FR2925053A3 - New lanthanide chelate compounds useful for marking bioactive substance e.g. oligopeptide, oligonucleotide, polynucleotide, oligosaccharide, polysaccharide, phospholipid or peptide nucleic acid, either in solution or on solid phase - Google Patents

Abstract

Lanthanide chelate compounds (I) for marking bioactive substance in solution, are new. Lanthanide chelate compounds of formula (I) for marking bioactive substance in solution, are new. A : reactive group for binding biomolecule, comprising isothiocyanate, bromoacetamido, iodoacetamido, maleimide, 4,6-dichloro-1,3,5-triazinyl-2-amino, pyridyldithiol, thioester, aminooxy, azide, alkyne, hydrazide, amino, carboxylic acid, ester or acid halide; Z 1>-Z 3>H or an electron donating group; Z 4>an electron donating group; Z 5>a substituent containing carboxylic acid group, phosphoric acid group or sulfonic acid group; X : linker formed from 1-10 groups of 3-12C alkyl or T; T : phenyl, ethyne-diyl, ethene-diyl, ether, thioether, amide (-CO-NH-, -CO-NR1aa-, -NH-CO- or -NR1aa-CO-), carbonyl, ester, disulfide, sulfonamide (-SO 2-NH-, -NH-SO 2-, -NR1aa-SO 2- or -SO 2-NR1aa-), sulfonyl, phosphate, diaza or tert-amine (-NR1aa-); R1aa : alkyl having less than five C; R1a, R1b, R1c, R1d : carboxylic acid or its salt, or an amide (-CO-NH 2or -CO-NHR1aa); and Ln : terbium, dysprosium, samarium or europium. An independent claim is included for a marking reagent comprising a substituted pyridine compound of formula (II) for marking bioactive substance on the surface of a hard phase. R : -COOR1b or -CONHR1b; R1b : phenyl or benzyl (both optionally substituted) or 1-4C alkyl; Z 5>a substituent containing carboxylic acid ester, sulfonic acid or sulfonic acid ester; X : 1-2C alkyl or T; A : reactive group for binding to biomolecule, comprising carboxylic acid or its salt, acid halide, ester or amino acid group of formula (-CH(NHR 2>)R 3>), or -E-O-PZ-O-R 4>, where O is substituted selectively by S; R 2>temporary protecting group; Z : Cl or NR 5>R 6>; R 4>protecting group; R 5>, R 6>alkyl; and E : absent or radical of purine or pyrimidine base, which is bond to O either by the hydrocarbon chain, which is substituted with a protected hydroxyethyl group, or by a furan or pyran ring or their modified derivatives, which enables oligonucleotide synthesis. [Image] [Image].

Description

MARKING REAGENTS

The present invention relates to novel labeling reagents capable of binding to bioactive substances either in solution or on the surface of a solid support.

Different specific physical and chemical properties are expected from u: l luminescent lanthanide chelate. In its basic state as in its excited state, the molecule must be stable from the thermodynamic, kinetic and photochemical point of view. The transfer of energy from the ligand to the central ion must be rapid and efficient. Frequently, the chelate must also be soluble in water. The chelate must retain its positive properties even after its coupling to a bioactive molecule. A good labeling reagent does not alter the properties of the bioactive substance to which it is bound. Many applications require covalent bonding of the tag molecule to the bioactive substance. For the labeling of target molecules such as proteins, oligonucleotides and oligopeptides, isothiocyanate, N-hydroxysuccinimide, haloacetamide, dichlorotriazine or maleimide derivatives of a chelate are often used. Since the labeling reaction takes place in the presence of excess activated labeling reagent, labor-intensive purification procedures are inevitable. The purification problems can be avoided when the labeling reaction is carried out on the surface of a solid phase. In this case, a large part of the 2 impurities can be washed away while the biomolecule conjugate is still adhering to its solid support. A multitude of reagents making it possible to couple chelates of lanthanides to bioactive molecules in the aforementioned manner has already been made available to the public. Labeling in solution is inevitable in the case of large bioactive molecules such as proteins. The labeling reaction should then be as efficient and as site specific as possible. The electron donor groups of an aromatic ring have been observed to improve the photo-physical properties of certain lanthanide (III) chelates derived from 4-phenyl-2,6-bis [N, N-di (carboxyalkyl) aminoalkyl ] pyridines (US 4,761,481). Subsequently, it was discovered that the above-mentioned properties can be improved by chelating several aromatic groups on a lanthanide ion (US patent application 11/004061). This also increases the stability of the chelate structure. Furthermore, aromatic ohromophores could also decrease the water solubility of the chelate. The main object of the present invention is to provide the use of labeling reagents derived from 4-phenylpyridine which make it possible to label bioactive substances both in solution and on the surface of a solid support. For labeling on the surface of the solid support, oligonucleotide and oligopeptide synthesizers can be used as standard models. The solubility in water of the 3 molecules of the invention could be improved by adding a single or a plurality of carboxylic acid, phosphoric acid or sulfonic acid groups to the chromophores. The bioconjugates thus obtained are particularly suitable for determination based on time-resolved fluorescence. The present invention relates to a chelate of formula (I) suitable for labeling a bioactive substance in solution. Another object of the present invention is the labeling reagent of formula (II) suitable for labeling a bioactive substance on the surface of a solid support. Therefore, the present invention relates to a chelate suitable for labeling a biospecific molecule in solution and having the structural formula (I). (^ '': ^ .. /

