New Cross-Linking Quinoline and Quinolone Derivatives for Sensitive Fluorescent Labeling

A variety of contemporary analytical platforms, utilized in technical and biological applications, take advantage of labeling the objects of interest with fluorescent tracers—compounds that can be easily and sensitively detected. Here we describe the synthesis of new fluorescent quinoline and quinolone compounds, whose light emission can be conveniently tuned by simple structural modifications. Some of these compounds can be used as sensitizers for lanthanide emission in design of highly sensitive luminescent probes. In addition, we also describe simple efficient derivatization reactions that allow introduction of amine- or click-reactive cross-linking groups into the fluorophores. The reactivity of synthesized compounds was confirmed in reactions with low molecular weight nucleophiles, or alkynes, as well as with click-reactive DNA-oligonucleotide containing synthetically introduced alkyne groups. These reactive derivatives can be used for covalent attachment of the fluorophores to various biomolecules of interest including nucleic acids, proteins, living cells and small cellular metabolites. Obtained compounds are characterized using NMR, steady-state fluorescence spectroscopy as well as UV absorption spectroscopy.


Introduction
Fluorescent labels are used in numerous applications that relay on sensitive detection of biological macromolecules (proteins, nucleic acids, polycarbohydrates, etc.), as well as for specific labeling of cells and tissues. In these applications, the fluorophore reporter groups are illuminated by visible or UV light, which leads to absorption of the light quantum and excitation of the molecule. The excited state is unstable and tends to relax either through dissipation of the absorbed energy by collision with other components in the medium or by emission of light. This light can be subsequently detected. Detection sensitivity is proportional to the number of the light quanta emitted by the fluorophore, which in turn is a linear function of the intensity of the excitation light. Therefore, for sensitive detection, high intensity light sources are employed. This creates the problem of discrimination of excitation and emission light, since even a small fraction of the excitation light that reaches the detector can cause significant background and decreases the detection sensitivity. This problem can be alleviated by using fluorophores with large spectral distances between excitation and emission light (Stokes shift). Quinolone [1] and quinoline fluorophores, discovered in the course of present study, possess the desired property. In this paper, we describe the synthesis and reaction mechanisms for new derivatives of these fluorophores that are suitable for attachment to biological macromolecules. We found that 7-aminoquinolones can be conveniently modified at either the 1-amido-or 2oxogroup, yielding corresponding quinolone and quinoline derivatives with preserved fluorescent properties and large Stokes shift (ca. 50-110 nm). Subsequent modification of the resulting compounds at the 7-amino group allows tuning of the fluorescence from deep blue to green emission. Using analogous modifications, we synthesized amine-reactive isothiocyano-derivatives, as well as azido derivatives capable to click-react with acetylenic counterparts [for review see [2][3][4]. Reactivity of the compounds was verified in reactions with cysteine and alkyne-derivatized DNA oligos. The results suggest suitability of the new reactive probes for fluorescent labeling with detection limit in the nanomolar range.

Investigation of the Reaction Mechanism for Quinolone and Quinoline Fluorophore Formation
In our previous study [5], during the synthesis of carbostyril derivatives along with expected quinolone compound cs-124-CF 3 [1], we detected a new fluorescent product which was not previously described. Since the compound was highly fluorescent and displayed large Stokes shift (120 nm), we set up to identify the compound and to study the reaction mechanism in more details in order to determine the influence of the reaction conditions on the product yield. The suggested mechanism for the reaction between ethyl 4,4,4-trifluoroacetoacetate with 1,3 phenylenediamine is shown in Scheme 1. Chromatographic analysis revealed that incubation of the starting compounds results in quick accumulation of an unknown fluorescent product (compound IVof Scheme 1, which we named Qin124-CF 3 ) with R f 00.9, along with the expected compound cs124-CF 3 (R f 00.44). Some non-fluorescent compounds, possibly reaction intermediates were also detected (compound V of Scheme 1, R f 00.62, and another product with R f 00.84). Continued incubation of these purified non-fluorescent products in the original reaction conditions showed that the compound V slowly converted into fluorescent cs124-CF 3 (compound VI of Scheme 1, R f 00.44). At the same time, incubation of purified compound V in the reaction conditions did not lead to compound IV, suggesting that compound IV originates from an earlier reaction intermediate (possibly from compound II). This is consistent with the fact that fluorescent compound IV stops accumulating after intermediate V is completely formed in the course of the synthetic reaction (Fig. S1). Finally, the non-fluorescent reaction product with R f 00.84 was stable at the incubation conditions and therefore represented a side-product. For compound II, elimination of ethanol would lead to the observed precursor of cs124-CF 3 (compound V), while dehydration would create compound III, which finally converts to fluorescent product IV. The identity of compounds IV, V, and VI was confirmed by NMR spectroscopy. In addition, we have shown that product IV was authentic to the compound obtained by O-ethylation of cs124-CF 3 (see below) as judged by chromatographic mobility, UV absorption, fluorescence, and NMR spectroscopy. The formation of a similar derivative related to compound IV has been reported before, when 3-aminophenol was incubated with trifluoroacetoacetate to yield 7-hydroxyquinoline [6], reflecting a common reaction mechanism. The structure of the initial reaction intermediate is more  There are also two nucleophilic centers in phenylenediamine (amino groups and carbons of the ring in positions 4 and 6) that can be potentially attacked by acetoacetate derivative. Thus initial reaction can proceed through acylation of the amino group (compound I) followed by intramolecular attack of the phenyl ring (product II). As indicated by UV absorption spectroscopy and chromatographic mobility, the same fluorescent compounds were formed at both 50°C and 110°C. However, high temperature dramatically accelerates the formation of the quinolone derivative (from intermediate V), while the amount of the produced quinoline compound was not effected in accordance with proposed kinetic scheme (Scheme 1).
We found that compound V-to-compound VI conversion is sharply accelerated (ca. 10 6 times) in the presence of NaOH. The same, but less pronounced effect (ca. 50 times) was observed upon the addition of a catalytic amount of trifluoroacetic acid to the starting reaction mixture. These findings allow dramatic reduction of both reaction temperature and the reaction time and nearly quantitative conversion of starting compounds to fluorophores IV and VI.

Synthesis of Reactive Quinolone and Quinoline Derivatives (Chart 1)
Our next goal was the synthesis of cross-linkable derivatives of cs124-CF 3 and the newly discovered quinoline fluorescent derivative Qin124-CF 3 . To this end, we investigated the possibility of a chemical modification of these fluorophores. By analogy with previous observations [7,8], we reasoned that the amide group of cs124-CF 3 in ionized form can undergo alkylation, thus allowing introduction of cross-linking groups into the core moiety (Scheme 2). Indeed, incubation of cs124-CF 3 with ethyl ester of p-toluenesulfonic acid in the presence of NaOH yielded two fluorescent products migrating with R f 00.44 and 0.9 on TLC, using an ethylacetate developing system. We proposed that these two products originate due to alkylation at the amido group nitrogen and oxygen that can assume a negative charge (providing high reactivity) as a result of lactim-lactam tautomery (see Scheme 2). These alkylation reactions would afford quinolone (VII) and quinoline (VIII) compounds, correspondingly. Indeed, NMR analysis confirmed the proposed structures. Next, we performed the same reaction, but with 1-iodo-3-azidopropane, alkylating compound bearing azido group (Scheme 3). In the other version we treated the fluorophore with bifunctional alkylating agent, 4,4′-bis(chloromethyl) biphenyl and then introduced the azido group by subsequent reaction with lithium azide. The final products (compounds IX and X) of Scheme 3 as well as compounds XIII and XIVof Scheme 4 can be used directly for coupling to the molecules of interest via the "click"-reaction with alkyne counterparts pre-attached to the molecules of interest. Alternatively, the azido group can be reduced to an amino group (compound XI) that can be converted to amine-reactive isothiocyano group (compound XII), or to thiol-reactive bromoacetamido group. The reduction of Structures of the synthesized reactive fluorophore derivatives the azido-compound was performed with high yield by treatment with triphenylphosphine followed by incubation with ammonium hydroxide. Isothiocyano derivative was obtained by reaction of products XI with thiocarbonyldiimidazole, and subsequent treatment with trifluoroacetic acid. The reactivity of the resulting compounds was confirmed by reaction with cysteine using TLC analysis.
We also used similar approaches for the synthesis of biphenyl derivatives of 4-methylquinolones and 4trifluoromethylquinolones (compounds XIII to XVI) (Scheme 4). The cross-linkable quinoline compounds could also be obtained using modified derivatives of ethyl 4,4,4trifluoroacetoacetate by adapting the protocols published in our previous research for the synthesis of analogous quinolone compounds [5]. In this way, we first performed the alkylation of ethyl 4,4,4-trifluoroacetoacetate with methyl ester of bromoacetic acid at methylene carbon (Scheme 5). The resulting intermediate was incubated with 1,3-phenylenediamine at moderate temperature that favors formation of quinoline compound (product XVII of Scheme 5) that was converted to cadroxylate (compound XVIII) by saponification. This compound was treated with carbodiimide and 4-nitrophenole resulting in an activated ester that was introduced in reaction with mono-tritylated 1,4-diaminobutane yielding compound XIX. Deprotection followed by treatment of the resulting amino-derivative with N,N-thiocarbonyldiimidazole and trifluoroacetic acid afforded product compound XX, an isothiocyanate derivative.

UV Absorption and Fluorescent Spectra of the Synthesized Compounds
As seen from Figure 1 and Table 1, the synthesized compounds have absorption maxima in the register 200-300 nm, as well as longer wavelength absorption (300-400 nm), which is optimal for fluorescence excitation. The molar extinction for the compounds in this far UV region vary from 6000 to ca. 19 000 M −1 sm −1 . Generally, quinolone compounds in the range 300-400 nm have extinction coefficients two to three fold greater than corresponding quinoline compounds (compare compounds IX and X; XIII and XIV). Quantum yields for compounds XIII and XIV are comparable, while quantum yield for X is half of that for IX. Among corresponding quinolone and quinoline derivatives of 4-methyl substituted compounds, quantum yields differ insignificantly as well (compare comp. XV and XVI).
In order to generate fluorophores with various colors, we examined how modification of 7-amino group of quinoline OEt Et-X

Et
Et-X X = p-toluenesulfonyl VIII Scheme 2 N-and O-alkylation of a quinolone fluorophore compounds effect their emission spectrum. Thus we obtained mono-and dimethylamino-derivatives (compounds XXI and XXII correspondingly) and related acetamido-derivative (compound XXIII). As seen from Table 1 and Figure 2 these modifications significantly changed fluorescent properties of the original fluorophore (compound X). Thus methylation of the aminogroup caused red shift, while acylation resulted in strong blue shift of the emission spectrum. While methylation did not affect quantum yield of the fluorophore, acetylation caused a two fold reduction in quantum yield. Comparing to trifluoromethyl fluorophores (compounds IX and X) corresponding 4-methyl derivatives (compounds XV and XVI) displayed a blue shift of the fluorescence emission (Table 1 and Fig. 2). Generally, Stokes shifts were larger for 4trifluoromethyl compounds comparing to the corresponding 4-methyl fluorophores. The emission spectra of the synthesized reactive fluorescent compounds cover all colors from deep blue to green (Figs. 2 and 3), which makes them very useful as labels in biochemical and technical applications.

Chemical Reactivity of Synthesized Compounds
In the present study, we obtained amine and thiol-reactive isothiocyano (ITC), as well as azido-derivatives of fluorophores, which are suitable for click-reaction with acetylenic counterparts, catalyzed by copper complexes. The reactivity of ITC compounds was examined with cysteine at weakly alkaline conditions favoring ionization of thiol group. The reaction proceeded quickly and quantitatively at room temperature, yielding dithiocarbamate derivatives as was evidenced by strongly reduced mobility of the reaction products on TLC.
The same effect was observed in reaction of ITC compounds with ethylenediamine at 50°C. Click reactivity of azido-fluorophores was confirmed by incubation with alkyne-derivatized oligonucleotides in the presence of copper complex and ascorbate. HPLC analysis of the reaction mixture revealed nearly quantitative coupling of the fluorophores to the oligos (Fig. S2). These results suggest suitability of the synthesized compounds for fluorescent DNA labeling. It should be mentioned that attachment of fluorophores to DNA was accompanied by considerable quenching (Table 2), which was most likely due to stacking interaction of the fluorophores with DNA bases.

Conclusion
In the present research, we explored new synthetic strategies to obtain fluorescent derivatives of quinolone fluorophores. In the course of optimization of the reaction conditions for the synthesis of 4-trifluoromethyl quinolone compound, we observed Scheme 4 Synthesis scheme of cs124 reactive derivative with a biphenyl spacer a previously unknown product with useful fluorescent properties. This prompted us to investigate the reaction mechanism in more details. NMR spectrum analysis, along with kinetic data of the product accumulation suggested that this new compound represents 7-amino-4-trifluoromethyl-2-ethoxyquinoline. The identity of the product was confirmed by its independent synthesis route through ethylation of 7-amino-4-trifluoromethylquinolone. Further characterization of this compound (Table 1) revealed its valuable fluorescent properties, comprising a large Stokes shift (104 nm) and a high quantum yield (ca. 0.3). Next, we explored the possibility to modify quinolone compounds with the aim to introduce cross-linkable groups for fluorescent labeling. We observed that quinolones can be easily modified by alkylation either at the amide nitrogen, or oxygen to yield N-1 derivatives of quinolone, or O-2 derivatives of quinoline, correspondingly. The reaction proceeds with a high yield under alkaline conditions that promote ionization of the amido group. Using this reaction, we further obtained cross-linkable derivatives containing isothiocyano-, or azido groups. In addition, we explored alternative ways to introduce cross-linking group into quinoline moiety using a previously described reaction of 1,3-phenylenediamine with trifluoromethylmethylethylsuccinate that proved useful for corresponding quinolone derivatives. The desired compound was obtained with reasonable yield by optimization of the reaction temperature. Structurally related 4-methyl quinolone and quinoline fluorescent derivatives were obtained using analogous derivatization reactions. These derivatives exhibit considerable blue emission shift compared to 4-trifluoromethyl counterparts. The obtained fluorescent compounds can be further modified at the 7-amino group, allowing tuning of fluorescence emission, so that altogether the synthesized reactive compounds span emission register from deep blue to green. These fluorescent compounds can be efficiently crosslinked to DNA and proteins, which makes them valuable probes for biochemical applications with detection limit in the nanomolar range.

1-iodo-3-azido propane.
Nine grams of 1,3-diiodopropane were mixed with 15 ml of DMF and supplemented with 1.5 g of lithium azide. After 1 h incubation at 37°C, TLC analysis using hexane as developing solvent revealed one reaction product with R f 00.2 along with starting 1,3-diiodopropane (R f 00.4). The mixture was poured into 150 ml of water and extracted with an equal volume of ether. The organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was dissolved in hexane and subjected to silicagel column chromatography using the same solvent as eluent. The fractions corresponding to product were collected and evaporated to dryness. Yield02.4 g, (30%).