University of California

Akwasi Minta

Roger Y Tsien

Grzegorz Grynkiewicz

The invention discloses fluorescent chelator compounds that are especially useful to monitor and measure cytosolic concentrations of alkali metal cations such as Na.sup.+, K.sup.+, and Li.sup.+. The new compounds are comprised of: (1) crown ethers (that may or may not have substituent groups attached to the core carbons, but will always contain at least one core nitrogen) that are linked via the core nitrogen(s) to at least one (2) fluorophore that contains an additional heteroaromatic liganding center. In the currently preferred dye, SBFI, the core compound is crown ether 1,7-diaza-4,10,13-trioxacyclopentadecane and the heteroaromatic fluorophores are benzofurans that are linked to isophthalate groups. Selectivities for Na.sup.+ over K.sup.+ of about 20 are observed, resulting in effective dissociation constants for Na.sup.+ of about 20 mM against a background of 120 mM K.sup.+. Increasing �Na.sup.+ ! increases the ratio of excitation efficiency at 330-345 nm to that at 370-390 nm with emission collected at 450-550 nm, so that ratio fluorometry and imaging work at the same wavelengths as used with the well-known Ca.sup.2+ indicator fura-2. If the macrocyclic ring is increased in size to a 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane, the chelators become selective for K.sup.+ over Na.sup.+. If the ring is decreased in size, for example to a 1,7-diaza-4,10-dioxacyclododecane, the chelators become selective for Li.sup.+ over Na.sup.+.

Fluorescent indicator dyes for alkali metal cations

The invention discloses fluorescent chelator compounds that are especially useful to monitor and measure cytosolic concentrations of alkali metal cations such as Na.sup.+, K.sup.+, and Li.sup.+. The new compounds are comprised of: (1) crown ethers (that may or may not have substituent groups attached to the core carbons, but will always contain at least one core nitrogen) that are linked via the core nitrogen(s) to at least one (2) fluorophore that contains an additional heteroaromatic liganding center. In the currently preferred dye, SBFI, the core compound is crown ether 1,7-diaza-4,10,13-trioxacyclopentadecane and the heteroaromatic fluorophores are benzofurans that are linked to isophthalate groups. Selectivities for Na.sup.+ over K.sup.+ of about 20 are observed, resulting in effective dissociation constants for Na.sup.+ of about 20 mM against a background of 120 mM K.sup.+. Increasing [Na.sup.+ ] increases the ratio of excitation efficiency at 330-345 nm to that at 370-390 nm with emission collected at 450-550 nm, so that ratio fluorometry and imaging work at the same wavelengths as used with the well-known Ca.sup.2+ indicator fura-2. If the macrocyclic ring is increased in size to a 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane, the chelators become selective for K.sup.+ over Na.sup.+. If the ring is decreased in size, for example to a 1,7-diaza-4,10-dioxacyclododecane, the chelators become selective for Li.sup.+ over Na.sup.+.

The present invention discloses a new class of calcium specific fluorescent indicator dyes having visible excitation and emission wavelengths. The new fluorescent indicator dyes combine at least one tricyclic chromophore with a tetracarboxylate parent CA.sup.2+ chelating compound having the octacoordinate pattern of liganding groups characteristic of BAPTA to give a rhodamine-like or fluorescein-like fluorophore. Binding of calcium.sup.2+ increases the fluorescence of the new compounds by up to 40-fold. The calcium.sup.2+ dissociation constants are in the range 0.37-2.3 microM, so that the new indicators give better resolution of high [CA.sup.2+ ] levels than were previously obtainable with predecessor compounds such as quin-2 or fluo-2. The visible excitation wavelengths of the new compounds are more convenient for fluorescent microscopy and flow cytometry than the UV required by previous indicators.

Fluorescent indicator dyes for calcium working at long wavelengths

The present invention discloses a group of calcium chelating compounds which have a descreased affinity for calcium following illumination. These new compounds contain a photolabile nitrobenzyl derivative coupled to a tetracarboxylate Ca.sup.2+ chelating parent compound having the octacoordinate chelating groups characteristic of EGTA or BAPTA. In a first form, the new compounds are comprised of a BAPTA-like chelator coupled to a single 2-nitrobenzyl derivative, which in turn is a photochemical precursor of a 2-nitrosobenzophenone. In a second form, the new compounds are comprised of a BAPTA-like chelator coupled to two 2-nitrobenzyl derivatives, themselves photochemical prcursors of the related 2-nitrosobenzophenones. The present invention also discloses a novel method for preparing 1-hydroxy- or 1-alkoxy-1-(2-nitroaryl)-1-aryl methanes. Methanes of this type are critical to the preparation of, or actually constitute, the photolabile Ca.sup.2+ chelating compounds disclosed and claimed herein.

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Eugene, OR, United States of America

Akwasi Minta