3326-32-7

Fluorescein 5-isothiocyanate (5-FITC) is an isomer of fluorescein isothiocyanate (FITC), which is commonly used as a mixture of the 5- and 6-isothiocyanate isomers. It reacts with amine and thiol groups to form conjugates with proteins, lipids, and other molecules for detection by a variety of fluorescent-based applications. 5-FITC displays excitation/emission maxima of 494/519 nm, respectively.

Fluorescein isothiocyanate(3326-32-7) is an orange-yellow crystal that is hygroscopic. It can be combined with various antibody proteins, and the combined antibody does not lose its specificity for binding to a certain antigen, and still has strong green fluorescence in alkaline solution, precipitates out after adding acid, and the fluorescence disappears. It is slightly soluble in acetone and ether and petroleum ether.

Fluorescein isothiocyanate is used as fluorescent labeling reagent for proteins and for rapid identification of pathogens in fluorescent antibody technique. It is also used as a reagent for microsequencing of proteins and peptides (HPLC). On celite it is used for the labeling of fibrinogen.

Fluorescein -5-isothiocyanate is a kind of fluorescent marker for biochemical applications. Reacts under mild conditions with primary amines and it is also used in modification of actin at lys-61, labelling of FAB and the modification of thiol groups.

ChEBI: Fluorescein 5-isothiocyanate is the 5-isomer of fluorescein isothiocyanate. Acts as a fluorescent probe capable of being conjugated to tissue and proteins; used as a label in fluorescent antibody staining procedures as well as protein- and amino acid-binding techniques.

Fluorescein 5-isothiocyanate (5-FITC) was first prepared as a hydrochloride in 1958 by the reaction of aminofluorescein and thiophosgene in acetone. However, only the neutral form of FITC can be used as a microbiological marker. To obtain neutral FITC, Boldyreva et al. introduced calcium carbonate, amides or tertiary amines into the synthesis (Figure 1)[4].

Store at 2-8°C, protect from light, stored under nitrogen

It is made from the pure 5-amino isomer. Purify it by dissolving it in boiling Me2CO, filtering and adding pet ether (b 60-70o) until it becomes turbid. If an oil separates, then decant it and add more pet ether to the supernatant and cool. Orange-yellow crystals separate, collect and dry them in vacuo. It should give one spot on TLC (silica gel) in EtOAc/pyridine/AcOH (50:1:1) and in Me2NCHO/CHCl3/28% NH4OH (10:5:4). IR (Me2SO): 2110 (NCS) and 1760 (C=O) cm-1. The max 1HNMR spectra in Me2CO-d6 of the 5-and 6-isomers are distinctly different for the protons in the *benzene ring; the UV in phosphate buffer pH 8.0 shows a at ~490nm. [Sinsheimer et al. Anal Biochem 57 2271974, McKinney et al. Anal Biochem 7 74 1964, Beilstein 19 III/IV 4337.]

[1] VOLKER SCHNAIBLE  Michael P. Identification of Fluorescein-5‘-Isothiocyanate-Modification Sites in Proteins by Electrospray-Ionization Mass Spectrometry[J]. Bioconjugate Chemistry Bioconjugate, 1999, 10 5: 861-866. DOI: 10.1021/bc990039x
[2] 4811218 Real time scanning electrophoresis apparatus for DNA sequencing[J]. International Journal of Biochemistry, 1989, 21 8: Page iii. DOI: 10.1016/0020-711x(89)90318-2
[3] SABNIS R W. Fluoroscein‐5‐isothiocyanate (FITC)[C]. 2015: 0. DOI: 10.1002/9781119007104.ch80

492/517 nm