Colorimetric and fluorimetric analysis of carbohydrates

Details Colorimetric and fluorimetric analysis of carbohydrates Colorimetric and fluorimetric analysis of carbohydrates

2001-02-05

2003-03-18

Warden, Jill (Department: 1743)

Chemistry: analytical and immunological testing

Heterocyclic carbon compound

Hetero-o

C436S093000, C436S095000, C436S164000, C436S166000, C436S169000, C436S172000, C568S765000

Reexamination Certificate

active

06534316

ABSTRACT:

This invention pertains to the detection of carbohydrates, and to synthetic compounds that exhibit colorimetric or fluorimetric responses in the presence of sugars and other carbohydrates.
Several efficient methods are available for analyzing amino acids or nucleic acids. By contrast, no single method is available that is suitable for the quantitative or qualitative analysis of saccharides generally. The high degree of structural similarity between different sugars hinders their selective detection. The direct visible detection of sugars is especially challenging, since unmodified saccharides generally do not absorb light in the visible region. See generally M. Chaplin, “Monosaccharides,” pp. 1-41 in M. Chaplin et al. (Eds.),
Carbohydrate Analysis. A Practical Approach
(Oxford University Press 1994); and J. Kennedy et al., “Oligosaccharides,” pp. 43-67 in M. Chaplin et al. (Eds.),
Carbohydrate Analysis. A Practical Approach
(Oxford University Press 1994).
Color assays for saccharides have been reported, including those based on certain synthetic molecules and those based on certain enzymes. Color assays based on synthetic molecules are typically less expensive than enzymatic methods, and their reagents are generally more resistant to degradation. Enzymatic assays can offer greater specificity than the non-enzymatic color tests, but they are generally more expensive, and their reagents are less stable. The inherently unstable enzymes must be protected from extreme conditions during manufacture, storage, and use. An ideal detection technique for sugars would be highly specific, and would employ relatively inexpensive and stable, non-enzymatic reagents.
Problem 1. Selective Visible Detection of Fructose.
Fructose is a nearly ubiquitous component of nutrient products. Non-enzymatic glycosidation products form more rapidly in vivo from fructose than from glucose. Fructose is absorbed by the gastrointestinal tract more slowly than is glucose, and does not require insulin for entry into the liver. These features make it appealing for use by diabetics. However, fructose has a higher tendency to be converted to fat rather than glycogen, thereby producing elevated blood triglyceride levels. High D-fructose intake has been implicated in the pathogenesis of hypertriglyceridaemia, atherosclerosis, and insulin resistance.
A glucose-fructose syrup is used in many food and beverage products. The worldwide production of high fructose syrup (HFS) is currently about 8×10
9
kg per year.
Carbohydrates comprise >98% of the soluble solids in fruit juices such as apple juice and orange juice. For example, fructose and glucose are the main carbohydrate constituents of apple juice, in a ratio greater than 2 fructose: 1 glucose.
There is an unfilled need for a simple and rapid color test that is highly specific for fructose, even in the presence of other monosaccharides such as glucose, a color test that does not require the corrosive, expensive, or degradable materials that are the basis of current monosaccharide color assays. Such a test could be of global benefit to industry and biomedicine.
The selective determination of fructose in plasma is especially challenging. Glucose is typically present in plasma in 100-fold excess of fructose. The determination of fructose levels in human plasma cannot currently be performed reliably, largely due to the “excess” glucose levels. Measured levels of plasma fructose thus vary greatly among laboratories, and vary by the technique employed.
The AOAC (formerly the Association of Official Analytical Chemists) official methods for the analysis of fructose rely principally on gas chromatography (GC), and high performance liquid chromatography (HPLC) with refractive index detection. The gas chromatography analysis typically employs a prior derivatization of the sugars (e.g., methylation or trimethylsilation). Refractive index detection in HPLC is subject to significant cross-sensitivity to other, non-specific sugars and biomolecules. More recently, an electrochemical method, pulsed amperometric detection (PAD), has gained widespread use for monosaccharide detection in conjunction with HPLC; however, PAD is limited by the need to operate at high pH which, in turn, limits the choice of solvents and conditions. Mass detectors can be very useful when coupled to HPLC or GC systems; however, mass spectroscopy adds a further degree of complication and expense to the analysis. Simple reducing sugar assays can be used in automated post-column detection systems for monosaccharides including fructose; however, they also require harsh reagents and conditions. Enzyme-based assays have also been used for specific fructose determination; however, enzymes are expensive and are readily degradable.
Recent studies have described the visual color sensing of monosaccharides, including fructose, by boronic acid-appended dyes. These techniques rely on sensing changes in color promoted either by the perturbation of an aggregation-disaggregation equilibrium (i.e, the addition of saccharides promotes the disaggregation of the boronic acid-functionalized dye); or by the perturbation by a sugar of the interaction of the boronic acid with a neighboring amine (attached to an azo dye), producing charge transfer effects. See, e.g., T. James et al., “Saccharide Sensing with Molecular Receptors Based on Boronic Acid,”
Angew. Chem. Int Ed. Engl
., vol. 35, pp. 1910-1922 (1996); K. Koumoto et al., “Design of a Visualized Sugar Sensing System Utilizing a Boronic Acid-azopyridine Interaction,”
Supramolecular Chemistry
, vol. 9, pp. 203 ff(1998); and K. Koumoto et al., “Colorimetric Sugar Sensing Method Useful in ‘Neutral’ Aqueous Media,”
Chem. Lett
., pp. 856-857 (2000).
In one modification, an optical wavelength shift has been observed following the binding of glucose in aqueous methanol; however, this modified system operates only at high pH (>12). See C. Ward et al., “A Molecular Colour Sensor for Monosaccharides,”
J. Chem. Soc., Chem. Commun.,
pp. 229-230 (2000).
Our research group has previously reported certain boronic acid-containing resorcinol condensation products (compounds 1 and 2 below), and their use in the non-selective color detection of sugars. See P. Lewis et al., “Tetraarylboronic Acid Resorcinarene Stereoisomers. Versatile New Substrates for Divergent Polyfunctionalization and Molecular Recognition,”
J. Org. Chem.,
vol. 62, pp. 6110-6111 (1997); and C. Davis et al., “Simple and Rapid Visual Sensing of Saccharides,”
Organic Letters
, vol. 1, pp. 331-334 (1999). Although different colors were observed for reactions with different sugars in the latter paper, the method of this paper is considered non-selective in the sense that it does not allow the selective detection of a single sugar of interest when it occurs in a background of other sugars.
Problem 2. Mild and Selective Detection of Sialic Acid.
Sialic acids are common components of glycoproteins, glycopeptides and glycolipids. Sialic acids play a role in cell-to-cell communication in humans and other animals, and have been implicated in increased virulence in some bacteria. Imbalances in sialic acid levels can alter cell adhesion, which may have an effect in certain cancers and in some types of graft rejection. An increase in either soluble or cellular sialic acid levels can be a diagnostic marker for cancer. The function of sialic acids is incompletely understood. They appear to act as amphiphilic donors of negative charge to the cell surface. Improved methods for analyzing sialic acids would greatly aid in the elucidation of their biochemistry and in the detection of certain cancers.
The most commonly used assays for sialic acid are probably the Warren assay and the Svennerholm color test. These assays require high temperatures, harsh and toxic reagents, and are subject to interference from other carbohydrates. Free sialic acids for the Warren and Svennerholm assay are obtained by either acidic or enzymatic (e.g., neuraminidase-promoted) hydrolysis. Acidic hydrolysis leads to the liberation of both sialic

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