# US 7,745,229 B2 · Chemoselective fluorgenic molecular linkers and methods for their preparation and use > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,745,229 B2 - Document ID: US7745229B2 - Status: Granted - Kind: B2 - Application number: 11/578,565 - Filed: 2005-04-15 - Published: 2007-09-27 - Granted: 2010-06-29 - Earliest claimed: 2004-04-16 - Assignees: University of South Carolina - Inventors: Qian Wang; Krishnamoorthy Sivakumar - Web page: https://patentgrep.com/patent/US7745229B2 ## CPC - G01N 33/533: with fluorescent label - C07D 405/04: directly linked by a ring-member-to-ring-member bond - C07D 405/14: containing three or more hetero rings - C07D 491/14: Ortho-condensed systems - C07K 1/13: Labelling of peptides - G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching - C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer - Y10S 436/80: Fluorescent dyes, e.g. rhodamine - Y10T 436/13: Tracers or tags - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## Abstract The present invention describes a bioconjugation strategy and compounds that are useful therein in which a fluorescent signal is produced when two molecular or supramolecular entities are linked by chemoselective combination of one linker having an azido or halide substituent group with another linker having a cyano or an alkyne substituent group. A kit is also provided. ## Claims ### Claim 1 A method of forming a linkage complex having a fluorescent signal, the method comprising: reacting a fluorophore with a linkage compound to form a linkage complex having a fluorescent signal that is different than a fluorescent signal of the fluorophore; wherein the fluorophore comprises coumarin substituted at position 3 or 4 with at least one azide group (—N3); and wherein the linkage compound has the structure where: m is 1 and LB comprises a linking group bonded to a biomolecule. ### Claim 2 (depends on 1) The method according to claim 1, wherein the linkage complex comprises a triazole. ### Claim 3 (depends on 1) The method according to claim 1, wherein the linkage complex comprises 1,2,3-triazole. ### Claim 4 (depends on 1) The method according to claim 1, wherein the fluorophore comprises a coumarin compound having the structure: where: n is an integer from 0 to 4; each R1 is the same or different and is any chemically possible substituent group, optionally two or more R1 groups join to form one or more substituted or unsubstituted rings; and at least one of R2 and R3 is —N3, and the other is any R1 group. ### Claim 5 (depends on 4) The method according to claim 4, wherein: each R1 is the same or different and is selected from hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, halo, carboxyl, carbonyl, alkyloxyl, alkoxyl, alkoxyalkyl, amino, alkylamino, azido, hydroxyalkyl, sulfonyl, sulfinyl, phospho, phosphino, or optionally two or more R1 groups join to form one or more substituted or unsubstituted rings. ### Claim 6 (depends on 4) The method according to claim 4, wherein R2 comprises —N3, and R3 comprises any R1 group. ### Claim 7 (depends on 1) The method according to claim 1, wherein the fluorophore comprises 3-azidocoumarin. ### Claim 8 (depends on 1) The method according to claim 1, wherein the fluorophore comprises 3-azido-7-dimethylaminocoumarin. ### Claim 9 (depends on 1) The method according to claim 1, wherein the fluorophore comprises 3-azido-7-hydroxycoumarin. ### Claim 10 (depends on 1) The method according to claim 1, wherein the fluorophore comprises 3-azido-7-methoxycoumarin. ### Claim 11 (depends on 1) The method according to claim 1, wherein the fluorophore comprises 7-acetic-3-azidocoumarin. ### Claim 12 (depends on 1) The method according to claim 1, wherein the fluorophore comprises 3-azido-7-diethylaminocoumarin. ### Claim 13 (depends on 1) The method according to claim 1, wherein the fluorophore comprises 3-azido-6-bromocoumarin. ### Claim 14 (depends on 1) The method according to claim 1, wherein the fluorophore comprises 4-azidocoumarin. ### Claim 15 (depends on 1) The method according to claim 1, wherein the fluorophore is chemically bonded to a second biomaterial. ### Claim 16 (depends on 15) The method according to claim 15, wherein the fluorophore is chemically bonded to the biomaterial according to the structure: where: n is an integer from 0 to 3; m is 1; each R1 is the same or different and is any chemically possible substituent group, optionally two or more R1 groups join to form one or more substituted or unsubstituted rings; at least one of R2 and R3 is —N3 and the other is any R1 group; and LA is a linking group chemically bonded to the second biomaterial. ### Claim 17 (depends on 16) The method according to claim 16, wherein LA is an organic substituent group having at least one reactive group that is selected from