NXN i A in which A is a reactive group for binding to the bioactive substance, selected from a group consisting of isothiocyanate, bromacetamido, iodoacetamido, maleimide, 4,6-dichloro-1,3,5-triazinyl-2-amino radicals , pyridyldithiol, thioester, aminooxy, azide, N Ra Rb Ln3 + R Rd (r) alkyne, hydrazide, amino, carboxylic acid, acid ester or halide, Z1 is hydrogen or an electron donating group, Z2 is l 'hydrogen or an electron donating group, Z3 is hydrogen or an electron donating group, Z9 is an electron donating group, Z5 is a substituent which contains a carboxylic acid group, a phosphoric acid group or a group sulfonic acid, X is a linker formed from one to ten parts, each of which is selected from a group consisting of phenyl, alkyl radicals of 3 to 12 carbon atoms, ethynediyl (-CC-), ethenediyl (-C = C- ), ether (-0-), thioether (-S-), amide (-CO-NH-, -CO-NR'-, -NH-CO- and - NR'-CO-), carbonyl (-CO- ), ester (-COO- and - O0C-), disulfide (-SS-), sulfonamide (-SO2-NH-, -NH-SO2-, -NR'-SO2- and -SO2-NR'-), sulfone (-SO2-), phosphate (- 0-PO2-O-), diaza (-N = N-) and tertiary amine (-NR'-) where R 'is an alkyl comprising less than cino carbon atoms. Ra, Rb, Rd and Rd are identical or different substituents selected from a group consisting of a carboxylic acid (-COOH; or its salt and an amide (-CO-NH2 and -CO-NHR ', where R' is a alkyl having less than five carbon atoms Ln is terbium, dysprosium, samarium or europium.

Z1, z2, Z3 and / or Z4 are methyl, ethyl, i-propyl, n-propyl, n-butyl, sec-butyl, t-butyl, methoxy, ethoxy, i-propoxy, n-propoxy or tert- butoxy.

The Z5 substituent is a carboxymethoxy group. The invention also relates to a labeling reagent of formula (II) which is suitable for labeling bioactive substances on the surface of a solid support: I ~ ~ I Â N r RRRR (II) in which R is -COOR " or -CONHR ", where R" is an alkyl, phenyl or benzyl radical of 1 to 4 carbon atoms, the phenyl or benzyl being substituted or unsubstituted, Z1 is hydrogen or an electron donor group, z2 is l 'hydrogen or an electron donating group, Z3 is hydrogen or an electron donating group, Z4 is an electron donating group, r "~ Z5 is a substituent which contains a carboxylic acid ester group, ester p: -losphoric acid, sulfonic acid or sulfonic acid ester, X is a linker formed from one to ten parts each of which is selected from a group consisting of phenyl or alkyl radicals of 1 to 12 carbon atoms, ethynediyl (-C = C-), ethenediyl (-C = C-), ether (-0-), thioether (-S-), amide (-CO-NH-, -CO-NR'-, -NH- CO-and - NR'-CO-), carbonyl (-CO-), es ter (-COO- and -O0C-), disulfide (-SS-), sulfonamide (-SO2-NH-; -NH-SO2-, -NR'-SO2- and -SO2-NR'-), sulfone (-SO2-), phosphate (-0-P02-O-), diaza (-N = N-) and tertiary amine (-NR'-), where R 'is an alkyl comprising less than five carbon atoms, A is a reactive bioactive substance binding group, selected from a group consisting of a carboxylic acid or its salt, a halide of acid, ester or amino acid residue -CH (NHR2) R3, where R2 is a transient protecting group and R3 is a carboxylic acid or its salt, an acid halide or an active ester, or A is

-EO-PZ-0-R4, where an oxygen atom is optionally replaced by a sulfur atom, Z is chlorine or NR5R6, R9 is a protecting group, R5 and R6 are alkyl groups, E is absent or is a radical of a purine or pyrimidine base bound to the oxygen atom i) either through the hydrocarbon chain substituted by a protected hydroxyethyl group, ii) or through a furary or pyran nucleus or their modified derivatives allowing the synthesis of an oligonucleotide. The R "group is preferably methyl, ethyl, tert-butyl, allyl or benzyl.

