Aldehyde- and Ketone-Reactive Coumarin Derivatives

DNA and RNA may be modified with hydrazide-reactive probes by reacting their cytosine residues with bisulfite to form reactive sulfone intermediates. These derivatives can undergo transamination reactions with hydrazide- or amine-containing probes to yield covalent bonds (Draper and Gold, 1980) (Chapter 27, Section 2.1). 

AMCA-Hydrazide 7-Amino-4-methylcoumarin-3-acetyl hydrazide MW 247. 1 Excitation = 345 nm Emission = 440-460 nm 

AMCA-hydrazide has a maximal excitation wavelength of 345 nm and a maximum emission wavelength in the range of 440-460 nm. A solution of AMCA in PBS at a concentration of 16.7ng/ml (71.61 nmoles/ml) gives an absorbance at 345 nm of about 1.28. This translates into a molar extinction coefficient at this wavelength of about 13,900M em-1. Different solvents and conditions may alter this value somewhat. 

AMCA-hydrazide is soluble in DMSO or DMF and may be dissolved as a concentrated stock solution in either of these solvents prior to the addition of a small aliquot to an aqueous reaction medium. The solid and all solutions made from the fluorophore must be protected from light to avoid photo-decomposition. Prepare the stock solution fresh immediately before use. A suggested protocol on the use of this fluorescent probe may be obtained from the following method on the labeling of periodate-oxidized IgG. Optimization may be necessary to achieve the best level of fluorescent modification (F/P ratio) for a particular application. 

Oxidation of IgG Carbohydrate Residues with Sodium Periodate 

The hydrazide derivative can be used to modify aldehyde- or ketone-containing molecules, including cytosine residues using the bisulfite activation procedure de- 

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Several derivatives of (57) have been studied as phototriggers for alcohols and phenols. All of them are reactive, with varying quantum yields for the release of the alcohol component from decomposition of the carbonate moiety. The best of these was found to be (58), which releases phenol with a quantum yield of 0.067. Dore and co-workers have described the photochemical activity of the related coumarin derivative (59). Irradiation brings about release of the aldehyde or ketone unit from the side chain affording (60). 

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