Tis invention relates to fluorescent 7-hydroxy coumarin compounds with substitutions in the 4 position having a length greater than one carbon atom. The compounds thus are derivatives of 4-methylumbelliferone (7-hydroxy-4-methyl coumarin, or 4-MU), the detectable label used in the IM.sub.x .RTM. instrument assays (Abbott Laboratories, Abbott Park, Ill.).
A number of fluorometric labels are known to one of ordinary skill in the art. However, for compatibility reasons, applicants desired a fluorophore label that had electronic properties substantially similar to the 4-MU utilized in the IM.sub.x .RTM. instrument. Otherwise, the label might fluoresce at a wavelength the instrument could not detect absent special filters and the like. A label optimized to the existing instrument was necessary.
The search began for a coumarin or umbelliferone nucleus that had an activated or activatable tether that could be coupled to a desired molecule. Such a tether to a coumarin nucleus had been obtained in the past by a Pechmann condensation to give either a 4-position methyl group or a 3-position alkyl substitution on the coumarin [H. V. Pechmann and C. Duisberg, Chem. Ber. 16, 2119 1883)]: ##STR2## Where R represents the activatable tether.
The 4-methyl product does not provide an activatable tether group. The effect of 3-position substitution on electronic structure in coumarins is shown in the .sup.13 C NMR spectra complied by Parmar and Boll [Mag. Res. Chem., 26, 430-433 (1988)]. If R of the product above is H, the .sup.13 C NMR chemical shift of C-3 is 110 ppm; while if R is CH.sub.2 COOEt, C-3 resonates at 115 ppm. This means that the relative electron density on C-3 has decreased upon alkyl substitution, disturbing the electronic structure of the coumarin nucleus. Thus, the Pechmann condensation was not useful since it did not produce 4-substituted materials, exclusive of having substitution at the 3-position, and since a compound having no substituent at the 3-position was desired because of the need for substantially similar electronic properties as 4-MU.
USPTO Backgrounds
USPTO Backgrounds