Abstract: Boronic acid protecting groups have revolutionized the use of organoborons in synthetic chemistry, enabling organoboron functional groups to be carried through multistep protocols, no longer limited to introduction of boron at a late stage or subject to. A new class of air-stable boronic acid derivatives is trifluoroborates, which are offering a unique alternative to most boronic acids, esters and organoboranes for use in Suzuki-Miyaura and other transition-metal-catalyzed cross-coupling reactions.
There remains a need in clinics and research to have simple and sensitive detection systems that allow the detection and quantification of sugar markers of biomedical relevance such as sugars lactulose and mannitol for noninvasive gut permeability assessment. We have prepared a new class of boronic acid-appended naphthyl-pyridinium receptor compounds as chemosensors. These were studied for their ability to act as modular internal charge transfer (ICT) fluorescent probes or donor/acceptor pair ensembles where the receptor compound can act as a quencher for an anionic dye. As an ICT sensor, fluorescence intensity increased upon diol recognition, which stems from the neutralization of the pyridinium nitrogen that is perturbing the chromophoric properties. We found these ICT probes provide good sensitivity for disaccharide lactulose with low micromolar detection and quantification limits.
![]()
In addition, their ability to form a non-fluorescent ground state complex with anionic reporter dyes, such as HPTS or TSPP, was examined as probes for various sugars. We have identified three receptor/quencher compounds with high quenching efficiency for anionic dyes. Subsequently, a range of sugars and sugar derivatives were tested for chemosenstivity of our probes. This study illustrates an approach for designing boronic acid-based chemoreceptors for the recognition and quantification of sugars and sugar derivatives. The molecular recognition of carbohydrates and diol-containing compounds by boronic acid systems is a growing area of research interest, and such fluorescent probes based on boronic acid recognition for sugars and sugar derivatives have been explored. Most notable and popular probes are classified as “one component systems” that utilize an internal charge transfer mechanism for signal generation. Various fluorophores that are amenable to boronic acid tethering, e.g., anthracene, naphthalene, naphthalimide, indol, quinolinium, benzo-thiophene, and fluorene, containing boronic acid tethered dyes have been reported to measure sugars and sugar derivatives in aqueous media.
Similarly, boronic acid containing fluorescent receptors have been developed for sugar alcohols, sugar acids, and recently, sugar markers such as lactulose and mannitol for clinical applications. Due to the structural nature of these sugars, boronic acids can adopt different conformations when binding to mannitol (sugar alcohol) as compared with cyclic aldoses (glucose) or ketoses (fructose). Consequently, designing selective boronic acid receptors for these sugar alcohols and lactulose poses a difficult challenge.
Because lactulose and mannitol are important sugar markers for gut permeability assessment, designing sensitive and selective sensors for sugar alcohols has attracted considerable attention in the field of sensor design. There is an increasing need for a simple and easy to use method for analyzing the sugar gut permeability markers that offers good sensitivity and alleviates wavelength interference from urine components (i.e. Riboflavin, urobilin, urobilinogen etc.) during sample analysis.Indicator displacement assays (IDA) have been reported as competitive binding assay systems, where the receptor and indicator (or reporter) units are two discrete entities which can provide advantages, such as signal modulation and ease of modification of receptor system over direct sensing ( i.e., one-component systems) (Fig. ). Singaram et al. Demonstrated a two-component system and designed a series of boronic acid appended bipyridinium compounds that utilized an anionic dye (reporter) and a boronic acid-appended viologen (receptor) to develop a continuous glucose sensor.
Two-component systems can be incorporated into a standard IDA, where an analyte displaces the indicator via allosteric interaction and the analyte cannot compete at the same binding site as the indicator. Instead, the analyte binds at another site, inducing a decrease in the affinity of the indicator for the receptor. This type of system refers to as an allosteric indicator displacement assay (AIDA). The two-component system relies on the cationic nature of the receptor compound to form a non-fluorescent ground state complex with the anionic fluorescent dye, 8-hydroxypyrene, 1,3,6-trisulfonic acid trisodium salt (HPTS), and a boronic acid–appended viologen (BBV) that acts as both a quencher and receptor. In the absence of sugar, a ground state complex forms by the coulombic attraction between the anionic dye and cationic quencher with a decrease of fluorescence intensity (as compared with free HPTS).