When the Z5 substituent contains carboxylic acid esters, a preferred substituent is -OCH2C00-t-Bu, -OCH2COOEt, -OCH2COOMe or -OCH2COOCH2Ph. The lanthanide Ln is preferably terbium, dysprosium, europium or samarium. Particularly advantageous lanthanides are teroium and dysprosium. The electron donor groups Z1, Z2, Z3 and / or Z4 are preferably linear or branched alkyl groups such as methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, t- butyl or alkoxy groups such as methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, sec-butoxy, t-butoxy. Other groups such as hydroxy groups, amino groups, carboxamido groups, phosphoric acid groups and their esters, carboxylic acid groups and their esters or sulfonic acid groups and their esters can also be attached to the alkyl and alkoxy groups. . When the Z5 substituent contains a carboxylic acid group, a preferred substituent is a carboxymethoxy group.

The carboxylic acid ester R is preferably a methyl, ethyl, allyl, benzyl or tert-butyl ester. When the group A as defined above is an acid halide, the most preferred halide is a chloride. The preferred transient protecting group R2 is a fluorenylmethoxycarbonyl (Fmoc), nitrobenzenesulfonyl (Ns), tert-butoxycarbonyl (Boc) or 1,1-dioxobenzo [b] thiophen-2-ylmethyloxycarbonyl (Bsmoc) group. When R3 is an active ester of a carboxylic acid, this ester is preferably N-hydroxysuccinimide, p-nitrophenol or pentafluorophenol. When R3 is a halide of a carboxylic acid, this halide is preferably a chloride or a fluoride. The protecting group R4 is preferably a cyanoethyl group. The alkyl groups R5 and R6 are preferably isopropyl groups. When A is -EO-PZ-O-R4, it is preferably chosen from the groups 9 DMTrO DMTrO 0 (1-Pr) 2 NP.OruCN 0 (, Pr) 2 NOivC o 0 (i Pr) 2N "P ' Or ~ CN DMTrO 1 o N, i'-1 JCHa ON / ù (O OM7ro DMTr 0 DMTrO DMTrO O CN (i Pr) ZN.PO / CCN (i Pr) 2N "PRO - / CN (i-Pr) 2Nk-0, .`-, CN (i-Pr) N 0 "- in which - represents the binding site of linker X and DTMr is a dimethoxytrityl group.

The bioactive molecule to be labeled is for example an oligopeptide, an oligonucleotide, a polypeptide, a polynucleotide, a protein, an oligosaccharide, a polysaccharide, a phospholipid, a PNA, an LNA, an antibody, a hapten, a drug, a molecule which binds to a receptor, or lectin. The invention is illustrated by means of the non-limiting experimental part below. The invention is explained in more detail by means of the following examples.

Example 1. Preparation of chromophores The synthesis of compound la used as starting material is described in publication US Pat. No. 5,055,578 (column 14, line 35-47, compound 24; cf. scheme VI, figure 6). Compound Ib was prepared by the same method using 2,6-dimethoxy-4-hydroxybenzaldehyde as a starting material. The o O ~ N N% ~ CH3 hydroxy group at position 4 of compounds la and lb was alkylated with tert-butyl bromoacetate in acetonitrile in the presence of potassium carbonate. Diesters 2a and 2b were reduced to dimethanols 3a and 3b with sodium borohydride, as described in the publication [Acta Chem. Scand, 1988, B42, 373]. Bromination with phosphorus tribromide in anhydrous DMF gave the dihalides 4a and 4b. This process is described in the publication [Bioconjugate Chem. 2005, 16, 700]. OH R OMe BrCH2000-t-Bu ./ MeOOC N COOMe ia, b 2a, b Ot-Bu R -OMe a: R = H b: R = OMe Example 2. Preparation of terbium chelates Compounds 4a and 4b were reacted with an equivalent amount of di-tertbutyl iminodiacetate as described in the publication of US patent application 11/004061 (cf. the synthesis of compound 24, page 15, chapter [0101]). The PBr3 11 reaction of products 5a and 5b with hexane-1,6-diamine as described in Sci. 12, 2006, 199], gave 6b. Removal of the acid protecting groups followed by terbium chloride treatment gave chelates 7a and 7b, which were activated with iodoacetic anhydride to give compounds 8a and 8b. A detailed description of the process can be found in the publication [Bioconjugate Chem. 1994, 5, 278]. NHMMTr protected by MMTr, publication [J. Peptide compounds 6a and 4a, b, - K2CO3 MeCN HN 2. TbCl3 iodoacetic anhydride X 1.TFA X ('' 'L --- Te (N, l 000'000- 6a.b 000-000- 6a XC l K2CO3 XN MeCN (NI II NR 6a, b RR = COC-t-Bu Example 3. Preparation of an oligopeptide labeling reagent The MMTr group was removed from compound 6a by short-term acid treatment. , as described in the publication [Bioconjugate Chem. 2002, 13, 870] The deprotected amino group was reacted with Fmoc protected allyl glutamate using HATU as a catalyst [Bioconjugate Chem. 2006, 17, 855] Finally, the allyl protection was removed under palladium catalysis.

