A New Saccharides and Nnucleosides Sensor Based on Tetrathiafulvalene-anthracene Dyad with Two Boronic Acid Groups

A new saccharides sensor based on the TTF-anthracene dyad with two boronic acid (2) groups was designed and synthesized. This new saccharides sensor shows selectivity towards D-glucose while its analogue with one boronic acid group (1) was reported to bind D-Fructose selectively. Moreover, reaction of compound 2 with uridine induced even larger fluorescence enhancement under the same condition.

Boronic acid is a good receptor for saccharides since boronic acid is able to react with 1,2-or 1,3diols to form five-or six-membered rings respectively. Shinkai and co-workers have described a series of saccharides sensors based on the fact that the corresponding photoinduced electron transfer (PET) process can be tuned after the binding of boronic acid with saccharides [8][9][10][11][12][13]. The Shinkai's saccharides sensors usually contain three components: a fluorophore, an amine group and a boronic acid group. As shown in Scheme 1, the nitrogen-boron interaction modulates the PET process and leads to fluorescence change before and after the interaction of the boronic acid group with saccharides.
We have recently reported that the amine group of Shinkai's saccharide sensors can be replaced by other electron donors, such as tetrathiafulvalene (TTF) [21]. By this way, a new saccharides sensor (1, Scheme 1) based on a tetrathiafulvalene-anthracene dyad with one boronic acid group was designed and synthesized. Compound 1 shows weak fluorescence mainly due to the PET process between excited anthracene and TTF units as reported by us previously [22][23][24][25]. After reaction with saccharides, the fluorescence intensity of 1 was enhanced (see scheme 1). Moreover, compound 1 shows selectivity towards fructose. We report herein the synthesis and fluorescent spectral studies of a new saccharides sensor based on TTF-anthracene dyad with two boronic acid groups (2, Scheme 2) because it is reported that the diboronic acid sensor could recognize D-glucose with the formation of a 1:1 complex [26][27][28][29]. The result indicates that compound 2 shows selectivity towards glucose at physiological pH. Moreover, even larger fluorescence enhancement was observed after the reaction of 2 with nucleosides such as uridine.

Synthesis
Compound 2 was synthesized through 5 steps starting from compound 3, which was prepared based on the unusual reaction of Zn(dmit) 2 anion reported by us recently [30,31]. Deprotection of 2cyanoethyl group in 3 with CsOH•H 2 O and further reaction with 9-(2-bromoethoxyl)anthracene afforded compound 4. Coupling reaction of 4 and 5 in the presence of tri(isopropyl)phosphite led to compound 6, which was transformed into compound 7 by reaction with excess of 1,5-dibromoheptane in the presence of CsOH•H 2 O. Finally, reaction of 7 with potassium 4-(5,5-dimethyl-1,3,2dioxaborinan-2-yl)benzoate afforded compound 8 and sequential hydrolysis of compound 8 led to compound 2 in a total yield of 15.1% after purification with column chromatography.  The fluorescence enhancement was also observed for 2 after reaction with D-galactose, D-mannose, and D-fructose as shown in Figure 1 (b) where the plot of the relative fluorescence intensity I/I o at 420 nm vs. the concentration of saccharides (D-glucose, D-galactose, D-mannose, and D-fructose) was displayed. The degree of fluorescence enhancement observed for 2 after the respective reactions with D-galactose, D-mannose, and D-fructose is relatively small compared to that of 2 in the presence of glucose. By plotting I o /(I-I o ) against the reciprocal of the concentration of saccharides, the apparent association constants (K a ) between 2 and saccharides were determined assuming the formation of a 1:1 complex ( Table 1). The order of the binding constants of 2 with saccharides is as follows: D-glucose > D-mannose > D-fructose > D-galactose (see Table 1). Apparently, compound 2 shows relatively strong binding tendency towards D-glucose. Nucleosides commonly possess a ribose moiety, in which the cis-2,3-diol group should be able to bind boronic acid. Nucleosides also contain base units (e. g. A, T). Thus, when a nucleoside is linked to compound 2 through the binding of the boronic acid group and the cis-2,3-diol group, the TTF unit of 2 will be flanked with three acceptor units: anthracene, base and boronate. As a result, the PET reaction between the excited anthracene and TTF units would be further prohibited compared to that after binding of 2 with saccharides. Accordingly, large fluorescence enhancement is anticipated to occur for 2 after reaction with nucleosides. For instance, the degree of the fluorescence enhancement was found to be larger for 2 after reaction with uridine than that after the reaction of 2 with glucose under the same condition (see Figure 2).

Spectral studies
In summary, a new saccharides sensor based on the TTF-anthracene dyad with two boronic acid groups was designed and synthesized. Fluorescent spectral studies showed that the fluorescence intensity of 2 was enhanced after binding saccharides. The association constants of compound 2 with four saccharides were estimated implying that compound 2 displayed selectivity towards D-glucose. It is interesting to note that compound 1 described previously by us shows selectivity towards D-Fructose [21]. Furthermore, reaction of 2 with uridine induced even larger fluorescence enhancement under the same condition. Further investigations along this vein are in progress.

Experimental Section
1H and 13C NMR spectra were recorded on BRUCK 300 MHz and BRUCK 400 MHz instruments. EI-MS and TOF-MS were determined with AEI-MS 50 and BEFLEX III, respectively. Fluorescence spectra were recorded on a JASCO FP6000 spectrofluorometer in a 1-cm quartz cell. Column chromatography was performed by using silica gel (200-300 mesh). Glucose, fructose, galactose, and mannose were purchased from Acros. THF was distilled from Na and benzophenone immediately prior to use.
Synthesis of compound 4 [23]: To a solution of 3 (1.10 g, 5.02 mmol) in anhydrous degassed THF ( 100 mL) was added a solution of CsOH•H2O ( 0.86 g, 5.05 mmol) in anhydrous degassed MeOH (5 mL) over a period of 30 min. The mixture was stirred for an additional 30 min, to which a solution of 9-(2-bromoethoxy)-anthracene (1.58 g, 5.25 mmol) in anhydrous degassed THF (10 mL) was added. The solution was stirred overnight. After removing solvents and separation by column chromatography, compound 3 was obtained in 75% yield.
Synthesis of compound 6: To a solution of 4 (1.40 g, 3.60 mmol) in 20 mL P(OiPr)3 was added 5 (2.1 g, 7.2 mmol) in N2 atmosphere. The mixture was stirred at 120 oC for 4 hours. After cool to room temperature, the sovent was removed under reduced pressure and the residue was purified through column chromatography to give compound 5 in 56% yield.  Synthesis of compound 8: To a solution of 7 (0.657 g, 0.8 mmol) in 2 mL dry DMF was added 0.653 g (2.4 mmol) of potassium 4-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)benzoate. The mixture was stirred at 75oC for 2 hours. After cool to the room temperature, the solution was poured into water and extracted with CH2Cl2 (3×30 mL). The organic phase was washed with water, dried by angydrous Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography to give compound 8 in 90% yield.