Several kynurenine derivatives including N'-formylkynurenine were prepared in high purity by the ozonization of the corresponding indole compounds. The fluorescence characteristics of those derivatives were examined in connection with the use of their fluorophores as reporters for the local environment of tryptophan in proteins. Kynurenine is a weak emitter of fluorescence, with an emission maximum at 480 nm on excitation at 365 nm. With decreasing solvent polarity, the fluorescence intensity increases logarithmically and the emission maximum shifts to blue. A linear relation between these fluorescence characteristics and solvent polarity exists when the polarity is shown in terms of dielectric constant. N'-Formylkynurenine is a somewhat stronger emitter of fluorescence than kynurenine. The emission maximum is 434 nm on excitation at 325 nm and it shifts to blue in solvents of low polarity. This blue shift is also linear with respect to the dielectric constant of the solvent. Other factors influencing kynurenine fluorescence and N'-formylkynurenine fluorescence examined were neighboring groups, ionic strength, temperature, and protein denaturants. Based on the results of the present investigation, the local environment of tryptophan 62 in hen egg-white lysozyme was examined using Kyn 62-lysozyme.

A pair of novel fluorophores (N-diacetates of crosslines A and B), (3/?,4S)-3,4-dihydroxy-5-[(1 S or 1 R,2 S,3/?)-1,2,3,4-tet ra hyd roxybutyll-1,7-bis[6-( N-acetyl-~-norleucyl )]-I ,2,3,4-tetra hyd ro-I ,7-nap ht hyrid i ni u m chloride and its epimer, were isolated from the Maillard reaction mixture of a-N-acetyl-L-lysine and D-glucose, and their properties are similar to those of fluorophores in age-and diabetes-related cross-linked proteins, and a pair of homologues derived from n-pentylamine.

We employed Raman microspectroscopy to measure the Raman spectra of phosphate in sound and carious tooth substance. The peak intensity at 960 cm −1 of the phosphate (PO 4 3− ) symmetric stretching vibrational mode (u 1 ) in sound enamel was stronger than that of sound dentin, which indicated that sound enamel contained more phosphate than sound dentin. Furthermore, the element analysis of phosphate in sound teeth substance, measured using a scanning electron microscope (SEM) equipped with an energy dispersive X-ray spectroscope (EDX), gave similar results to those of the Raman measurement. In addition, the border between sound enamel and dentin was clearly demonstrated by mapping the image of the Raman spectrum of phosphate. The mapping image of phosphate in the carious enamel region revealed a heterogeneous low Raman spectrum intensity of phosphate in the area surrounding carious enamel; this finding indicates that phosphate had dissolved from the tooth substance in such areas. In contrast with the decrease in the Raman spectrum intensity of phosphate, the intensity of amide I increased mainly in the low-phosphate area. Although it remains very difficult to clinically identify the accurate border between sound and carious tooth substance, this distinction may be enabled by using the Raman spectrum of carious tooth substance.

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