Fmoc-Glu-OAJI HATUI DIPEA IN N 9 RRRR FmocHN 0 ü XX / I ~ SO2Na '/ ~ N ^ i ~~ NV NI 11 (N` RR lR B Pd (Ph3P) 4 1% TFA HN OH X = 10 ( N1 RRR = C00-t-Bu Example 4. Preparation of an oligonucleotide labeling reagent The oligonucleotide labeling reagent 15 was prepared by the method described in the publication [Bioconjugate Chem. 2005, 16, 700]. Briefly, reaction of compound 4a with met: zyl iminoacetate gave compound 12, which was reacted with rMMT-protected aminohexanol in anhydrous acetonitrile in the presence of potassium carbonate. .

The MMTr group of compound 13 was removed by short-term acid treatment and then the free hydroxy group was phosphitylated. OMMTr HNRR 4a K2CO3 McCN Of course, the invention is not limited to the embodiments described above and 14 shown, from which other embodiments and other embodiments can be provided, without necessarily departing from within the scope of the invention.

Claims (14)

1. Chelate of formula (I) which is suitable for labeling a bioactive substance in solution: i z2Z Zz Z5 Z3 t Z5 Z3 \ Z4 ZX ("1 Ra Rb Ln3 + Rd Rd (1) characterized in that A is a reactive group binding to a biomolecule selected from a group consisting of isothiocyanate, bromacetamido, iodoacetamido, maleimide, 4,6-dichloro-1,3,5-triazinyl-2-amino, pyridyldithiol, thioester, aminooxy, azide, alkyne, hydrazide radicals , amino, carboxylic acid, acid ester or halide, Z1 is hydrogen or an electron donor group, Z2 is hydrogen or an electron donor group, Z3 is hydrogen or an electron donor group, Z3 is hydrogen or an electron donor group 'electrons, Z4 is an electron donor group, Z5 is a substituent which contains a carboxylic acid group, a phosphoric acid group or a sulfonic acid group, X is a linker formed from one to ten parts each of which is chosen from a group consisting of phenyl, alkyl of 3 to 12 carbon atoms, ethynediyl (-C ° C-), ethenediyl radicals e (-C = C-), ether (-0-), thioether (-S-), amide (-CO-NH-, -CO-NR'-, --NH-CO- and - NR'-CO -), carbonyl (-CO-), ester (-COO-and -OOC-), disulfide (-SS-), sulfonamide (-SO2-NH-, -NH-SO2-, -NR'-SO2- and - SO2-NR'-), sulfone (-SO2-), phosphate (-0- P02-0-), diaza (-N = N-) and tertiary amine (--NR '-) where R' is an alkyl comprising less than five carbon atoms, Ra, Rb, Rd and Rd are the same or different and are chosen from a group comprising a carboxylic acid (-COOH) or its salt and an amide (-CO-NH2 and -CO-NHR ', where R 'is an alkyl comprising less than five carbon atoms. Ln is terbium, dysprosium, samarium or europium. 2. Chelate according to claim 1, characterized in that Z1, Z2, Z3 and / or Z9 are methyl, ethyl, i-propyl, n-propyl, n-butyl, sec-butyl, t-butyl, methoxy, ethoxy, i-propoxy, n - propoxy or tert-butoxy. 3. Chelate according to claim 1, characterized in that the substituent Z5 is a carboxymethoxy group. 4. Labeling reagent of formula (IIr), which is suitable for labeling a bioactive substance: ive on the surface of a solid support: 30 characterized in that λ (1 \ 1.1 RRR ROI) R is -COOR " or -CONHR ", where R" is an alkyl, phenyl or benzyl group of 1 to 4 carbon atoms, the phenyl or benzyl being substituted or unsubstituted, Z1 is hydrogen or an electron donor group, Z2 is 1 'hydrogen or an electron donating group, Z3 is hydrogen or an electron donating group, Z4 is an electron donating group, Z5 is a substituent which contains a carboxylic acid, sulfonic acid or ester ester group sulfonic acid, X is a linker formed from one to ten