Chemistry: analytical and immunological testing – Metal or metal containing – Li – na – k – rb – cs – fr – be – mg – ca – sr – ba – ra
Reexamination Certificate
1998-07-30
2001-01-30
Ceperley, Mary E. (Department: 1641)
Chemistry: analytical and immunological testing
Metal or metal containing
Li, na, k, rb, cs, fr, be, mg, ca, sr, ba, ra
C435S007210, C435S040500, C436S519000, C436S537000, C436S805000, C548S242000, C548S525000, C549S060000, C549S467000, C549S468000, C540S461000, C546S089000
Reexamination Certificate
active
06180411
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to methods of determining analyte concentrations, and more particularly, to photolabile chelators.
BACKGROUND OF THE INVENTION
Although Ca
2+
is a well established intracellular messenger, there are still many unanswered questions concerning the kinetics and spatial localization of its effects. Because of the importance of calcium as an intracellular messenger, a variety of methods have been developed for measuring intracellular Ca
2+
levels. The most successful of these use dye-linked chelators which change absorbance or fluorescence upon binding Ca
2+
ions. Existing indicators for physiological cations such as Ca
2+
, as well as, Mg
2+
, H
+
, and Na
+
change their optical properties reversibly in response to changes in cation concentrations. See, for example, U.S. Pat. No. 4,603,209, and EP 314,480. Chelating compounds have been developed that facilitate changing the intracellular calcium concentration. See, for example, U.S. Pat. Nos. 4,689,432, 4,806,604, and 5,141,627; and EP 177,202, in which a photochemical reaction causes the release or uptake of Ca
2+
and thereby alters the ambient Ca
2+
.
SUMMARY OF THE INVENTION
In general, the invention features optical indicators which memorize the spatial location and/or concentration of an analyte at a particular chosen point in time. This can allow for time-resolved analysis of analyte concentration. Analysis of the temporal and spatial resolution of the intracellular concentrations of physiologically significant analytes, such as calcium and other cations, can be facilitated by freezing a snapshot of the analyte concentration in the intracellular matrix. The snapshot can allow one to postpone the analysis of the analyte concentration at that particular moment to a later time.
In one aspect, the invention features a compound including a chromophore carrying a photolabile group capable of undergoing an irreversible and detectable chemical transformation upon irradiation by light, the chromophore being linked to or including a binding or chelating site for an analyte. Binding of the analyte to the binding site alters an optical property of the chromophore, thus altering the ability of the photolabile group to undergo the chemical transformation upon irradiation of the compound.
The analyte can be a cation, such as calcium and the chelator can be a BAPTA or BAPTA-like derivative. The chromophore can be any chemical grouping, such as an aromatic, heteroaromatic, or polyene group which absorbs light. One type of photolabile group that photolyzes with high efficiency to an isoxazole is a &bgr;-azido, &agr;,&bgr;-unsaturated carbonyl group.
In another aspect, the invention relates to a method of memorizing an intracellular concentration of an analyte. The method includes the steps of: (a) loading a cell with an effective amount of a compound as described above under conditions which do not alter the chromophore; (b) irradiating the cell with a wavelength of light which selectively photolyzes the compound to yield a photolyzed product; (c) detecting the presence of the photolyzed product and/or unphotolyzed compound; and (d) relating the presence of the photolyzed product and/or unphotolyzed compound to the intracellular concentration of the analyte. The cell is briefly irradiated. The compound can be bound to the analyte when photolyzed to form the product. Only one form of the compound (i.e., bound or not bound) forms the photolyzed product.
In another aspect, the invention features kits for practicing the above method.
The term “hydrocarbyl” refers to an organic radical comprised of carbon chains to which hydrogen and other elements are attached. The term includes alkyl, alkenyl, alkynyl and aryl groups, groups which have a mixture of saturated and unsaturated bonds, carbocyclic rings and includes combinations of such groups. Hydrocarbyl can refer to straight chain, branched-chain, cyclic structures or combinations thereof.
The term “alkyl” refers to a branched or straight chain acyclic, monovalent saturated hydrocarbon radical of one to twenty carbon atoms.
The term “lower-alkyl” refers to an alkyl radical of one to six carbon atoms. This term is further exemplified by such radicals as methyl, ethyl, n-propyl, isopropyl, isobutyl, sec-butyl, n-butyl and tert-butyl, n-hexyl and 3-methylpentyl.
The term “alkenyl” refers to an unsaturated hydrocarbon radical which contains at least one carbon—carbon double bond and includes straight chain, branched chain and cyclic radicals.
The term “alkynyl” refers to an unsaturated hydrocarbon radical which contains at least one carbon-carbon triple bond and includes straight chain, branched chain and cyclic radicals.
The term “acyl” refers to the group R—C(O)—, wherein R represents a hydrocarbyl group or a heterocyclic or homocyclic aromatic group.
The term “lower” referred to herein in connection with organic radicals or compounds respectively defines such with up to and including six, preferably up to and including four carbon atoms. Such groups may be straight chain or branched.
The term “aryl” refers to an aromatic carbocyclic radical having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl, anthracenyl). Aryl radicals can be optionally substituted with substituents exemplified, but not limited, to those listed below.
The term “heterocyclic aromatic” refers to an aromatic mono- or poly-cyclic radical having at least one heteroatom within a ring, e.g., nitrogen, oxygen, or sulfur. For example, typical heteroaryl groups with one or more nitrogen atoms are tetrazoyl, pyrrolyl, pyridyl (e.g., 4-pyridyl, 3-pyridyl, 2-pyridyl), pyridazinyl, indolyl, quinolyl (e.g., 2-quinolyl, 3-quinolyl, etc.), imidazolyl, isoquinolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridonyl or pyridazinonyl; typical oxygen heteroaryl radicals with an oxygen atom are 2-furyl, 3-furyl or benzofuranyl; typical sulfur heteroaryl radicals are thienyl, and benzothienyl; typical mixed heteroatom heteroaryl radicals are furazanyl, oxazolyl, isoxazolyl, thiazolyl, and phenothiazinyl. Further the term also includes instances where a heteroatom within the ring has been oxidized, such as, for example, to form an N-oxide or sulfone. Heterocyclic aromatic radicals can be optionally substituted with substituents exemplified, but not limited, to those listed below.
The term “optionally substituted” refers to the presence or lack thereof of a substituent on the group being defined. When substitution is present, the group may be mono-, di- or tri-substituted, independently, with alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl, aminolower-alkyl, hydroxyl, thiol, amino, halo, nitro, lower-alkylthio, lower-alkoxy, mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl, lower-alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl, lower-alkylsulfonyl, lower-alkylsulfinyl, trifluoromethyl, cyano, tetrazoyl, carbamoyl, lower-alkylcarbamoyl, and di-lower-alkylcarbamoyl. The substituent can be a carboxylic acid, ester, sulfonamides, phosphono or phosphoryl group, or arsono group.
The term “pharmaceutically acceptable salt” refers to those salts which retain the biological effectiveness and properties of the free bases or free acids and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, benzoic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid and the like; or inorganic or organic bases.
The term “pharmaceutically acceptable ester” refers to those nontoxic readily hydrolyzable esters conventionally used in the pharmaceutical industry, especially &agr;-acyloxyalkyl esters, such as acetoxymethyl esters. The invention contemp
Adams Stephen R.
Tsien Roger Y.
Ceperley Mary E.
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
The Regents of the University of California
LandOfFree
Light-triggered indicators that memorize analyte concentrations does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Light-triggered indicators that memorize analyte concentrations, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Light-triggered indicators that memorize analyte concentrations will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2533931