HK1186472B - New iridium-based complexes for ecl - Google Patents

Description

Novel iridium-based complexes for ECL

Technical Field

The present invention relates to novel iridium ir (iii) -based luminescent complexes, conjugates comprising these complexes as labels and their use in e.g. electrochemiluminescence-based analyte detection.

Background

Electrochemiluminescence (also known as electrochemiluminescence and abbreviated as ECL) is a process in which a substance generated at an electrode undergoes a high-energy electron transfer reaction to form an excited state that emits light. Detailed ECL studies were initially described by Hercules and Bard et al in the mid sixties of the twentieth century. After about 40 years of research, ECL has now become a very powerful analytical technique and is widely used in fields such as immunoassays, food and water testing, and biological warfare agent detection.

For use in Organic Light Emitting Devices (OLEDs), there are a large number of compounds that seem to be of interest. These compounds are suitable for use in solid materials or are soluble in organic fluids. However, no conclusions have been drawn as to their utility in aqueous media, e.g., for detecting analytes from biological samples.

Generally, ECL-based detection methods are based on the use of water-soluble ruthenium complexes comprising Ru (II +) as metal ion.

Despite significant improvements over the past several decades, there is still a pressing need for more sensitive in vitro diagnostic assays based on electrochemiluminescence.

It has now surprisingly been found that certain iridium Ir (III +) based luminescent complexes represent very promising labels for future high-sensitivity ECL-based detection methods.

Disclosure of Invention

The invention discloses iridium-based luminescent or electrochemiluminescent compounds of formula I

Wherein R1-R12 are hydrogen, halogen, cyano, nitro, hydrophilic groups or R19, wherein R19 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, hydrophilic groups,

wherein R13-R16 are hydrogen, halogen, cyano, nitro, hydrophilic groups, -Q-Y or R19, wherein R19 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, hydrophilic groups,

wherein R17-R18 represent hydrogen, alkyl, aryl, substituted aryl and alkyl, heteroaromatic ring systems, non-aromatic rings or ring systems, imidazolium, cyclodextrin or-Q-Y, or

Wherein in R1-R12 or/and R13-R16, respectively, two adjacent R may form an aromatic ring or a substituted aromatic ring, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, hydrophilic groups, or

Wherein in R1-R12 or/and R13-R16, respectively, two adjacent R may form an aliphatic ring or a substituted aliphatic ring, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, hydrophilic groups,

wherein X represents C or N, and the compound is shown in the specification,

wherein Y represents C or N, or a salt thereof,

wherein at least one of R13-R18 is-Q-Y,

wherein Q represents a linking group and Y is a functional group.

Also disclosed are conjugates comprising the above compounds and an affinity binding agent covalently bound thereto.

The invention also relates to the use of the presently disclosed compounds or conjugates for performing luminescence measurements or electrochemiluminescence reactions in aqueous solutions, in particular in electrochemiluminescence devices or electrochemiluminescence detection systems.

The present invention also discloses a method for measuring an analyte by an in vitro method, the method comprising the steps of: (a) providing a sample suspected or known to contain said analyte, (b) contacting said sample with a conjugate of the invention under conditions suitable for the formation of a conjugate complex of the analyte, and (c) measuring the complex formed in step (b) and thereby obtaining a measurement of the analyte.

Detailed Description

The invention relates to iridium-based luminescent or electrochemiluminescent compounds of formula I

Wherein R1-R12 are hydrogen, halogen, cyano, nitro, hydrophilic groups or R19, wherein R19 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, hydrophilic groups, wherein R13-R16 are hydrogen, halogen, cyano, nitro, hydrophilic groups, -Q-Y, or R19, wherein R19 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, substituted alkenyl, alkynyl, and R3978, Halogen, cyano, nitro, hydrophilic groups, wherein R17-R18 represent hydrogen, alkyl, aryl, substituted aryl and alkyl, heteroaromatic ring systems, non-aromatic rings or ring systems, imidazolium, cyclodextrin or-Q-Y, wherein in R1-R12 or/and R13-R16, respectively, two adjacent R's may form an aromatic ring or a substituted aromatic ring, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, hydrophilic groups, wherein in R1-R12 or/and R13-R16, respectively, two adjacent R may form an aliphatic ring or a substituted aliphatic ring, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, hydrophilic groups, wherein X represents C or N, wherein Y represents C or N, wherein at least one of R13-R18 is-Q-Y, wherein Q represents a linking group and Y is a functional group.