parts, each of which is selected from a group consisting of phenyl, alkyl of 1 to 12 carbon atoms, ethynediyl (-C = C-), ethenediyl (- C = C-), ether (-O-), thioether (-S-), amide (-CO-NH-, -CO-NR'-, -NH-CO- and -NR'-CO-), carbonyl (-CO-), ester (-COO- and -O0C-), disulfide (-SS-), sulfonamide (-SO 2-NH-; -NH-SO2-, -NR'-SO2- and -SO2-NR'-), sulfone (-SO2-), phosphate (-0- P02-0-), diaza (-N = N-) and tertiary amine (--NR '-) where R' 18 is an alkyl comprising less than five carbon atoms, A is a reactive group for binding to a biomolecule, selected from a group consisting of: killed by a carboxylic acid or its salt, a halide acid, an ester or an amino acid residue -CH (NHR2) R3, where R2 is a transient protecting group and R3 is a carboxylic acid or its salt, an acid halide or an active ester, or A is - EO-PZ-O-R4, where an oxygen atom is optionally replaced by a sulfur atom, Z is chlorine or NR5R6, R4 is a protecting group, R5 and R6 are alkyl groups, E is absent or is a radical of a purine or pyrimidine base which is attached to the oxygen atom i) either through the hydrocarbon chain substituted by a protected hydroxyethyl group, ii) or by a furan or pyran ring or by their modified derivatives that allow synthesis of an oligonucleotide. 5. Labeling reagent according to claim 4, characterized in that R "is a tert-butyl, methyl, ethyl, allyl or benzyl group. 6. Labeling reagent according to claim 4, characterized in that the Z5 substituent is -OCH2COO-t-Bu, -OCH2COOEt, -OCH2COOMe or -OCH2CO0CH2Ph. 7. Labeling reagent according to claim 4, characterized in that Z1, Z2, Z3 and / or Z4 are methyl, ethyl, i-propyl, n-propyl, n-butyl, sec-butyl, t-butyl groups, methoxy, ethoxy, i-propoxy, n-propoxy or tert-butoxy. 8. Labeling reagent according to claim 4, characterized in that the transient protective group R2 is a fluorenylmethoxycarbonvle (Fmoc), nitrobenzenesulfonyl (Ns), tert-butoxycarbonyl (Boc) or 1-1-dioxobenzo [b] thiophén-2 group. -ylmethyloxycarbonyl (Bsmoc). 9. Labeling reagent according to claim 4, characterized in that the protecting group R4 is a cyanoethyl group. 10. Labeling reagent according to claim 4, characterized in that the alkyl groups R 'and R6 are isopropyl groups. 11. Labeling reagent according to claim 4, characterized in that A is -E-O-PZ-O-R4 and is preferably chosen from the groups 20DMTrO 0 (1.Pr) 3N. P.O ~ vCN O P ~ ~~ CN (i-Pr) .N ~ OMTrO. 0 (i Pf) 1N, PO ~~ CN 0MTrO O 0 NOO ~ N ~ O ~ NiIYCH3 pl, v ~ Otk) N `~~ CHs N rù \ / DMT Ô 1 DMTrO 0 OMTrO 0 DMTrb ~ CN P. CN P . ^ / CN (i-Pr) 2N F ',. CN O ^ ~ (r • Pr) 2N (j.Pr) zN 0 ~~ (1'Pr) 2N, 0 in which - represents the binding site of the linker X and DTMr is a dimethoxytrityl group. 12. Labeling reagent according to claims 4 to 11, characterized in that R "is a methyl, ethyl or tert-butyl group. 13. Chelate according to claims 1 to 3, characterized in that the bioactive substance is an oligopeptide, an oligonucleotide, a polypeptide, a polynucleotide, a protein, an oligosaccharide, a polysaccharide, a phospholipid, a PNA, an LNA, an antibody , a hapten, a drug, a molecule that binds to a receptor, or a lectin. 14. Labeling reagent according to claims 4 to 11, characterized in that the bioactive substance is an oligopeptide, an oligonucleotide, a polypeptide, a polynucleotide, a protein, an oligosaccharide, a polysaccharide, a phospholipid, a PNA, an LNA, an antibody, a hapten, a drug, a nolecule that binds to a receptor, or a lectin.