Fluorescent labels hydrazide derivatives

Fluorescein-5-thiosemicarbazide is a hydrazide derivative of fluorescein that can spontaneously react with aldehyde- or ketone-containing molecules to form a covalent, hydrazone linkage (Fig. 208) (Pierce). It also can be used to label cytosine residues in DNA or RNA by use of the bisulfite activation procedure (Chapter 17, Section 2.1). The resulting fluorescent derivative exhibits an excitation maximum at a wavelength of 492 nm and a maximal emission wavelength of 519 nm when dissolved in buffer at pH 8.6. In the same buffered environment, the compound has an extinction coefficient of approximately 78,000 M-1cm 1 at 492 nm. 

Texas Red hydrazide is a derivative of Texas Red sulfonyl chloride made by reaction with hydrazine (Invitrogen). The result is a sulfonyl hydrazine group on the No. 5 carbon position of the lower-ring structure of sulforhodamine 101. The intense Texas Red fluorophore has a QY that is inherently higher than either the tetramethylrhodamine or Lissamine rhodamine B derivatives of the basic rhodamine molecule. Texas Red s luminescence is shifted maximally into the red region of the spectrum, and its emission peak only minimally overlaps with that of fluorescein. This makes derivatives of this fluorescent probe among the best choices of labels for use in double-staining techniques. 

Hydrazide groups directly react with aldehyde and ketone functional groups to form relatively stable hydrazone linkages (Chapter 2, Section 5.1). Two fluorescein derivatives are commonly available that contain hydrazide groups off their No. 5 carbons on the lower ring structure. Both may be used to label fluorescently aldehyde- or ketone-... 

Where reducing (aldehydic) terminals are available for reaction, they can be labelled by reduction with agents such as tritiated sodium borohydride or by conversion into a cyanohydrin with " C-labelled sodium cyanide. Alternatively, labelling with reagents such as dansyl hydrazide is possible, to yield a fluorescent derivative. 

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