In one embodiment, at least one of R17 or R18 is-Q-Y.

In one embodiment, one of R13 to R18 is a cyclodextrin. Preferably, the cyclodextrin is β -cyclodextrin. It is also preferred that the cyclodextrin is permethylated.

In one embodiment, at least one of R1 to R16 of the compound of formula I is substituted with at least one hydrophilic group.

Preferred hydrophilic groups are amino, alkylamino (said alkyl representing a straight chain such as methyl, ethyl, propyl, butyl, pentyl chain or a branched alkyl chain such as isopropyl, isobutyl, tert-butyl, preferably a straight alkyl chain such as methyl or ethyl), substituted alkylamino (which contains one or two, for example branched or straight, chains bound to the N atom substituted by another hydrophilic group such as hydroxyl or sulfo, preferably such substituted alkylamino contains two hydroxypropyl or hydroxyethyl residues), arylamino (aryl means an aromatic residue such as phenyl, or naphthyl, preferably phenyl), substituted arylamino (having an aryl group as defined above and a further residue formed by a hydrophilic group), alkylammonium (alkyl as defined above, and alkylammonium preferably a trimethylammonium residue or a triethylammonium residue) or mixtures thereof, Substituted alkylammonium groups, carboxyl groups, carboxylate groups (preferably alkyl ester groups such as methyl or ethyl ester groups), carbamoyl groups, hydroxyl groups, substituted or unsubstituted alkyloxy groups (alkyl and substituted alkyl groups as defined above) or aryloxy groups or substituted aryloxy groups (aryl and substituted aryl groups as defined above), sulfanyl groups, alkylsulfonyl groups, arylsulfonyl groups, sulfo groups, sulfino groups, sulfoxyl groups, sulfonamido groups, sulfoxo groups, sulfone groups, phosphonate groups, phosphinate groups.

Preferably, such hydrophilic groups are selected from amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, hydroxyl, sulfo, sulfoxyl, sulfonamido, sulfoxido, sulfonyl and phosphonate groups, where applicable, each preferably as defined in the preceding paragraph.

In one embodiment, the hydrophilic group is selected from amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carbamoyl, hydroxyl, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonamido, sulfoxido, sulfone, phosphonate, phosphinate.

In one embodiment, a preferred substituent of a substituted alkyloxy group is an ethyleneoxy chain comprising from 1 to 40 ethyleneoxy units, alternatively from 1 to 20 ethyleneoxy units, alternatively from 1 to 10 ethyleneoxy units.

In a further embodiment, the hydrophilic group is selected from sulfo, sulfonamido, sulfone.

In one embodiment, at least one of R1 through R12 of formula I is a sulfo group.

In one embodiment, at least one of R1 to R12 of the phenylphenanthridine residues comprised in formula I is substituted with at least one hydrophilic group.

In one embodiment, the phenylphenanthridine residues comprised in formula I are selected from the substituted phenylphenanthridines given below.

In the compounds of the present invention, the linking group Q preferably has a main chain length of 1 to 20 atoms. In other words, the shortest connection between the pyridine ring of formula I and the functional group Y consists of 1 to 20 atoms. In one embodiment, the linking group Q in the electrochemiluminescent complex of the invention is a linear or branched, saturated or unsaturated, unsubstituted, substituted C1-C20 alkyl chain, or a 1-20 atom containing chain with a backbone consisting of carbon atoms and one or more heteroatoms selected from O, N and S.

In one embodiment, the linking group Q in the compounds of the invention is a saturated C1-C12 alkyl chain or a 1-12 atom containing chain having a backbone consisting of carbon atoms and one or more heteroatoms selected from O, N and S.

In one embodiment, the functional group Y comprised in the iridium-based complex of the invention is selected from the group consisting of carboxylic acid group, N-hydroxysuccinimide ester group, amino group, halogen, thiol group, maleimido group, alkynyl group, azido group and phosphoramidite group.

