Functional group assays

Tables (1 - 3) give an overview on the methods described in the literature. Most of the procedures listed in the first two tables are methods determining total phenolics, whereas table three lists the functional group assays, which are specific for proanthocyanidins.

Procyanidin contents are commonly determined using the more specific functional group assays. If purified extracts have to be analyzed, procedures determining phenolics in general may nevertheless be quite useful. A distinction between polyphenol and non-polyphenol "contents" can be made by performing a general phenolic assay before and after... 

The following functional group assays are used proanthocyanidin assay, vanillin assay and dimethylaminocinnamaldehyde (DMACA) assay. Details to the experimental designs are listed in Tab. (3.1) to (3.3). 

Table 3.1. Conventional Methods Specific for Proanthocyanidins (Functional Group Assays)...

A major drawback of all functional group assays is that a satisfactory standard does not exist. For a given sample the most appropriate standard is a purified procyanidin fraction prepared from the same matrix. The isolation and characterization of such purified fractions are laborious. Added to that procyanidins undergo oxidation, complexation and self-polymerization very easily, rendering such purified fractions only reproducible to a limited degree. At least in the proanthocyanidin assay the color reaction depends not only on the polyphenols themselves, but also on the matrix. The use of specified proanthocyanidins as a standard in a suitable blank matrix is an attempt to correct for such effects [67],... 

A suitable functional group is assayed in the same sample. In general chemistry and many polymer applications, this is merely the titration of acid groups with a base, or vice versa. Note that only volumetric glassware and a method for end point determination are required to do this. 

The method described is of particular value in the determination in complex matrices of metabolites or impurities which differ in the presence of functional groups. Notable examples of this are assays of compounds with free amino or acidic groups. 

Assays of ciguatoxin. Determination of ciguatoxin levels in fish was carried out in many laboratories by mouse assays. Enzyme immunoassay to screen inedible fish has been proposed by Hokama (9). No specific chemical assay has been developed, as information on functional groups suitable for fluorescence labeling is not available. Analyses conducted in the authors laboratory on remnant fish retrieved from patients meals indicated that ciguatoxin content as low level as 1 ppb could cause intoxication in adults. An extremely high sensitivity and a sophisticated pretreatment method will be required for designing a fluorometric determination method for the toxin. 

As far as we know, this is the first molecular probe that includes two different types of reporter units activated upon on a specific stimulus. The other option to achieve dual detection would be to use two separate probes. However, in this case there could be a problem of competitive catalysis (circumstances in which the Km of the two substrate is not identical). In our probe, 6-aminoquinoline and 4-nitrophenol, detected by fluorescence and absorbance spectroscopy, respectively, were used as reporter units. Due to the synthetic flexibility of our approach, other reporter molecules with different types of functional groups, like amine or hydroxyl, can be linked to our molecular probe. The two assays must be orthogonal to each other, in order to prevent disturbances in the detection measurement. Another advantage of the probe is the aqueous solubility... 

On Pd/C, carbon-carbon double bonds and halogens are usually reduced before the N-oxides. The final selectivity in the hydrogenation of N-oxides has been shown to depend on the pH, the catalyst system used, and the functional groups present and their position. PtSx was a superior catalyst with a higher assay and yield in the selective hydrogenation of 6-chloro-2(lH)-hydroxyqui-noxaline-4-oxides (43) (Scheme 4.139).534... 

Fusion of a barbituric acid motif and a pyrone ring afforded compounds containing a novel pyranopyrimidine core (L) that were discovered as GPR109A agonists [92,93]. This core appears to be distinct from other fused bicyclic cores such as xanthine and anthranilide based on their poor overlap. Furthermore, several compounds, exemplified by 39 and 40, provided remarkable potency in the cAMP assay. The critical acidic functional group is present as the N-PI of barbituric acid motif which has a calculated pKa of 8 [82]. 

Chemists and biologists have long known that certain chemical moieties are likely to produce false positives in biochemical assays because of their chemical reactivity [86]. Software filters are now routinely used at Vertex [24, 87] and other companies to flag compounds containing functional groups known empirically to contribute to reactivity, insolubility, toxicity, or poor ADME. Table 18.4 lists examples of such undesirable functional groups. 

An alternative approach is provided by NMR spectroscopy. Separate NMR signals can be in principle obtained for stable or short-lived diastereomeric derivatives of the enantiomeric mixtures, the intensities of which are correlated with the enantiomeric composition and their relative stereochemistry to the absolute configuration. For this reason, great effort has been continually devoted to the development of new chiral auxiliaries for NMR spectroscopy. The majority of these are dedicated to the chiral assay of molecules having polar functional groups. 

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