# US 8,541,604 B2 · Process for the functionalization of biological molecules > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,541,604 B2 - Document ID: US8541604B2 - Status: Granted - Kind: B2 - Application number: 12/905,393 - Filed: 2010-10-15 - Published: 2011-05-19 - Granted: 2013-09-24 - Earliest claimed: 2009-10-15 - Assignees: Commissariat A L'energie Atomique Et Aux Energies Alternatives - Applicants: Aude Bernardin; David Bonnaffe; Isabelle Texier-Nogues - Inventors: Aude Bernardin; David Bonnaffe; Isabelle Texier-Nogues - Web page: https://patentgrep.com/patent/US8541604B2 ## CPC - C07C 221/00: Preparation of compounds containing amino groups and doubly-bound oxygen atoms bound to the same carbon skeleton - C07D 311/82: Xanthenes - C07F 7/1804: {Compounds having Si-O-C linkages (Si-O-acyl linkages C07F7/1896)} - C07B 2200/11: Compounds covalently bound to a solid support - C07C 2601/18: with a ring being at least seven-membered - C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS - C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08) - C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22) - C07B: GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR (preparation of carboxylic acid esters by telomerisation C07C67/47; telomerisation C08F) ## Abstract The present invention is targeted at a novel coupling method of 1,3-dipolar cycloaddition type which is of particular use for the functionalization of biological molecules, such as, for example, proteins, enzymes or nucleic acids, insofar as it can be carried out in an aqueous medium and at ambient temperature. ## Claims ### Claim 1 A process of grafting at least one molecule of interest to an additional entity comprising at least the stages consisting in having available said molecule provided, at the surface, with at least one group G1 which is reactive according to a 1,3-dipolar cycloaddition reaction, and bringing together said molecule and said entity provided, at the surface, with at least one group G2 complementary to the group G1 from the viewpoint of said 1,3-dipolar cycloaddition, under conditions favorable to the cycloaddition of said groups G1 and G2, wherein said groups G1 and G2 are respectively a nitrone and a strained cycloalkynyl radical or vice versa. ### Claim 2 (depends on 1) The process according to claim 1, wherein the cycloaddition is carried out in an aqueous medium. ### Claim 3 (depends on 1) The process according to claim 1, wherein the cycloaddition is carried out at ambient temperature or at body temperature. ### Claim 4 (depends on 1) The process according to claim 1, wherein said molecule of interest is chosen from labels, colorants, fluorophores, radiolabeled molecules, contrast agents (X-ray, MRI), linkers, toxins, therapeutic, cosmetic or plant-protection active principles, members of a specific binding pair, peptides, amino acids and amino acid residues, polypeptides, sugars and sugar residues, photosensitizers, porphyrins, or compounds exhibiting cis-trans isomerism. ### Claim 5 (depends on 4) The process according to claim 4, wherein the compounds exhibiting cistrans isomerism are chosen from diarylethylene derivatives, spiropyrans, spiroxazines, fulgides and azobenzene. ### Claim 6 (depends on 1) The process according to claim 1, wherein said molecule of interest is biological. ### Claim 7 (depends on 1) The process according to claim 1, wherein the additional entity is also a molecule of interest. ### Claim 8 (depends on 7) The process according to claim 7, wherein one of the molecules of interest is a contrast agent. ### Claim 9 (depends on 1) The process according to claim 1, wherein the additional entity is a substrate. ### Claim 10 (depends on 9) The process according to claim 9, wherein the substrate is chosen from surfaces of metal, semiconductor, oxide, organic and biological type. ### Claim 11 (depends on 10) The process according to claim 10, wherein the surfaces of oxide type are chosen from aluminas, SiO2 or TiO2. ### Claim 12 (depends on 10) The process according to claim 10, wherein the surface is nanostructured. ### Claim 13 (depends on 1) The process according to claim 1, wherein the cycloalkynyl radical is a cyclooctynyl radical. ### Claim 14 (depends on 1) The process according to claim 1, wherein the strained cycloalkynyl group is covalently bonded via a spacer arm to the molecule of interest or to the additional entity. ### Claim 15 (depends on 14) The process according to claim 14, wherein the spacer arm has the formula —X-E-A-G (I) wherein: X, bonded to the molecule of interest or the additional entity under consideration, is the reaction product of two reactive functional groups, E is an organic spacer group, A represents a single bond or a group chosen from —CONH—, —NHCO—, —OCH2CONH—, —NHCOCH2O—, —O— or —S—, and G is the strained cycloalkynyl group. ### Claim 16 (depends on 15) The process according to claim 15, wherein E is a poly(ethylene)glycol chain. ### Claim 17 A process of labeling a molecule of interest or of a substrate by a contrast agent comprising at least the stages consisting in: having available said molecule of interest or said substrate provided, at the surface, with at least one group G1 which is reactive according to a 1,3-dipolar cycloaddition reaction, and bringing together said molecule of interest or said substrate and said contrast agent provided, at the surface, with at least one group G2 complementary to the group G1 from the viewpoint of said 1,3-dipolar cycloaddition, under conditions favorable to the cycloaddition of said groups G1 and G2, said groups G1 and G2 respectively being a nitrone and a strained cycloalkynyl radical or vice versa.