that of the base compound that also confirms the presence of the tautomeric forms of compounds (data not presented).

Spectral properties of dyes in buffer and in the presence of native proteins
The fluorescent characteristics of the 9 β-ketoenole compounds in buffer solution and in the presence of insulin, lysozyme and BSA in their native form are presented in Table 2.
The excitation maxima for the studied dyes in the free state are located in the   bearing the methyl ester as tail group possesses better sensitivity to insulin and BSA than to lysozyme.

Spectral properties of dyes in the presence of fibrillar proteins
Insertion of the dye molecule into the groove of the amyloid fibril is suggested to be the most possible model of the dye-fibril binding. With the help of the computer simulation, the dimensions of the dye 14 were estimated to be about 23Å×7.8Å×4.8Å, that is characteristic for the dyes of the studied series. Thus the studied dyes fit to the fibrillar groove formed by the β-pleated structure of the fibril, the width of which is believed to be equal to 6.5-7Å [12]. Thus, we can expect the formation of the dye-fibril fluorescent complex due to the fixation of the dye molecules in the fibril groove.
The fluorescent characteristics of the 9 β-ketoenole compounds in the presence of fibrillar aggregates of insulin and lysozyme are presented in Table 3.
For the majority of the studied β-ketoenole dyes, the addition of the fibrillar insulin results in the long-wavelength shift of the excitation maximum wavelength for up to 22 nm (except of the dye 88 with the 3 nm short-wavelength shift). The short-wavelength shifts of the emission maximum wavelength for up to 26 nm were also observed for the majority of the dyes except 50 (almost no change), 88 (7-nm long-wavelength shift) and 54 (two maxima shifted to the short-and longwavelength region with respect to the free dye were observed).  Generally the β-ketoenole molecules are uncharged, so its affinity to the charged amino acid residues would be higher in the case of the molecule's transition to the zwitterionic tautomer form (Fig. 3).

Fig. 3. Zwitterionic tautomer form of β-ketoenole dye
It was observed that some of β-ketoenoles are even more sensitive to the native lysozyme than to its well-structured beta-pleated aggregates that formed the binding places in the fibrillar grooves. Thus we could suggest that electrostatic interaction with charged groups mainly drive the interaction of the dye molecules to the lysozyme. The reorganization of the protein structure to beta-pleated aggregates does not lead to the essential increase in the number of tightly bound and thus fluorescent molecules, and can even decrease their number.

Application of dye 14 for fibrillar insulin detection: detection limit and linear detection range
To examine the applicability of the dye 14 bearing 2-hydroxyethyl group as amyloid-sensitive probe for the quantification of amyloid insulin, we performed titration of the 2 µM dyes solutions with increasing amounts of the aggregated protein (Fig. 4). The lower limit for the fibrillar insulin detection by the dye 14 was  It should be mentioned also that the large shifts between the excitation and emission maxima for the fibril-bound dye (about 100 nm), good fluorescence intensity increase, rather high quantum yield and sufficient detection range make the dye 14 promising as probe for fluorescent detection of the fibrillar aggregates.

CONCLUSION
The series of β-ketoenole dyes was firstly synthesized and characterized, their fluorescent properties as potential probes for the sensing of amyloid aggregates of proteins were studied. Thus we suggest the β-ketoenole dyes as prospective fluorescent molecules for the design on their base of the probes for the detection of the β-pleated protein aggregates and investigation of the aggregation reaction.