Carbostyril Derivatives as Antenna Molecules for Luminescent Lanthanide Chelates

Abstract

Luminescent lanthanide complexes consisting of a lanthanide-binding chelate and organic-based antenna molecule have unusual emission properties, including millisecond excited state lifetimes and sharply spiked spectra, compared to standard organic fluorophores. We have previously used carbostyril (cs124, 7-amino-4-methyl-2(1H)-quinolinone) as an antenna molecule (Li and Selvin, J. Am. Chem. Soc., 1995) attached to a polyaminocarboxylate chelate such as DTPA. Here, we report the chelate syntheses of DTPA conjugated with cs124 derivatives substituted on the 1-, 3-, 4-, 5-, 6-, and 8-position. Among them, the DTPA chelate of cs124-6-SO(3)H has similar lifetime and brightness for both Tb(3+) and Eu(3+) compared to the corresponding DTPA-cs124 complexes, yet it is significantly more soluble in water. The Tb(3+) complex of DTPA-cs124-8-CH(3) has significantly longer lifetime compared to DTPA-cs124 (1.74 vs 1.5 ms), indicating higher lanthanide quantum yield resulting from the elimination of back emission energy transfer from Tb(3+) to the antenna molecule. Thiol-reactive forms of chelates were made for coupling to proteins. These lanthanide complexes are anticipated to be useful in a variety of fluorescence-based bioassays.

Minocycline‐Based Europium(III) Chelate Complexes: Synthesis, Luminescent Properties, and Labeling to Streptavidin

“…As described for 9, with 0.1m Tris · HCl (pH 8.0; 2 ml) containing 12 (2.5 mg, 2.63 mmol) and a soln. (12), N-hydroxysuccinimide (NHS), and DCC were vacuum-dried for 24 h before use. To a soln.…”

“…The titrations suggested also that an 1 : 1 ligand Eu 3þ complex is formed from 12, whereas an 1 : 2 complex was formed from 8 minocycline-DTPA. The H 5 (minocycline-TTHA) (12) was successfully conjugated to streptavidin (SA) (Scheme 5), and thus the applicability of the corresponding Eu 3þ complex to label a protein was established.…”

“…Ligands H 4 (minocycline-DTPA) (8) and H 5 (minocycline-TTHA) (12). From all of the above unsuccessful attempts to link H 4 EDTA or H 6 TTHA to the amide N-atom of Scheme 2 tetracycline, we concluded that the amide group at the 2-position of tetracycline is not suitable to link amino groups of other molecules, and we moved to minocycline (5) instead of tetracycline (1) since amination at the 9-position of 5 had been reported [31], and H 5 DTPA (¼ diethylenetriaminepentaacetic acid ¼ N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine) and H 6 TTHA were used as the chelating groups.…”

“…Both spectra are very similar to each other, and suggest that the spectra are dominated by the minocycline moiety and are not appreciably affected by the aminopolycarboxy moiety. The absorption maxima and the molar extinction coefficients are listed in the Table, together with those of free minocycline, free H 4 (minocycline-DTPA) (8), and free H 5 (minocycline-TTHA) (12). The absorption bands of 8 or 12 at ca.…”

“…Streptavidin was selected as a typical protein to see whether the new labeling reagent can be labeled to proteins without any inconvenience, since our past experience showed that for some labeling reagents for Eu 3þ , the conjugation reaction causes precipitation of the protein. If SA can be conjugated successfully to H 5 (minocycline-TTHA) (12), it means that the reagent can be used in many bio-analytical applications taking advantage of the selective and strong binding of the conjugated SA to biotin. Thus, one of the carboxy groups of compound 12 was succinimidylated (ester formation with N-hydroxysuccinimide (NHS)) to give 14, which was treated with SA in 0.1m phosphate buffer (pH 8.0) at room temperature for 6 h. The details of the purification of the conjugated streptavidin, H 4 (SA-minocycline-TTHA), is described in the Exper.…”