Conjugation agent useful e.g. for bioassay and in medical diagnosis, comprises protected Staudinger component that is convertible into unprotected Staudinger component before conjugation reaction, and is linked with carrier by spacer group

摘要

Conjugation agent comprises a protected Staudinger component (Ia), which is convertible into an unprotected Staudinger component (I) before conjugation reaction, and is linked with a carrier (IIa)-(IIm) by at least one spacer group (IIIa) or by up to not 3 spacer groups (IIIa), (IIIb), (IIIc) or (IIId). Conjugation agent comprises a protected Staudinger component of formula (L2-M1(PG)(L3)-L4-X1) (Ia), which is convertible into an unprotected Staudinger component of formula (L2-M1(L3)-L4-X1) (I) before conjugation reaction, and is linked with a carrier of formula (Pol-L1-L2-M1(L3)-L4-X1) (IIa), (L2-M1(L3-L1-Pol)-L4-X1) (IIb), (L2-M1(L3)-L4(L1-Pol)-X1) (IIc), (Pol-L1-L2-M1(L3-L11-Pol)-L4-X1) (IId), (Pol-L1-L2-M1(L3)-L4(L11-Pol)-X1) (IIe), (L2-M1(L3-L1-Pol)-L4(L11-Pol)-X1) (IIf), (Pol-L1-L2-M1(L3-L11-Pol)-L4(L11-Pol)-X1) (IIg), (Pol-L1-L2-M1(L3)(LP1-Pol)-L4-X1) (IIh), (L2-M1(L3-L1-Pol)(LP1-Pol)-L4-X1) (IIi), (L2-M1(L3)(LP1-Pol)-L4(L1-Pol)-X1) (IIj), (Pol-L1-L2-M1(LP1-Pol)(L3-L11-Pol)-L4-X1) (IIk), (Pol-L1-L2-M1(LP1-Pol)(L3)-L4(L11-Pol)-X1) (IIl) or (L2-M1(LP1-Pol)(L3-L1-Pol)-L4(L11-Pol)-X1) (IIm) by at least one spacer group of formula (L1) (IIIa) or by up to not 3 spacer groups of formula (IIIa), (L11) (IIIb), (L21) (IIIc) or (LP1) (IIId). M1 : P, As, Sb, Bi or V, which selectively reacts with electrophilic nitrogen reagents and thus allows a Staudinger coupling; PG : removable protecting group for M1, which protects M1 from oxidation, and is linked only with M1 or additionally with (IIId); L2, L3 : mono- or bifunctional groups, which are linked only with M1 or additionally with (IIIa)-(IIIc), preferably 1-400C (hetero)aliphatic ring or 1-400C (hetero)aliphatic chain (optionally branched) (both optionally saturated) or 1-400C (hetero)aromatic ring (all contain 0-400 heteroatoms); L4 : bi- or trifunctional group, which connects M1 with X1, and is linked with (IIIa)-(IIIc), preferably 1-400C (hetero)aliphatic ring or 1-400C (hetero)aliphatic chain (both optionally saturated) or 1-400C (hetero)aromatic ring (all optionally contain 0-400 heteroatoms); X1 : acylatable group or leaving group for nucleophilic substitution at an aliphatic group or at an activated aromatic group, which is a component of L4, preferably 1-50C (hetero)aliphatic ring or 1-50C (hetero)aliphatic chain (both optionally saturated) or 1-50C (hetero)aromatic ring (all optionally contain 0-50 heteroatoms) Pol : phase, on which (I) or (Ia) are immobilized, where Pol separates the conjugation agent from the solvents and reagents; L1, L11, L21 : 0-400C (hetero)aliphatic ring or 0-400C (hetero)aliphatic chain (optionally branched) (both optionally saturated) or 0-400C (hetero)aromatic ring (all optionally contain 0-400 heteroatoms); and LP1 : protecting group for M1 (linked with Pol), preferably 1-400C (hetero)aliphatic ring or 1-400C (hetero)aliphatic chain (optionally branched) (both optionally saturated) or 1-400C (hetero)aromatic ring (all contain 0-400 heteroatoms). Full definitions are given in the