![]()
When a sugar binds, the boronic acids convert to tetrahedral anionic boronate ester, which neutralizes the cationic viologen, diminishing its quenching efficiency and liberating HPTS. The fluorescent signal generated upon dissociation of the ground state complex is directly proportional to sugar concentration. Previous work demonstrated that the 4,4′- o-BBV bidentate receptor exhibited increased binding affinity due to the ortho substitution of the boronic acid motif. The boronic acid sits close to the quaternary nitrogen, thus, able to participate in a favorable electrostatic interaction.
Studies of several boronic acid-appended viologens that possess both bipyridinium or phenanthrolinium cores have been previously reported. The ability of these viologens to quench the fluorescence of various anionic reporters is directly proportional to the number of cationic charges on the quencher.
To alleviate the need for a large number of cationic groups, we investigated new boronic acid-appended quenchers with ortho substitution for improved ability to (1) quench the fluorescence of an anionic reporter and (2) improve the sensitivity for the recognition of sugars and sugar derivatives. Herein, we report new boronic acid-appended naphthyl-pyridinium receptor compounds as discrete two-component fluorophore-receptor probes and their ability to quench the fluorescence of anionic dyes. The combination of each probe (receptor-dye) was investigated for their performance in recognizing various sugars and sugar derivatives. An indicator displacement “two-component” system can be used to detect sugars and sugar derivatives, as it provides flexibility in signal modulation and is less sensitive to pH changes.
The flexibility of the two-component system is that it allows for tailoring of the quencher/receptor compound’s properties without affecting the photophysical properties of the reporter dye. This can improve the sensitivity, and also the selectivity of measuring sugar and sugar derivatives in complex media, such as urine or blood. With the ability to overcome sensitivity and/or selectivity challenges that come with boronic acid-based recognition systems, we pursued a naphthalene moiety as a new quencher along with anionic dyes such as HPTS and tetrakis (4-sulfophenyl) porphine (TSPP). Since naphthalene has its intrinsic fluorescence properties, we first investigated these naphthyl-pyridinium receptors as potential new one-component systems to act as chemosensors for sugars and sugar derivatives.
The previous study reported a naphthalene-based sensor for fructose recognition which utilizes the internal charge transfer (ICT) process for the generation of a fluorescent signal. This sensor relies on the sp 2-hybridized boron atom directly attached to the naphthalene chromophore, which can form a conjugated system and act as an electron acceptor. We envisioned designing a new type of boronic acid-based chemosensor that relies on quaternary nitrogen that is separate from the fluorophore unit, and primarily responsible for the ICT mechanism. To design this, we incorporated a pyridinium tethered to the fluorophore moiety acting as the electron acceptor to facilitate the excited state charge transfer.
Upon converting the boron atom from sp 2- to sp 3-hybridization, it switches off the ICT process resulting in an increase of fluorescence with increasing sugar concentration. We hypothesized that a quarternized nitrogen could serve as an excellent electron acceptor for the excited singlet electron and this would alter the emissive properties of naphthalene upon the charge neutralization event from sp 2- to sp 3-hybridization change, thereby further inducing spectroscopic changes (Fig. ). Additionally, because of its conjugated π-system and cationic character, we hypothesized that these naphthyl-pyridinium receptor compounds would be excellent quenchers for the anionic dye HPTS or TSPP. Upon boronic acid-diol recognition of sugar, it would form boronate, thus neutralizing the quarternized nitrogen and liberating the anionic fluorophore for detection (Fig. ). Starting with commercially available 1-bromo- or dibromo-naphthalene, each coupled precursor was obtained via Suzuki-Miyaura coupling, followed by nucleophilic substitution to afford the boronic acid appended naphthyl-pyridinium receptor salt compound (Fig. ).
The linking of naphthalene and pyridine aromatic systems provides a unique environment for complex formation with its anionic partner that would enhance the electron transfer, improving the ability to quench the fluorophore with minimal quencher concentration. Furthermore, this unique structural feature enables the receptor compound to undergo an ICT process, acting as a one-component system and facilitating participation in dual probe systems.