The conjugates comprise an iridium-based electrochemiluminescent compound of formula I as disclosed and defined herein above and a biological substance covalently bound thereto. Examples of suitable biological substances are cells, viruses, sub-cellular particles, proteins, lipoproteins, glycoproteins, peptides, polypeptides, nucleic acids, Peptide Nucleic Acids (PNA), oligosaccharides, polysaccharides, lipopolysaccharides, cell metabolites, haptens, hormones, pharmacological substances, alkaloids, steroids, vitamins, amino acids and sugars.

In one embodiment, the biological substance of the conjugate of the invention (i.e. covalently bound to the compound of formula I) is an affinity binding agent. It will be appreciated by those skilled in the art that in the conjugates of the invention, the functional group Y of the compound of formula I is used to form a covalent bond with a group on the affinity binding agent. In case the affinity binding reagent itself does not contain an appropriate group to bind or react with the group Y, such a group can be easily introduced into the affinity binding agent by relying on established procedures.

Without wishing to be further limited, but for clarity, the affinity binding agent may comprise any of the following: antigens, proteins, antibodies, biotin or biotin analogues and avidin or streptavidin, sugars and lectins, enzymes, polypeptides, amino groups, nucleic acids or nucleic acid analogues and complementary nucleic acids, nucleotides, polynucleotides, Peptide Nucleic Acids (PNAs), polysaccharides, metal ion chelators, receptor agonists, receptor antagonists, or any combination thereof. For example, an affinity binding agent can be one partner of a specific binding pair, wherein the other partner of the binding pair is associated with or is a target on a cell surface or intracellular structure.

Preferably, the affinity binding agent is a partner or member of an affinity binding pair, or a partner or member of a specific binding pair also known to those skilled in the art.

An affinity binder has at least 10 to its target (e.g., one member of a specific binding pair, e.g., an antibody, and the other member of the specific binding pair, e.g., its antigen)⁷Affinity in l/mol. The affinity binder and its target preferably have a binding capacity of 10⁸l/mol affinity or even more preferably 10⁹Affinity in l/mol.

In one embodiment, the invention relates to a conjugate wherein the affinity binding agent is selected from the group consisting of an antigen, an antibody, biotin or a biotin analogue, avidin or streptavidin, a sugar, a lectin, a nucleic acid or nucleic acid analogue and a complementary nucleic acid, a receptor and a ligand.

In one embodiment, the invention relates to a conjugate wherein the affinity binding agent is selected from the group consisting of an antibody, biotin or a biotin analogue, avidin or streptavidin and a nucleic acid.

In one embodiment, the conjugate of the invention comprises a covalently linked compound of formula I as disclosed and defined herein above and an affinity binding agent which is an oligonucleotide or an antibody.

The biotin analogue is aminobiotin, iminobiotin or desthiobiotin.

The term "oligonucleotide" or "nucleic acid" as used herein generally refers to a short, usually single-stranded polynucleotide comprising at least 8 nucleotides and at most about 1000 nucleotides. In preferred embodiments, the oligonucleotide is at least 9, 10, 11, 12, 15, 18, 21, 24, 27, or 30 nucleotides in length. In preferred embodiments, the oligonucleotide is no more than 200, 150, 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length.

The term oligonucleotide is to be understood broadly and includes DNA and RNA as well as analogues and modifications thereof.

The nucleic acid analog may, for example, contain substituted nucleotides carrying substituents at the standard bases deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), deoxythymidine (dT), deoxyuridine (dU). Examples of such substituted nucleobases are: 5-substituted pyrimidines such as 5-methyl dC, aminoallyl dU or dC, 5- (aminoethyl-3-acryloylimino) -dU, 5-propynyl-dU or-dC, 5-halo-dU or-dC; n-substituted pyrimidines such as N4-ethyl-dC; n-substituted purines such as N6-ethyl-dA, N2-ethyl-dG; 8-substituted purines such as 8- [ 6-amino) -hex-1-yl ] -8-amino-dG or-dA, 8-halogenated dA or dG, 8-alkyl dG or dA; and 2-substituted dAs such as 2-amino dAs.

The nucleic acid analog may contain a nucleotide or a nucleoside analog. That is, a naturally occurring nucleobase can be replaced by using a nucleobase analog, such as 5-nitroindole d-inosine; 3 nitropyrrole d inosine, deoxyinosine (dI), deoxyxanthosine (dX); 7-deaza-dG, -dA, -dI or-dX; 7-deaza-8-aza-dG, -dA, -dI or-dX; 8-aza-dA, -dG, -dI, or-dX; d-m-type mycin; simulating dU; pseudo-iso-dC; 4-sulfur dT; 6-sulfur dG; 2-sulfur dT; iso-dG; 5-methyl-iso-dC; n8-linked 8-aza-7-deaza-dA; 5, 6-dihydro-5-aza-dC; and vinylidene-dA or pyrrole-dC (pyrollo-dC). It is clear to the person skilled in the art that the nucleobases in the complementary strand need to be selected in such a way that: such that duplex formation is specific. For example, if 5-methyl-iso-dC is used in one strand (e.g., (a)), then iso-dG needs to be in the complementary strand (e.g., (a')).

In nucleic acid analogs, the oligonucleotide backbone may be modified to contain substituted sugar residues, sugar analogs, modifications in the internucleoside phosphate moiety, and/or be PNA.

The oligonucleotide may, for example, contain a nucleotide having a substituted deoxyribose sugar such as 2 ' -methoxy, 2 ' -fluoro, 2 ' -methylseleno, 2 ' -allyloxy, 4 ' -methyl dN (where N is a nucleobase, e.g., A, G, C, T or U).

Sugar analogs such as xylose; 2 ', 4' bridged ribose such as (2 '-O, 4' -C methylene) - (oligomers known as LNA) or (2 '-O, 4' -C ethylene) - (oligomers known as ENA); l-ribose, L-d-ribose, hexitol (oligomer known as HNA); cyclohexenyl (an oligomer known as CeNA); altritol (oligomer known as ANA); tricyclic ribosaccharide analogs in which the C3 'and C5' atoms are connected by an ethylene bridge fused to a cyclopropane ring (referred to as oligomers of tricyclic DNA); glycerol (oligomer known as GNA); glucopyranose (oligomer called Homo DNA); carbaribose (replacement of tetrahydrofuran subunits with cyclopentane); hydroxymethyl-morpholine (an oligomer known as morpholino DNA).

It is also known that a large number of modifications of the internucleoside phosphate moiety do not interfere with the hybridization properties, and that such backbone modifications can also be combined with substituted nucleotides or nucleotide analogs. Examples are phosphorothioate (phosphorodithioate), phosphorodithioate (phosphorodithioate), phosphoramidate and methylphosphonate oligonucleotides.

PNA (with a backbone free of phosphate and d-ribose) can also be used as a DNA analog.

In the sense of the present invention, the above-described modified nucleotides, nucleotide analogs and oligonucleotide backbone modifications can be combined as desired in the oligonucleotide.

The term "antibody" is used in the broadest sense in the present application and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

An "isolated" antibody is one that has been identified and separated and/or recovered from a component of the natural environment. Contaminant components of the natural environment are substances that interfere with the research, diagnostic, or therapeutic uses of antibodies, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95 wt%, in some embodiments greater than 99 wt% by weight of the antibody as determined, for example, by Lowry methods, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using, for example, a spinning cup sequencing analyzer, or (3) to homogeneity by using, for example, coomassie blue or silver stained SDS-PAGE under reducing or non-reducing conditions. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, an isolated antibody will typically be prepared by at least one purification step.

"native antibodies" are typically heterotetrameric glycan proteins of about 150,000 daltons containing two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide linkages varies among heavy chains with different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains.

The "variable domain" or "variable region" of an antibody refers to the amino-terminal region of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These regions are typically the most variable parts of an antibody and contain antigen binding sites.

