Bioconjugation with Stable Luminescent Lanthanide(III) Chelates

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Bioconjugate Chem. 2009, 20, 404–421

REVIEWS Bioconjugation with Stable Luminescent Lanthanide(III) Chelates Comprising Pyridine Subunits Jari Hovinen*,† and Pamela M. Guy‡ PerkinElmer Life and Analytical Sciences, Turku Site, POB 10, FI-20101 Turku, Finland, and PerkinElmer Incorporated, Winter Street, Waltham, Massachusetts 02451. Received August 30, 2008; Revised Manuscript Received October 30, 2008

The use of long-lifetime emitting lanthanide(III) chelate labels or probes together with time-resolved fluorometry in detection provides a method to generate sensitive bioaffinity assays. However, the use of stable chelates demands very complicated optimization of the chelate structure. A great number of chelates have been synthesized, but usually, only the most prominent structures were then converted to corresponding biomolecule labeling reactants. This review covers the syntheses of luminescent lanthanide chelates comprising a pyridine subunit that allow solution and solid-phase bioconjugation.

INTRODUCTION The idea of using lanthanide chelates as labels in timeresolved fluoroimmunoassays was described almost three decades ago (1, 2). The use of long-lifetime emitting lanthanide(III) chelate labels or probes together with timeresolved fluorometry in detection has proven its value in the development of highly sensitive bioaffinity assays (3). The heterogeneous DELFIA time-resolved fluorometry assay method uses non-luminescent lanthanide(III) chelates as the labels (4-6). After immunoreaction, lanthanide(III) ions are dissociated from the non-luminescent chelates by lowering pH, and luminescence is enhanced with a mixture of β-diketone, detergent, and trioctylphosphine oxide (TOPO). The resultant chelates have a very high luminescence, giving excellent detection sensitivity. However, the DELFIA timeresolved fluorometry technology is not useful for applications requiring site-specificity. Furthermore, the use of the additional chelator TOPO is essential for high luminescence. In contrast, the FIAgen time-resolved fluorometry system uses a fluorescent 4,7-bis(chlorosulfophenyl)-1,10-phenanthonine2,9-dicarboxylate europium(III) as the label, and thus in contrast to the DELFIA system, the signal enhancement step is avoided (7-10). Due to limited chelate stability, FIAgen assays are performed in the presence of an excess of lanthanide ion. Another assay system, sold under trademark TRACE, was the first real homogeneous time-resolved fluorometry system and is based on an energy transfer between a europium(III) chelate of tris(bipyridine)cryptate and an organic fluorophore (11). However, detection sensitivity of the FIAgen and TRACE technologies is considerably lower than in assays based on DELFIA technology. To overcome these limitations, better luminescent lanthanide(III) chelates have to be developed. * Corresponding author. Dr. Jari Hovinen. Tel + 358 2 2678 513; fax + 358 2 2678 380; [email protected]. † PerkinElmer Life and Analytical Sciences. ‡ PerkinElmer Incorporated.

For time-resolved fluorometry assay systems, a luminescent lanthanide(III) chelate should fulfill several requirements: (a) the molecule has to be photochemically stable, in both the ground and excited states; (b) the molecule has to be kinetically stable; (c) the molecule has to be chemically stable; (d) the excitation wavelength has to be as high as possible, preferably over 330 nm; (e) the molecule must have a high excitation coefficient in the excitation wavelength; (f) the energy transfer from the ligand to the central ion has to be efficient; (g) the luminescence decay time has to be long; (h) the chelate should be readily soluble in water; and (i) the bioactive molecules have to retain their affinities after the coupling to the lanthanide chelate. Although organic chelators and their substituents have a significant effect on the photophysical properties of lanthanide(III) chelates, no general rules for the estimation of these effects are available. Thus, finding a highly fluorescent chelate structure to fulfill all the requirements for a label with respect to signal, conjugation, stability, and biocompatibility remains as a challenge. Hundreds of stable fluorescent chelates have been developed at numerous laboratories. A great number of chelates synthesized are basic structures that do not allow biomolecule derivatization; usually, only the most prominent structures have been converted to the corresponding biomolecule labeling reactants. Although there are several excellent review articles on the photophysical properties and bioanalytical applications of lanthanide(III) chelates (12-23), syntheses of luminescent lanthanide(III) chelates has not been reviewed. Stable luminescent lanthanide(III) chelates consist of a ligand with a reactive group for covalent conjugation to bioactive molecules, an aromatic structure, which absorbs the excitation energy and transfers it to the lanthanide ion, and additional chelating groups such as carboxylic acid moieties and amines. Unlike organic chromophores, these molecules do not suffer from Raman and Rayleigh scattering or concentration quenching. This allows multilabeling and development of chelates bearing several light-absorbing moieties. The pyridine moiety is by far the most common chromophoric subunit in luminescent lanthanide chelates. When appropriately