carboxylic acid esters, sulfonyl halide, acyl halide, aldehydes, reactive ketones, aromatic halides, bromoacetamides, idoacetamides, maleimides, disulfides, hydroxyl, amines, diazonium compounds, active alkylating reagents, active acylating reagents, or diketones. ### Claim 18 (depends on 16) The method according to claim 16, wherein LA is an organic substituent group that is selected from branched or unbranched C1-C12 alky, C1-C12 alkenyl, C1-C12 alkynyl, or aryl, heteroaryl, or heterocyclyl, and which has at least one substituent reactive group that is selected from carboxylic acid esters, sulfonyl halide, acyl halide, aldehydes, reactive ketones, aromatic halides, bromoacetamides, idoacetamides, maleimides, disulfides, hydroxyl, amines, diazonium compounds, active alkylating reagents, active acylating reagents, or diketones. ### Claim 19 (depends on 15) The method according to claim 15, wherein the second biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces. ### Claim 20 (depends on 1) The method according to claim 1, wherein the biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces. ### Claim 21 (depends on 1) The method according to claim 1 further comprising measuring the fluorescent signal of the linkage complex. ### Claim 22 A kit for forming a linkage complex having a fluorescent signal, the kit comprising a fluorophore comprising coumarin substituted at position 3 or 4 with at least one azide group (—N3); and a linkage compound, wherein the linkage compound has the structure where: m is 1 and LB comprises a linking group bonded to a biomolecule. ### Claim 23 (depends on 22) The kit according to claim 22, wherein the fluorophore comprises a coumarin compound having the structure: where: n is an integer from 0 to 4; each R1 is the same or different and is any chemically possible substituent group, optionally two or more R1 groups join to form one or more substituted or unsubstituted rings; and at least one of R2 and R3 is —N3, and the other is any R1 group. ### Claim 24 (depends on 23) The kit according to claim 23, wherein: each R1 is the same or different and is selected from hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, halo, carboxyl, carbonyl, alkyloxyl, alkoxyl, alkoxyalkyl, amino, alkylamino, azido, hydroxyalkyl, sulfonyl, sulfinyl, phospho, phosphino, or optionally two or more R1 groups join to form one or more substituted or unsubstituted rings. ### Claim 25 (depends on 23) The kit according to claim 23, wherein R2 comprises —N3, and R3 comprises any R1 group. ### Claim 26 (depends on 22) The kit according to claim 22, wherein the fluorophore comprises 3-azidocoumarin. ### Claim 27 (depends on 22) The kit according to claim 22, wherein the fluorophore comprises 3-azido-7-dimethylaminocoumarin. ### Claim 28 (depends on 22) The kit according to claim 22, wherein the fluorophore comprises 3-azido-7-hydroxycoumarin. ### Claim 29 (depends on 22) The kit according to claim 22, wherein the fluorophore comprises 3-azido-7-methoxycoumarin. ### Claim 30 (depends on 22) The kit according to claim 22, wherein the fluorophore comprises 7-acetic-3-azidocoumarin. ### Claim 31 (depends on 22) The kit according to claim 22, wherein the fluorophore comprises 3-azido-7-diethylaminocoumarin. ### Claim 32 (depends on 22) The kit according to claim 22, wherein the fluorophore comprises 3-azido-6-bromocoumarin. ### Claim 33 (depends on 22) The kit according to claim 22, wherein the fluorophore comprises 4-azidocoumarin. ### Claim 34 (depends on 22) The kit according to claim 22, wherein the fluorophore is chemically bonded to a biomaterial. ### Claim 35 (depends on 22) The kit according to claim 22, wherein the fluorophore is chemically bonded to a second biomaterial. ### Claim 36 (depends on 35) The kit according to claim 35, wherein the fluorophore comprises a compound having the structure: where: n is an integer from 0 to 3; m is 1; each R1 is the same or different and is any chemically possible substituent group, optionally two or more R1 groups join to form one or more substituted or unsubstituted rings; at least one of R2 and R3 is —N3, and the other is any R1 group; and LA is a linking group chemically bonding the fluorophore to the second biomaterial. ### Claim 37 (depends on 35) The kit according to claim 35, wherein the second biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces. ### Claim 38 (depends on 22) The kit according to claim 22, wherein the biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces. ### Claim 39 (depends on 22) The kit according to claim 22 further comprising a catalyst, wherein the catalyst comprises copper(I). ### Claim 40 (depends on 1) The method according to claim 1 wherein reacting the fluorophore with the linkage compound to form the linkage complex is catalyzed with copper(I).