The term "variable" refers to the fact that: in antibodies, certain portions of the variable domains differ severely in sequence and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain of the antibody. In the light and heavy chain variable domains, it is concentrated in three segments called the hypervariable domains (HVRs). The more highly conserved portions of the variable domains are called the Framework Regions (FR). The variable domains of native heavy and light chains each comprise 4 FR regions largely adopting a β -sheet configuration, connected via 3 HVRs that form a loop connection, and in some cases form part of a β -sheet structure. The HVRs in each chain are held together in close proximity by the FR region and, together with HVRs from other chains, promote the formation of antigen binding sites for antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, national institute of Health, Bethesda, Md. (1991)). Constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as participation of antibodies in antibody-dependent cellular cytotoxicity.

The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be assigned to one of two distinct types, termed kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

Antibodies (immunoglobulins) can be assigned to different species depending on the amino acid sequence of the constant domains of their heavy chains. There are 5 major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al, Cellularand mol. The antibody may be part of a larger fusion molecule formed by covalent or non-covalent association (association) of the antibody with one or more other proteins or peptides.

The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably in this application and refer to an antibody in its substantially intact form, rather than the antibody fragment defined below. The term particularly denotes antibodies in which the heavy chain contains an Fc region.

An "antibody fragment" comprises a portion of an intact antibody, preferably, the antigen-binding region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; a double body; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments (each with a single antigen-binding site) and a residual "Fc" fragment (the name of which reflects its ability to crystallize readily). Pepsin treatment yields F (ab') 2 fragments with two antigen binding sites and still be able to cross-link the antigen.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In single chain Fv (scfv) species, one heavy chain variable domain and one light chain variable domain may be covalently linked by a flexible peptide linker such that the light and heavy chains may associate in a "dimeric" structure similar to that in a two-chain Fv species. It is in this configuration that the 3 HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, 6 HVRs confer antigen binding specificity on the antibody. However, even a single variable domain (or half of an Fv comprising only 3 HVRs with specificity for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

The Fab fragment contains both the heavy and light chain variable domains, and also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues (including one or more cysteines from the antibody hinge region) at the carboxy terminus of the heavy chain CH1 domain. Fab '-SH is the name used in this application for Fab' in which one or more cysteine residues of the constant domain carry a free thiol group. F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

"Single chain Fv" or "scFv" antibody fragments comprise the VH and VL regions of an antibody, wherein these regions are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL regions that enables the scFv to form the desired structure for antigen binding. For a review of scfvs, see, e.g., Plueckthun, In; the pharmacological of Monoclonal Antibodies, Vol.113, RosenburgardMoore (eds.), Springer-Verlag, New York (1994), pp.269-315.

The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragment comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between two regions on the same strand, the regions are forced to pair with complementary regions of the other strand and two antigen binding sites are created. The diabodies can be bivalent or bispecific. Doublets are described in more detail in, for example, EP 0404097; WO 1993/01161; hudson, p.j.et., nat. med.9(2003) 129-; and Holliger, P.et., PNASA 90(1993) 6444-. Trisomy and tetrasomy are also described in Hudson, p.j.et al, nat. med.9(2003) 129-.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations that may be present in minor amounts, such as naturally occurring mutant forms. Thus, the modifier "monoclonal" indicates that the antibody is characterized as not being a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies generally include an antibody comprising a target-binding polypeptide sequence, wherein the target-binding polypeptide sequence is obtained by: the method comprises selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection method can be to select a unique clone from a combination of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the selected target binding sequence may be further altered, for example, to improve affinity for the target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. In contrast to polyclonal antibody preparations, which typically comprise different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are also advantageous in that they are generally uncontaminated by other immunoglobulins.

As mentioned above, the compounds and conjugates disclosed herein have quite advantageous properties. For example, the disclosed compounds or conjugates, respectively, exhibit high ECL potency. This high potency is also present if the corresponding measurements are carried out in an aqueous system, in contrast to many ECL-markers which show high ECL potency only when analyzed in organic solvents. For example, many OLED dyes are typically analyzed in acetonitrile and are not soluble in water or, if soluble in water, are not expected to exhibit efficient electrochemiluminescence.

In a preferred embodiment, the invention relates to the use of the compounds or conjugates disclosed in the invention, respectively, for performing an electrochemiluminescence reaction in aqueous solution. An aqueous solution is any solution comprising at least 90% water (by weight). Obviously, such an aqueous solution may contain further ingredients, such as buffer compounds, detergents and, as electron donors in the ECL reaction, for example tertiary amines such as tripropylamine.

In one embodiment, the invention relates to the use of a compound or conjugate, respectively, disclosed in the invention in an electrochemiluminescence based detection method.

In one embodiment, the invention relates to the use of a compound or conjugate disclosed herein, respectively, for the detection of an analyte.

The analyte of the present invention may be any inorganic or organic molecule, including any biological substance of interest. Examples of suitable biological substances which represent analytes in the sense of the present invention are cells, viruses, subcellular particles, proteins, lipoproteins, glycoproteins, peptides, polypeptides, nucleic acids, oligosaccharides, polysaccharides, lipopolysaccharides, cell metabolites, haptens, hormones, pharmacological substances, alkaloids, steroids, vitamins, amino acids and sugars.

The analyte may be selected from polypeptides, carbohydrates and inorganic or organic drug molecules.

A polypeptide or protein is a molecule consisting essentially of amino acids and having at least two amino acids connected by a peptide linker. In the case of studying an analyte of interest in the methods disclosed herein, the polypeptide preferably consists of at least 5,6, 7, 8, 9, 10, 12, 15, 20, 25, and 30 up to about 10,000 amino acids. Preferably, the polypeptide will contain 5-2,000 amino acids, still preferably 10-1,000 amino acids.

Where the analyte is a nucleic acid, these are preferably naturally occurring DNA or RNA oligonucleotides.

In one embodiment, the present invention relates to a method of measuring an analyte by an in vitro method comprising the steps of: (a) providing a sample suspected or known to contain said analyte, (b) contacting said sample with a conjugate of an affinity binding agent disclosed in the present invention and a compound of formula I under conditions suitable for the formation of a conjugate complex of the analyte, (c) measuring the complex formed in step (b) and thereby obtaining a measurement of the analyte.

In one embodiment, the measurement in the above method for detecting an analyte is carried out by using an electrochemiluminescence-based detection operation. It is also preferred that the process is carried out in an aqueous solution.

The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications may be made to the operations set forth without departing from the spirit of the invention.

Example 1:

synthesis of substituted phenyl-phenanthridines

Example 1.1:

general procedure for the synthesis of substituted 2-aminobiphenyls:

using a Suzuki-Miyaura coupling reaction between a commercially available 2-bromoaniline derivative and the corresponding arylboronic acid as described by Youn, s.w. in Tetrahedron lett.50(2009)4598-4601, the appropriate 2-aminobiphenyl required for further reaction to phenanthridine can be synthesized.

Typical operation:

a:10mol％PdCl₂(PPh₃)₂,K₂CO₃,DMF/H₂o (5/1),80 ℃,24 hours

Example (b):

example 1.2:

general procedure for the synthesis of substituted phenanthridines:

to an ice-cooled solution of 2-arylaniline 1(0.01mol) in chloroform (20ml) was added arylacid chloride 2(0.01mol) and stirred at room temperature for 30 minutes under inert conditions. The resulting mixture was then refluxed with stirring for 2 hours. The reaction mixture was treated over 60 minutes by dropwise addition of pyridine (0.02mol in 10ml of chloroform). The mixture was cooled to room temperature and stirred overnight. The mixture was washed thoroughly with 0.5MHCl over MgSO₄Dried and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (3:2 hexanes/ethyl acetate) to give the pure product 3 in 66% yield.

Benzoylamino-2-biphenyl 3(0.01mol) and POCl₃(5ml) the solution in 20ml of toluene was refluxed under nitrogen and stirred for 18 hours, following the procedure described by Lion, C. in Bull. Soc. Chim. Belg.98(1989) 557-566. The cooled reaction mixture is treated with CH₂Cl₂Diluted (30ml) and poured into ice with 25% NH₄OH and distilled water. The organic layer was purified over MgSO₄Drying and concentration in vacuo, followed by purification by flash chromatography (silica gel, 1:1 hexanes/ethyl acetate) afforded the product 4, 6-phenylphenanthridine.

Yield: 52 percent. A white solid.¹H NMR(CDCl₃,400MHz)7.54-7.85(m,9H),8.10(d,J＝8.0Hz,1H),8.28(d,J＝7.9Hz,1H),8.62(d,J＝8.4Hz,1H),8.67(d,J＝8.4Hz,1H)。

Example 1.3:

procedure for the synthesis of 6- (2-sulfophenyl) phenanthridine:

the 6- (2-sulfophenyl) phenanthridine can be synthesized by the following method: arylanilines (0.01mol) were reacted with 2-sulfobenzoic acid cyclic anhydride (0.01mol) in CH using the procedure described in Nicolai, E.chem.Pharm.Bull.42 (1994)1617-₃CN is heated at moderate temperature for 6 hours.

After purification, the product can be converted into the appropriate phenanthridine based on the method described in example 1.2.

Example 1.4:

procedure for the synthesis of 6-phenyl-alkylsulfonylphenanthridine:

the 6-phenyl-alkylsulfonylphenanthridine can be synthesized by: the alkylsulfonyl-arylaniline (0.01mol) and benzoyl chloride (0.01mol) were heated in chloroform at moderate temperatures using the procedure described in Bull. Soc. Chim. Belg.98(1989)557-566, Lion, C.the procedure is described in example 1.2.

After purification, the product can be converted into the appropriate phenanthridine based on the method described in example 1.2.

6- (4-Methylsulfophenyl) phenanthridine was also prepared following the procedure described in Cymerman, J. in J. chem. Soc. (1949) 703-707.

Example 2:

general procedure for the synthesis of chloro-crosslinked dimer complexes:

general procedure is disclosed by Nonoyama, M., J.Organomet.Chem.86(1975) 263-267.

The iridium dimer was synthesized as follows: IrCl is added₃·3H₂O and 2.5 equivalents of 6-phenylphenanthridine were heated under nitrogen in a 2-ethoxyethanol/water mixture (3:1, v/v) at 120 ℃ for 18 hours. After cooling to room temperature, the precipitate is filtered off and successively treated with methanol and Et₂O washing and drying to obtain the expected dimer.

Example (b):

[ (6-Phenylphenanthridine)₂IrCl]₂. Yield: 71 percent. Brown solid.¹H NMR(DMSO-d₆,400MHz)6.45(d,J＝6.8,4H),6.58(t,J＝7.1,13.9Hz,4H),6.95(t,J＝7.1,14.2Hz,4H),7.56(t,J＝7.4,16.0Hz,4H),7.68(t,J＝8.1,16.2Hz,4H),7.93(t,J＝8.0,14.6Hz,4H),8.07-8.13(m,8H),8.80(d,J＝7.3Hz,4H),8.93-9.01(m,12H)。

Example 3:

general procedure for the Synthesis of Iridium complexes

0.5mmol of the chloro-crosslinked dimer complex, 1.25mmol of pyridine-2-formate and 3mmol of Na were added₂CO₃Mixed into 2-ethoxyethanol (12ml) and heated at 120 ℃ for 15 hours. Distilled water (25ml) was added to the cooled mixture, then the crude product was filtered off and washed with water, then with several portions of n-hexane and Et₂And O washing. The product was purified by column chromatography (silica gel, n-hexane/dichloromethane) to give a red powder.

(based on Lamansky, S., Inorg. chem.40(2001)1704-1711)

Ir (6-phenylphenanthridine)₂C₉H₁₀N₄And O. Yield: 68 percent. A red solid.¹H NMR(CDCl₃,400MHz)3.95-3.97(m,2H),4.53-4.55(m,2H),6.77-6.93(m,4H),7.03-7.30(m,5H),7.37-7.66(m,4H),7.82-7.95(m,5H),8.07(d,J＝8.0Hz,1H),8.23(d,J＝7.8Hz,1H),8.34(t,J＝7.8,14.4Hz,3H),8.46(d,J＝5.5Hz,1H),8.56(t,J＝7.6,14.2Hz,2H),9.07(dd,J＝8.2,16.0Hz,2H),9.46(s,1H)。

