Aldehydic Group detection

The R band characteristic for aromatic aldehyde groups (aldehyde n TT bands) occurs in the spectrum of A -methylcotarnine (9a) and that of JV-benzoylcotarnine (9c), which are real aldehydes, at 330 m/i in the form of an inflection. Even in alkaline solution the hypothetical amino-aldehyde form of cotarnine can only occur in amounts not detectable by spectroscopic methods. 

Isolation of Citronellal and Citral. At the close of each experiment (7 to 10 days), the nests were frozen intact. Groups of 200 workers were placed in a micro-Soxhlet apparatus and extracted for 8 hours with methylene chloride. A few milligrams of carrier citronellal and citral were added and the mixture was applied to a thin-layer chromatoplate (silica gel G) which was developed with hexane-ethyl acetate (92 to 8) to separate citronellal and citral (3). The aldehydes were detected by spraying with a solution of 2, 4-dini-trophenylhydrazine in tetrahydrofuran (20) and the citronellal and citral peaks were scraped off and allowed to react with excess dinitro-phenylhydrazine reagent for a further 12 hours. 

Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide (Chapter 4, Section 8.1) and the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) create activated proteins that can covalently bind to formyl residues. Hydrazide-modified enzymes prepared in this manner can bind specifically to aldehyde groups formed by mild periodate oxidation of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985). 

When heated with a strong acid, pentoses and hexoses are dehydrated to form furfural and hydroxymethylfurfural derivatives respectively (Figure 9.20), the aldehyde groups of which will then condense with a phenolic compound to form a coloured product. This reaction forms the basis of some of the oldest qualitative tests for the detection of carbohydrates, e.g. the Molisch test using concentrated sulphuric acid and a-naphthol. 

Identification of pyridoxal phosphate as coenzyme suggested the aldehyde group on pyridoxine might form an intermediate Schiff s base with the donor amino acid. Pyridoxamine phosphate thus formed would in turn donate its NH2 group to the accepting a-ketonic acid, a scheme proposed by Schlenk and Fisher. 15N-labeling experiments and, later, the detection of the Schiff s base by its absorption in UV, confirmed the overall mechanism. Free pyridoxamine phosphate however does not participate in the reaction as originally proposed. Pyridoxal phosphate is invariably the coenzyme form of pyridoxine. 

In both reactions with the meso substrates, no intermediary monoadduct could be detected. Consequently, a potential kinetic preference of the aldolase for either of the competing enantiotopic hydroxyaldehyde moieties within the starting substrates could not be investigated. No matter which of the enantiotopic aldehyde groups is attacked first, however, the second addition steps must be kinetically faster in each case, probably supported by the presence of an anionic charge in the intermediates, which should improve the substrate affinity to the enzyme. 

The absence of characteristic waves of the aldehydic group can, of course, be used as a proof of the absence of the group in a molecule. In this way, the absence of an aldehydic group has been proved (57) for absinthindiol, guajtriol C and artemazulene. The presence of a phenacyl group has been proved in the molecule of kynurenine (755). a,(3 unsatura-ted ketones can be distinguished from saturated ketones, e.g. A4-3-ketosteroids and A1 4-3-ketosteroids can be detected in the presence of 3-ketosteroids and can be even determined in a mixture (772). 

Table V, Entries 5 and 6). The catalyst tolerates both sulfur and nitrogen substituents (Table V, Entries 2, 4, and 8). Protected (1-ami no-alcohols are smoothly converted into the corresponding aldehydes without detectable racemisation (Table V, Entries 4 and 8) (28). It is also noteworthy that the reaction conditions are sufficiently mild as to be compatible with the Boc protecting group (Table V, Entry 8). 

The first consideration when setting up an AlphaScreen assay is the choice of an assay format In most cases, the decision will depend on the interaction partners under investigation and on the biological tools available for their detection. The interaction partners can be coupled to the beads directly via reductive amination of reactive aldehyde groups, similar to the immobilization on a Biacore sensor chip (see above). The usefulness of this approach is limited by the reaction conditions, which may not be appropriate for maintaining the biologically active conformation of the biomolecule. Therefore the biomolecule of interest is usually not coupled to the beads directly, but instead captured via an antibody, also preventing steric hindrance. While not strictly necessary, it is often convenient to use a biotinylated molecule which can be captured by streptavidin-coated donor beads. 

The carbinolamine bond observed in these crystal structures has not been detected biochemically. Nevertheless, its formation may explain the results of a number of previous biochemical experiments. Modification or removal of the aldehyde group from a macrolide results in 100-fold increases in minimum inhibitory concentrations (MIC) [22-28]. Furthermore, mutation of Hm A2103 (Ec 2062) to guanine (which has an 06 instead of N6) confers resistance specifically to macrolides that have an aldehyde group at C6, but not to others [29]. It seems likely that the carbinolamine bonds observed in these crystal structures also form in vivo, and are physiologically relevant to the inhibitory effects of macrolides. 

Ozonolysis of the corresponding meso-precursor 18 [97] gave the dialdehyde also as a complex mixture of isomeric forms, from which tandem aldolization with FruA expectedly delivered a non symmetrical, bisfuranoid undecodiulose 19 as the sole product which was isolated in 25% yield [56]. No intermediary mono adduct could be detected by t.l.c. from which follows that, no matter which of the enantiotopic aldehyde groups was attacked first, the second addition step must be kinetically faster, most likely due to steric reasons and the presence of anionic charge in the intermediates. 

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