Nile Red Dyes

Oxazine

Oxazine is a heterocyclic compound containing one oxygen and one nitrogen atom in a double unsaturated six-membered ring. The presence of isomers depends on the relative position of the heteroatom and the relative position of the double bond. By extension, these derivatives are also called oxazines. Examples include ifosfamide and morpholine (tetrahydro-1,4-oxazine). Commercially available dihydro-1,3-oxazine is a reagent in the Meyers synthesis of aldehydes. Fluorescent dyes such as Nile Red and Nile Blue are based on aromatic benzoxazines. Cinnabar and cinnabaric acid are two naturally occurring dioxazines derived from the biodegradation of tryptophan. Nile red is an oxazine derivative fluorescent dye.

Figure 1. The 8 existing isomers of oxazine.

Dioxazine

Dioxazine is a pentacyclic compound consisting of two oxazine subunits. A commercially important example is Pigment Violet 23.

Figure 2. Synthetic route to dioxazine dyes.

Benzoxazine

The benzoxazine formed by the reaction of phenol, formaldehyde, and primary amine polymerizes to form a polybenzoxazine network when heated to about 200 ° C (~ 400 ° F). The obtained high molecular weight thermosetting polymer-based composites can be used in applications requiring higher mechanical properties, flame resistance and fire resistance than epoxy resins and phenolic resins.

Nile Red

Nile Red (also known as Nile Blue Oxazolone) is a lipophilic dye. Nile red staining made intracellular lipid droplets yellow. In most polar solvents, Nile Red does not fluoresce; however, when in a lipid-rich environment, it can emit strong fluorescence with a color ranging from deep red (for polar membrane lipids) to intense the golden-yellow emission (for neutral lipids in intracellular storage) varies. The dye is highly solvated, its emission and excitation wavelengths vary with the polarity of the solvent, and it hardly emits fluorescence in polar media. Nile Red has applications in cell biology and can be used as a membrane dye. It can be easily seen using epi-fluorescence microscopy. Excitation and emission wavelengths are usually shared with red fluorescent proteins. Nile Red is also used as part of the sensitive detection process for microplastics in bottled water.

Figure 3. Ball-and-stick model of Nile red.

Synthesis

Nile Red can be prepared by boiling Nile Blue with sulfuric acid by acid hydrolysis. This method replaces imino with carbonyl. Alternatively, Nile Red and its analogs (naphthoxazine dyes) can be prepared by acid-catalyzed condensation of the corresponding 5- (dialkylamino) -2-nitrosophenol with 2-naphthol. Since no co-oxidant is used in this process, the yield is generally medium. Since the reaction to produce Nile Red usually does not completely deplete the supply of Nile Blue, an additional separation step is required if pure Nile Red is required.

Figure 4. Nile red synthesis.

References:

1. R Plenderleith.; et al. Highly-branched poly(N-isopropyl acrylamide)s with core–shell morphology below the lower critical solution temperature. RSC Advances. 2014, 4 (92): 50932–50937.
2. SD Fowler & P Greenspan. Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O. Journal of Histochemistry and Cytochemistry. 1985, 33 (8): 833–836.