NIR dyes for bioimaging applications.

Fluorescent dyes based on small organic molecules that function in the near infrared (NIR) region are of great current interest in chemical biology. They allow for imaging with minimal autofluorescence from biological samples, reduced light scattering, and high tissue penetration. Herein, examples of ongoing NIR fluorophore design strategies as well as their properties and anticipated applications relevant to the bioimaging are presented.

Capillary electrophoretic screening for the inhibition of homocysteine thiolactone-induced protein oligomerization.

We report the first demonstration of rapid electrophoretic monitoring of homocysteine thiolactone-induced protein oligomerization (HTPO), a unique type of post-translational protein modification that may have clinical significance as an indicator of cardiovascular and neurovascular diseases. HTPO of the model protein bovine cytochrome c was initiated in vitro. The relative monomer and aggregate levels of the resultant protein mixtures were determined following separation using capillaries coated with the cationic polymer, poly(diallyldimethylammonium chloride). UV detection provided adequate sensitivity for the monitoring of higher order species, which exist at relatively low concentrations in the protein reaction mixture as compared to the monomeric species. Separations performed under standard injection conditions were optimized on the basis of applied voltage and sample denaturation conditions. Separations performed using short-end injection allowed for more rapid analyses, typically in less than 70 s. Relative errors for run-to-run migration times were less than 0.5%. This novel oligomeric system provides a rapid and straightforward in vitro method to screen therapeutic agents for their ability to inhibit HTPO. Changes in peak area for monomer and aggregate species were used to assess HTPO inhibition as a function of pyridoxal 5-phosphate (PLP) concentration. PLP was shown to effectively inhibit HTPO in vitro. Rapid analysis times of approximately 1.5 min were achieved for inhibition screening.

Stereochemical and regiochemical trends in the selective detection of saccharides.

Several discreet sugar-boronate complexes exist in solution. This is due to the complex equilibria between isomeric species of even the simplest monosaccharides. In the current investigation, we determine the regio- and stereochemical features of the various equilibrating sugar isomers that induce signal transduction in boronic acid chemosensors such as 1 as well as 2 and 3. We present a unique example of a chemosensor (1) that is selective for ribose, adenosine, nucleotides, nucleosides, and congeners. As a result of this study, we are able to predict and achieve selective fluorescence and colorimetric responses to specific disaccharides as a consequence of their terminal sugar residue linkage patterns and configurations. We also find that the combined use of chemosensors exhibiting complementary reactivities may be used cooperatively to obtain enhanced selectivity for ribose and rare saccharides.

Lanthanide complexes as fluorescent indicators for neutral sugars and cancer biomarkers.

Simple water-soluble lanthanum and europium complexes are effective at detecting neutral sugars as well as glycolipids and phospholipids. In solutions at physiologically relevant pH the fluorescent lanthanum complex binds neutral sugars with apparent binding constants comparable to those of arylboronic acids. Interference from commonly occurring anions is minimal. The europium complex detects sialic acid-containing gangliosides at pH 7.0 over an asialoganglioside. This selectivity is attributed, in large part, to the cooperative complexation of the oligosaccharide and sialic acid residues to the metal center, based on analogous prior studies. In MeOH, lysophosphatidic acid (LPA), a biomarker for several pathological conditions including ovarian cancer, is selectively detected by the europium complex. LPA is also detected via a fluorescence increase in human plasma samples. The 2-sn-OH moiety of LPA plays a key role in promoting binding to the metal center. Other molecules found in common brain ganglioside and phospholipid extracts do not interfere in the ganglioside or LPA fluorescence assays.

Detection of homocysteine and cysteine.

At elevated levels, homocysteine (Hcy, 1) is a risk factor for cardiovascular diseases, Alzheimer's disease, neural tube defects, and osteoporosis. Both 1 and cysteine (Cys, 3) are linked to neurotoxicity. The biochemical mechanisms by which 1 and 3 are involved in disease states are relatively unclear. Herein, we describe simple methods for detecting either Hcy or Cys in the visible spectral region with the highest selectivity reported to date without using biochemical techniques or preparative separations. Simple methods and readily available reagents allow for the detection of Cys and Hcy in the range of their physiologically relevant levels. New HPLC postcolumn detection methods for biological thiols are reported. The potential biomedical relevance of the chemical mechanisms involved in the detection of 1 is described.

Macrocycle-derived functional xanthenes and progress towards concurrent detection of glucose and fructose.

The detection of saccharides in biological media is of great current importance for the monitoring of disease states. We have previously reported that solutions of boronic acid-functionalized macrocycles form acyclic oligomeric materials in situ. The oligomers contain fluorescent xanthene moieties. Current efforts are aimed at modulating the spectroscopic responses of these materials for the analysis of specific sugars. We describe conditions whereby the xanthene boronic acids exhibit high colorimetric fructose selectivity. In contrast, at physiological levels selective glucose monitoring can be achieved via fluorescence. Additionally, we describe a method which exhibits promise for detecting both glucose and fructose at dual wavelengths in the UV-Vis region. Mechanistic rationale for each of these findings is presented.

A supramolecular approach to protein labeling. A novel fluorescent bioassay for concanavalin a activity.

[reaction: see text] A new method for the bioassay of concanavalin A is based on the interaction of saccharide-substituted oligopyrrolic macrocycles with lectins. A general sensing mechanism involves the interaction of aggregated and primarily nonfluorescent labels with the target protein, label deaggregation, site-specific binding, and fluorescence signaling. Addition of saccharides to the fluorescent lectin-macrocycle complex leads to the release of the label and partial quenching of fluorescence due to reaggregation. Specificity of the protein-probe binding is discussed.

Visual detection of cysteine and homocysteine.

The determination of cysteine and homocysteine levels is of great current interest for the monitoring of desease states. A new colorimetric method for the simultaneous detection of l-cysteine and l-homocysteine has been developed. A fluorescein derivative reacts with the above amino acids, producing their respective thiazolidines resulting in color changes. Interference from other amino acids and proteins is minimal.

Postcolumn HPLC detection of mono- and oligosaccharides with a chemosensor.

Novel chromophoric compound 1 promotes the HPLC postcolumn detection of mono- and oligosaccharides. The detection of chromatographic peaks in the visible region for glucose, fructose, maltodextrins, sialic acid, and a ganglioside can be accomplished with a standard UV-vis detector. The use of selective, reversible binding agents in automated HPLC assays should allow for improved monitoring of specific analytes as well as material recovery. [structure: see text]

1,1'-Binaphthyl-substituted macrocycles as receptors for saccharide recognition.

The preparation of receptors for saccharide recognition in a natural environment has been an unmet goal for a long time. We present herein the synthesis and binding properties of (R,S)-1,1'-binaphthyl-substituted macrocycles as receptors for saccharide recognition in water/acetonitrile (1:1) and in DMSO. Porphyrin and metalloporphyrin macrocycles with two to four 1,1'-binaphthyl substituents and multiple hydroxy groups generate a binding site for saccharides that incorporates hydrogen-bonding hydroxy groups together with the aromatic hydrophobic pocket. The specificity for di- and trisaccharides is governed by the cavity size. The mechanism of binding has been studied by 1H NMR spectroscopy and the role of H-bonding and CH-pi interactions has been evaluated; the ability to bind saccharides has been demonstrated by the surface plasmon resonance (SPR) technique. The application of these macrocyclic receptors to sensor development is also presented.