Chiral Fluorescence Recognition by Anthracene Fluorescent Dyes ⊂ Water-Soluble Pillar[5] arene containing Phosphonic Acid Group (PWP[5])

Chirality plays a pivotal role in drugs, agrochemicals and food additives et al. The enantiomers of a chiral molecule often show huge difference in bioactivity, metabolism, and toxicity et al. thereby, the recognition of chiral molecules shows an increasingly important priority. In this paper, a novel method for chiral fluorescence recognition based on anthracene fluorescent dyes (AD) ⊂ water-soluble pillar[5] arene containing phosphonic acid group (PWP[5]) is developed. The AD as guest molecule can complex with PWP [5] to form 1:1 AD ⊂ PWP[5] assembly, and this assembly can be further used as a fluorescent probe to identify D/L-phenylalanine and D/L-phenylalaninol by fluorescent titration. The fluorescence intensity of the assembly was significantly reduced for D-phenylalanine and D-phenylalaninol, while L-phenylalanine or L-phenylalaninol was added to AD ⊂ PWP[5] assembly, the fluorescence intensity of the assembly almost unchanged. Hence, the chiral recognition based on assembly between the achiral fused ring fluorescent dye and achiral PWP[5] was developed.


Introduction
Chirality means that an object cannot coincide with its mirror image, such as the relationship between our left and right hands [1]. Chiral molecules not only play an important role in medicine. biochemistry and agrochemistry et. al. [2], but are also widely applied in asymmetric organocatalysis, molecular recognition, and material functionalization. A pair of enantiomers have different pharmacological activities [3]. R-thalidomide can alleviate pregnancy reaction; thus, S-thalidomide not only has antiemetic effect, but also has severe teratogenic effect [4]. D-penicillamine has strong toxicity and potential carcinogenicity, and L-penicillamine has antiviral activity. As an essential amino acids for humans and animals, L-phenylalanine( Fig. 1)has broad physiological activity, and can also be used in synthesis of antiviral and anticancer drugs and sweeteners. D-phenylalanine can enhance human immunity and shows an excellent analgesic effect.
Pillar(n)arenes as the 5th generation macrocyclic host after crown ethers [5], cyclodextrins [6], calixarene [7] and cucurbituril [8] were first reported by Ogoshi [9] in 2008, and synthesized by condensation of hydroquinone ethers and paraformaldehyde with Lewis acid as a catalyst. Compared with other macrocyclic hosts, they have higher symmetry and rigidity, and high binding selectivity to the guest. Modification of pillar(n) arenes at two ports with different substituents can provide assembling diversity, and adjust lipophilic/hydrophilic properties to realize assembling in organic solvents or water.
At present, the traditional method of chiral recognition includes HPLC [10], CE [11], GC [12], CD [13] et al. However, these methods have some shortcomings and cannot meet the requirements of real-time, in-situ detection and high-quality screening in drug development.
So far, there are few reports about fluorescence chiral recognition based on assembly. Previously, our group has studied the cis/trans and chiral fluorescence recognition by NA ⊂ CB [7] assembly [14,15]. On this basis, in order to enhance stability of binary assembly and the π-π interaction in recognizing process, PWP [5] as a substitute of CB [7], and anthracene dye as a substitute of naphthalimide dye, we further studied characteristics of the achiral AD ⊂ achiral PWP [5] to hope to achieve better selectivity and sensitivity in fluorescence recognition of chiral molecules. In this paper, the assembly of AD ⊂ PWP [5] was constructed and characterized by fluorescence titration and 1 H NMR. The results showed that AD can be included by PWP [5] with 1:1 stoichiometry and fluorescence intensity decreased 6 times. When D-phenylalanine or   -phenylalaninol was added to the AD ⊂ PWP [5], the fluorescence intensity of the assembly further reduced 2.5 times and 2.7 times, while L-phenylalanine or L-phenylalaninol was added to AD ⊂ PWP [5], the fluorescence intensity of the AD ⊂ PWP [5] assembly almost unchanged. Thereby, a new method to recognize chiral substances based on AD ⊂ PWP [5] was developed.

Materials and Methods
The absorption spectra were determined by Agilent Cary 60 UV-spectrophotometer. The fluorescence titration spectra were measured on Perkin Elmer ls-55 fluorescence spectrophotometer. The excitation and emission slits were set at 3 nm and 5 nm. 1 H NMR spectra were recorded on Bruker 400 MHz spectrometer. Tetramethyl silane (TMS) was used as internal reference.
D/L-phenylalanine and D/L-phenylalaninol used in the experiment were purchased from Aladdin Biochemical Technology Co., Ltd.
PWP [5] was synthesized according to the method provided in the literature [16], AD was synthesized from anthracene. and the other reagents could be purchased and used directly.
Synthesis of 9-anthracene formaldehyde: (Fig. 2) Anthracene (4 g, 22.4 mmol) was dissolved in a mixture of 4.4 mL of DMF and 5 mL of 1,2-dichlorobenzene, to the mixture POCl 3 (6 mL, 65.3 mmol) was added under stirring. The solution was then heated at 100 °C for 2 h, and cooled down to 0 °C, saturated sodium acetate solution was added until pH 5-6, and stirred at r. t. for 10 min, extracted with DCM. The organic phase was evaporated under reduced pressure and then triturated with hexane, a yellow solid was obtained (1.5 g, 32%). 1 (Fig. 3). 13 (Fig. 4).

Formation of AD ⊂ PWP[5] Assembly
The binding behaviors of AD ⊂ PWP [5] was characterized by fluorescence and UV-Vis spectroscopy. AD (concentration is 6 × 10 −5 mol/L) was titrated by different concentration of PWP [5] solution (0 ~ 9 × 10 −5 mol/L), and its fluorescence curves (Fig. 9a) and inclusion constant (Fig. 10) were obtained, respectively. With the increase of PWP [5], the emission wavelength changes from 498 to 495 nm, and the fluorescence intensity decreased 6 times. From Fig. 9b, red shift of the absorption wavelength from 375 to 395 nm appeared, this phenomenon indicated an interaction between PWP [5] and AD. The fluorescence emission mechanism of anthracene dye AD belongs to intramolecular charge transfer (ICT), that is, the charge is transferred from the aminohexyl chain to the anthracene ring in the molecule. When AD was titrated with PWP [5], the aminohexyl group of AD was encapsulated by the cavity of PWP [5], which blocked electron transfer from amino chain to anthracene ring, led to fluorescence decrease. When the concentration of AD was equal to that of PWP [5], the fluorescence intensity decreased to minimum, Even if more PWP [5] was added, the fluorescence intensity almost unchanged, which indicates that AD and PWP [5] form a 1:1 complex. (Fig. 9).
Here we define ΔF by ΔF = F-F 0 . Where F represented the fluorescence intensity of PWP [5] at different concentrations of PWP [5], and F 0 was the fluorescence intensity of the guest (AD) solution. Figure 10 showed the relationship between 1/ΔF and 1/PWP [5]. The ratio of the intercept of the regression line to the slope gave the inclusion constant K AD⊂PWP [5] = 2.05 × 10 5 L/mol, indicating that a more stable inclusion compound is formed. (Compared with the previously studied NA ⊂ CB [7] assembly, the inclusion constant is increased about 4 times. K NA ⊂ CB [7] = 4.94 × 10 4 L/mol [15]).
In order to further confirm formation of AD ⊂ PWP [5], the 1 H NMR was performed. As shown in Fig. 11, in the presence of PWP [5], the signal of the guest AD proton At the same time, due to the deshielding effect, the proton peaks in anthracene shifted down field (Δδ = 0.31, 0.10, 0.12, 0.13, 0.13 and 0.1 ppm for protons of 10, 1, 2, 4, 6 and 8, respectively). These results indicate that the aminoalkyl side chain of AD penetrates into the cavity of PWP [5].

Fluorescent Recognition of D/L-phenylalanine and D/L-phenylalaninol by AD ⊂ PWP[5]
A concentration of 6 × 10 −5 mol/L solution of AD ⊂ PWP [5] was titrated by different concentration (0 ~ 9 × 10 −5 mol/L) of D/L-phenylalanine, fluorescence spectra had been measured and recorded. 6 × 10 −5 mol/L of AD ⊂ PWP [5] solution and 0 ~ 9 × 10 −5 mol/L of D/L-phenylalanine were mixed, as shown in Fig. 12b, with the addition of different concentrations of L-phenylalanine solution to the AD ⊂ PWP [5] assembly, the emission wavelength and fluorescence intensity almost unchanged. However, when different concentrations of D-phenylalanine were added to AD ⊂ PWP [5], the fluorescence intensity decreased 2.5 times (Fig. 12a), at the same time, blue shift of emission wavelength from 504 to 500 nm appeared.
Therefore, AD ⊂ PWP [5] had ability to recognize the enantiomers of D/L-phenylalanine. We supposed that in the process of hydrogen bond formation between the amino and carboxyl groups of D-phenylalanine and phosphonic acid group at the port of PWP [5] (Fig. 14), AD ⊂ PWP [5] assembly could induce the benzene ring and anthracene ring to be parallel, thereby, a stable π-π interaction appeared, which led to the fluorescence decrease. However, Lphenylalanine was different from D-phenylalanine in the spatial configuration, the assembly could not induce the parallel between benzene ring and anthracene ring and a stable π-π interaction could not occur, so the fluorescence almost unchanged.
The same titration was also performed to D/L-phenylalaninol solution, as shown in Fig. 13. When D-phenylalaninol was added to AD ⊂ PWP [5], the fluorescence intensity decreased by 2.7 times, and the emission wavelength shifted from 504 to 498 nm (Fig. 13a). For L-phenylalanine, the emission wavelength and fluorescence intensity almost unchanged (Fig. 13b). The  Fig. 14 Interacting mechanism between AD ⊂ PWP [5] and D/L-phenylalanine recognition mechanism of D/L-phenylalaninol is similar to that of D/L-phenylalanine.

Conclusions
In summary, the assembly based on AD ⊂ PWP [5] had been successfully applied to the recognition of chiral molecule. AD ⊂ PWP [5] assembly was constructed and characterized by fluorescent titration and 1 H NMR with 1:1 ratio and K AD ⊂ PWP[5]= 2.05 × 10 5 mol/L. The AD ⊂ PWP [5] assembly was used as fluorescent probe to recognized D/L-phenylalanine and D/L-phenylalaninol, When assembly was titrated by D-phenylalanine or D-phenylalaninol, a stable π-π interaction occurred which led to the fluorescence decrease, and for L-phenylalanine or L-phenylalaninol, a stable π-π interaction could not occur, so fluorescence intensity almost not changed.
In a words, we have successfully recognized D/Lphenylalanine and D/L-phenylalanine by AD ⊂ PWP [5]. Thus we have provided a new method to recognize chiral molecule.