Absorption and fluorescence studies of boronic acid appended naphthyl-pyridinium receptor compoundsWith naphthalene as the core moiety of each receptor compound, we anticipated these compounds would undergo photophysical changes upon recognition of a sugar. Therefore, the compound was examined for its absorbance changes to demonstrate such ability to sense sugars. Representative absorption and emission spectra for each naphthyl-pyridinium receptor compound with increasing amounts of lactulose in buffer solution are shown in Figs. Change of absorbance for each receptor compound was monitored as a function of increasing lactulose (Fig. ). Owing to the nature of the boronic acid charge switch, perturbation of the naphthalene chromophore may occur through the charge-neutralization of the pyridine-boronic acid interaction. Incremental changes of absorbance were observed with increasing amounts of lactulose for each naphthyl-pyridinium receptor compound. Each compound provided absorbance maxima between 300–350 nm and isosbestic points between 330–350 nm for all four receptors.
The molar extinction coefficients of 1.17 × 10 4 (100 µM), 1.10 × 10 4 (100 µM), 2.77 × 10 4 (50 µM), and 5.01 × 10 4 (50 µM) M −1 cm −1 for 1– 4, respectively were obtained. The absorption changes upon sugar binding at physiological pH for each receptor is indicative of a charge transfer process. The effect of lactulose on the fluorescent properties of each receptor compound ( 1–4) examined in phosphate buffer at pH 7.4 is shown as Fig.Receptor compounds ( 1) and ( 2) exhibited similar emission profiles with emission maxima at 505 and 490 nm. Meanwhile, the emission maxima for ( 3) and ( 4) was 50 nm less, peaking at 440 nm. For these receptor compounds, a minimum of 6-fold increase in intensity was observed in the presence of lactulose at pH 7.4. We found that introduction of lactulose (0.063 mM) provided a drastic intensity change for ( 3) & ( 4) initially, and smaller incremental changes were observed afterward.
These types of fluorescent changes have been previously observed by Norrild et al., where a pyridinium moiety directly conjugates to an anthracene core. We speculate that the pyridinium unit in these receptor compounds can act as an electron sink for the singlet electron, thus diminishing the fluorescence intensity. Boronic acid-diol recognition and subsequent conversion of the hybridization of boron atom from neutral sp 2 to anionic sp 3 generated zwitterion induces an ICT-like mechanism for increasing the fluorescence intensity. It is well understood that the binding properties of boronic acid-based receptors are pH dependent and identifying the pK a of such sensor will provide the optimal window for sugar recognition. Therefore, we determined the pK a of each boronic acid receptor compound ( 2), ( 3), and ( 4) by monitoring the fluorescence-pH in the absence and presence of lactulose (see Supplementary Fig.
In the absence of lactulose, the fluorescence intensity of ( 2), ( 3), and ( 4) increased with changing pH resulting in pK a of 7.7, 8.2, and 7.8 respectively. This pK a value shifted to 5.5, 5.8, and 5.7 for ( 2), ( 3), and ( 4) in the presence of lactulose (30 mM), which is indicative of the boronate ester formation from an increased Lewis acidity of the boron atom with sugar binding. Given the higher fluorescence recovery for ( 3) and ( 4) in the presence of lactulose, we examined these boronic acid receptor compounds for other sugar and sugar derivatives to explore potential applications, for example in sugar gut permeability marker. The binding properties of ( 3) and ( 4) provided increased sensitivity (detection limit of 70 and 100 µM) for sugar markers of interest with good dynamic response in the concentration range that is pertinent to sugar gut permeability marker measurements between 100–900 μM (see Supplementary Fig. These data also suggest other potential application of these standalone-receptor compounds as fluorescent probes to the detection of various types of sugars and sugar derivatives without the need of a reporter dye.
Formation of a non-fluorescent complex with anionic reporter dyesGiven the cationic character of these naphthyl-pyridinium receptor compounds, we sought to investigate their ability to form a non-fluorescent ground state complex with anionic reporter dyes, such as HPTS and TSPP. The fluorescence quenching of each dye can be quantitatively monitored by Stern-Volmer analysis. Two types of quenching can exist: static quenching, due to complex formation, and dynamic quenching, due to collisional encounters between the dye and quencher. Based on previous work with bis-boronic acid-appended viologens (BBV) and their ability to quench HPTS, we reasoned a similar quenching mechanism for each anionic reporter dye through static quenching.
Figure shows the effects of adjusting the quencher to dye ratio for HPTS & TSPP with each naphthyl-pyridinium quencher compound, with monitoring the decreasing fluorescence for each dye. With each quencher having only two cationic groups, we anticipated a need for higher (400 μM) amounts to achieve at least 80% of quenched fluorescence. However, we found that a concentration of.
Comments are closed.
|
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |