Fluorescent probes for saccharrides

Abstract

The spectroscopic and photophysical properties of fluorescent probes comprising donor-acceptor derivatives comprising the boric acid group or a derivative of boric acid, B(OH)3 (or borate ion, BO(OH)2<-1>), arsenious acid, H3 AsO3 (or arsenite ion, H2AsO3<-1>), telluric acid, H6TeO6 (or tellurate ion, H5 TeO6<-1>) or germanic acid, Ge(OH)6 (or germanate ion, GeO(OH)3<-1>) are described. Method of using said probes are also provided.

Description

[0001] The present application is related to and claims priority under 35 U.S.C. .sctn. 119(e) to U.S. provisional patent application Serial No. 60 / 383,799, filed May 30, 2002 which is incorporated by reference herein in its entirety, along with the references cited therein.[0002] This invention was made with partial government support and as a result of this funding, the United States Government may have certain rights in this invention.[0003] This invention relates generally to the field of fluorescent probes. More specifically, the invention relates to electron-donor and electron acceptor pairs that possess a boronic acid group or boronic, arsenious, germanic and telluric acid derivatives and methods of use of such compounds as sensors for detecting the presence of sugars.[0004] The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice are incorporated by reference and for ease of reference...

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Benefits of technology

0093] pH and Sugar Effects on the Optical Spectra

0095] FIG. 4 shows the intensity changes after addition of sugars for STBA. A decrease of the fluorescence emission is observed after the addition of sugar. The overall decrease is relatively weak, about 15%. Since the same intensity change is observed by increasing the pH, this change is attributed to the formation of the anionic form of the boronic acid group. The pK.sub.a of STBA is 8.86 (Table 3) and decreases to 6.4 for the complex STBA:fructose. A PK.sub.a of 8.86 is similar to the pK.sub.a of a multitude of phenylboronic acid derivatives reported in the literature (47-49). The decrease of this pK.sub.a for the complex with sugar is also a general observation for this chelator group. For example, Yoon et al. reported a pK.sub.a change of 8.8 to 5.9 for the anthrylboronic acid and its complex with fructose. At pH 8.0, STBA is present mostly in its neutral form. After the addition of sugar, the complex exists under the anionic form due to its lower pK.sub.a. This change from the neutral to the anionic form is at the origin of the intensity changes observed for STBA and the other complexes presented in this study. Dissociation constants (K.sub.D) for the three different sugars are presented in Table 3. All titration curves against sugars have been taken at pH 8.0. This pH does not correspond necessarily to the maximum effect of the optical change but is an average pH that allows measurements of all derivatives at the same pH. The pH effect on the K.sub.D values is discussed below. Monophenylboronic acid groups are well known to be more sensitive to D-fructose and the sensitivity decreases for D-galactose and decreases again for D-glucose. All stilbene derivatives presented in this study follow this rule and show K.sub.D similar to the other monophenylboronic group. For example, phenylboronic acid shows K.sub.D of 0.2, 3.6 and 9.1 mM for D-fructose, D-galactose and D-glucose, respectively.

0096] Intensity changes observed for STBA are similar to the intensity changes reported for anthryl-2-boronic acid. For this anthracene derivative, the boronic acid group is also linked directly on the anthracene fluorophore. Intensity decreases of 30% and 10% were reported after the addition of fructose and glucose at pH 7.4, respectively. To explain these decreases, the authors suggested a photoinduced electron transfer (PET) mechanism where the negative charge on the boron atom acts as the quencher. This statement was based on the oxidizability of the borate. In the insert of FIG. 4, we show the emission spectra of STBA with different concentrations of fructose. We can observe that the emission is slightly blue shifted and an isosbestic point appears at about 338 nm after the addition of fructose. This could suggest that the effects of sugars on the emission spectra of STBA could be induced by the change of conformation of the boron atom from the neutral to the anionic forms, sp.sup.2 to sp.sup.3, as schematically shown in Scheme 1. Without being bound by theory, the loss of the empty p orbital of the boron atom could result in a partial loss of the resonance between the arom

Problems solved by technology

The use of enzymes shows some limitations in the development of implantable sensors for continuous glucose monitoring in blood or interstitial tissue.

Large variation in the glucose level in blood could result in important medical problems including cardiovascular disease, neuropathies and blindness.

Despite some promising results, these methods show limitation as important background with the NIR (Near infra red) technique and low optical rotation and important depolarization du to the tissue with the optical rotation technique.

Despite these advantages, they exhibit low stability (to heat and organic solvents), solubility problems and are difficult to modify.

Despite some promising results, enzymes and proteins show some stability problems against organic solvents and heat.

Until now, however, no system and analysis using the fluorescence lifetime could be found for these systems.

Despite these interesting approaches to use the combination of the boronic acid group and fluorophores, they are mostly restricted to a few fluorophores.

The PET mechanism is expected to be ineffective for long wavelength fluorophore use (43).

It is known in the art that the measure of absolute flourescent intensity can be a difficult and time and labor consuming endeavor.

Background noise from the sample can be present and the equipment requires frequent calibration and maintenance.

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