DEFINITIONS (Full Definitions) Field. Independent claims are also included for: (1) a Staudinger component without the carrier; (2) a metallocenyl group of formula (XIX) or (XX), which is linked with a group that acylates the group X1 of the conjugation agent, preferably carboxylic acid-, thiocarboxylic acid- or dithiocarboxylic acid group present in free form or in the form of its salts, a carboxylic acid chloride, carboxylic acid fluoride, carboxylic acid bromide, carboxylic acid cyanide, carboxylic acid azide, carboxylic acid hydrazide, carboxylic acid oxyimide, carboxylic acid anhydride, carboxylic acid imidazolide, carboxylic acid triazolide, carboxylic acid tetrazolide, carboxylic acid benzotriazolide, carboxylic acid pyrazolide, carboxylic acid benzimidazolide, carboxylic acid oxy-benzotriazolide, carboxylic acid aza-oxybenzotriazolide, carboxylic acid alkyl ester, carboxylic acid aryl ester, carboxylic acid thioalkyl ester, carboxylic acid thioaryl ester or derivatives that are substituted with halo, nitro, alkyl, aryl or cyano groups, through a bifunctional group R; (3) a product of the above Staudinger component, whose group M1 forms nitrogen bond with an azido-component or a reagent exhibiting an electrophilic nitrogen atom; (4) a kit comprising a Staudinger component bonded to polymer, which is safely transferable in a protected manner by its columns casing, comprising a device for absorbing unused azido component or carboxyl component; and (5) an assay, which is based on the use of the above conjugation agent. R12-R23 : H or 1-200C organic group, preferably 1-200C aliphatic ring or 1-200C aliphatic chain (both optionally saturated), 1-200C (hetero)aromatic ring, 1-200C heteroaliphatic ring or 1-200C heteroaliphatic chain (all contain 0-200 F, Cl, Br, I, S, O, N, Se, Te, Si, Al, Ge, P, As, Sb, B, Li, Na, K, Cs, Ca, Mg, Sr, Ba, Fe, Ce or its isotopes), where adjacent groups are optionally connected with each other directly or by bridges made of C or heteroatoms; X41, X42 : 1-200C organic group (optionally substituted by F), preferably 1-200C aliphatic ring or 1-200C (hetero)aliphatic chain (both optionally saturated), 1-200C (hetero)aromatic ring or 1-200C heteroaliphatic ring (all optionally contain 0-200 F, Cl, Br, I, S, O, N, Se, Te, Si, Al, Ge, P, As, Sb B, Li, Na, K, Cs, Ca, Mg, Sr, Ba, Fe, Ce or its isotopes) or H, where X41 and X42 are linked to M2 and in addition to C and heteroatoms, preferably carboxylate, thiocarboxylate, dithiocarboxylate, carbamate, thiocarbamate, dithiocarbamate, alkyl sulfide, aryl sulfide, heteroaryl sulfide, alkyl selenide, aryl selenide, heteroaryl selenide, alkyl telluride, aryl telluride, heteroaryl telluride, alkoxy, aryloxy, heteroaryloxy, OH, SH, Cl, Br, I, heterocyclic imidazole, triazole, tetrazole, thiazole, benzimidazole, benzotriazole, indole, imidazole, aniline, pyrazole, pyrrolidone, azetane, azepine, benzodiazepine, piperidine, purine, morpholine, piperazine, triazine, oxazole, hydantoin, aziridine, pyrrolidine, pyrrole, hexamethyleneimine, azaindole or groups, whose conjugated organic or inorganic acids exhibits a pKa of less than 12, when used in optionally substituted form; and M2 : Ti, Zr or Hf. 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