Ir (6-phenylphenanthridine)₂C₁₁H₁₃N₃And O. Yield: 71 percent. A red solid.¹H NMR(CDCl₃,400MHz)1.47-1.49(m,2H),2.35-2.49(m,2H),3.30-3.35(m,2H),5.76(s,1H),6.71-6.74(m,3H),6.81-6.99(m,3H),7.07-7.31(m,6H),7.37-7.41(m,1H),7.73-7.85(m,5H),8.25-8.35(m,5H),8.45-8.54(m,3H),9.09(d,J＝8.2Hz,1H),9.29-9.32(m,1H)。

Claims (11)

1. An iridium-based luminescent or electrochemiluminescent compound of formula I:

wherein R1-R12 are hydrogen, halogen, cyano, nitro, hydrophilic groups or R19, wherein R19 is aryl, substituted aryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, hydrophilic groups,

wherein R13-R16 are hydrogen, halogen, cyano, nitro, hydrophilic groups, -Q-Y or R19, wherein R19 is aryl, substituted aryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, hydrophilic groups,

wherein R17-R18 represent hydrogen, alkyl, aryl, substituted aryl and alkyl, heteroaromatic ring systems, non-aromatic ring systems, imidazolium, cyclodextrin or Q-Y, or

Wherein in R1-R12 or/and R13-R16, respectively, two adjacent R may form an aromatic ring or a substituted aromatic ring, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, hydrophilic groups, or

Wherein in R1-R12 or/and R13-R16, respectively, two adjacent R may form an aliphatic ring or a substituted aliphatic ring, wherein the substituents are selected from hydrogen, halogen, cyano, nitro, hydrophilic groups,

wherein X represents C or N, and the compound is shown in the specification,

wherein Y represents C or N, or a salt thereof,

wherein at least one of R13-R18 is-Q-Y,

wherein Q represents a linking group and Y is a functional group,

wherein the linking group Q is a linear or branched, saturated or unsaturated, unsubstituted or substituted C1-C20 alkyl chain, or a chain containing 1-20 atoms having a backbone consisting of carbon atoms and one or more heteroatoms selected from O, N and S,

wherein the functional group Y is selected from the group consisting of carboxylic acid group, N-hydroxysuccinimide ester group, amino group, halogen, mercapto group, maleimido group, alkynyl group, azido group and phosphoramidite group,

wherein the hydrophilic group is selected from the group consisting of amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carbamoyl, hydroxyl, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfino, sulfoxido, sulfonyl, phosphonate, phosphinate.

2. The compound of claim 1, wherein the linker Q is a saturated C1-C12 alkyl chain or a chain containing 1-12 atoms having a backbone consisting of carbon atoms and one or more heteroatoms selected from O, N and S.

3. The compound of claim 1, wherein R1-R12 are R19, wherein R19 is branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl.

4. The compound of claim 1, wherein R13-R16 are R19, wherein R19 is branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl.

5. A conjugate comprising a compound of any one of claims 1-4 and an affinity binding agent covalently bound thereto.

6. The conjugate of claim 5, wherein the affinity binding agent is selected from the group consisting of antigens and antibodies, biotin or biotin analogues selected from the group consisting of aminobiotin, iminobiotin and desthiobiotin and avidin or streptavidin, sugars and lectins, nucleic acids and complementary nucleic acids and receptors and ligands.

7. The conjugate of claim 5 or 6, wherein the affinity binding agent is a nucleic acid or an antibody.

8. Use of a compound according to any one of claims 1 to 4 or a conjugate according to any one of claims 5 to 7 for non-diagnostic purposes in performing an electrochemiluminescence reaction in aqueous solution.

9. Use of a compound according to any one of claims 1 to 4 or a conjugate according to any one of claims 5 to 7 in an electrochemiluminescence-based detection method for non-diagnostic purposes.

10. Use of a compound according to any one of claims 1 to 4 or a conjugate according to any one of claims 5 to 7 in the detection of an analyte for non-diagnostic purposes.

11. A method for non-diagnostic purposes of measuring an analyte by an in vitro method, the method comprising the steps of:

a) providing a sample suspected or known to contain the analyte,

b) contacting the sample with the conjugate of any one of claims 5-7 under conditions suitable for formation of an analyte conjugate complex,

c) measuring the complex formed in step (b) and thereby obtaining a measurement of the analyte.