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Bioconjugate Chem., Vol. 20, No. 3, 2009 405

Scheme 1. Derivatization of Pyridinea

a

X ) Br, I, or Cl; R ) an alkyl group.

derivatized, pyridine can serve as the chromophoric moiety alone. Alternatively, it can be present in multimeric forms in open-chain structures and macrocyclic cage-type complexes such as cryptates and cyclic Schiff bases. This review mainly describes synthesis of luminescent lanthanide chelates comprising pyridine subunits that can undergo bioconjugation. Accordingly, luminescent stable chelates with no pyridine subunits, such as those comprising DTPA and DOTA tethered to lightharvesting groups, and chelates based on β-diketones and phenolic compounds, are not discussed here.

THE CHROMOPHORE A single unsubstituted pyridine moiety is not efficient enough to serve as a light-absorbing and triplet-sensitizing aromatic group in stable luminescent chelates. Thus, pyridine has often been substituted with various energy-absorbing groups. Pyridine analogues conjugated to five-membered heteroaromatic rings have also been prepared. The chromophores of stable chelates are often composed from one to three conjugated pyridine rings, or the pyridine moieties are connected to each other via N, O, or S-containing hydrocarbon chains. While bipyridine and terpyridine moieties are commercially available, preparation of chromophores based on 4-substituted pyridines and pyridines conjugated to five-membered hetereocyclic rings require numerous synthetic manipulations. Derivatization of C4 of Pyridine. There are numerous methods for attaching additional light absorbing groups to 4-halopyridines (Scheme 1). Phenylethynyl substitution (1) has been performed using the Sonogashira reaction (24-26). Furyl (2) and thienyl (3) substituted pyridines have been synthesized using the palladium(0)-catalyzed Stille reaction with the corresponding stannyl derivatives of the additional light-absorbing groups (28-31). Suzuki coupling of the halopyridine with alkoxyphenylboronates gives rise to the alkoxyphenylpyridine

chromophore 4 (32). The trans-4-styrylpyridine derivative (5) has been synthesized by Heck coupling with 4-chloropyridine2,6-dicarboxylate using the mixture of palladium(II) acetate and triphenyl phosphine (29). Aromatic nucleophilic substitution has been exploited in the preparation the 4-(2-napthoxy)pyridine derivative (6). The 4-benzoylpyridine-2,6-dicarboxylate 7 was obtained under similar conditions using O-(tetrahydropyran-2yl)mandelonitrile followed by acid-catalyzed cleavage of the THP group (29). Derivatization of Terpyridine. This has most commonly been performed using Claisen-Schmidt condensation of various aromatic aldehydes (33-42) with 2-acetylpyridine to give the (E)-propen-2-enones 8 (Scheme 2). Their reaction with N-[2(pyrid-2-yl)-2-oxoethyl]pyridinium iodide and sodium acetate in methanol yielded the terpyridine derivatives 9a-d. Stille reaction between terpyridine 4-triflate 10 and tributylstannyl furan has been utilized in the preparation of 4-furyl terpyridine 11 (28). Pyridine Conjugated to Five-Membered Heteroaromatic Rings. A wide range of lanthanide chelates comprising a pyridine subunit conjugated to one or two heteroaromatic rings have been synthesized and claimed (29, 43-50). The conjugated chromophore often exhibits a high excitation coefficient, long excitation wavelength, and good energy transfer from the ligand to the lanthanide ion, giving rise to improvements in detection sensitivity. However, only few of these chelates have been converted to the corresponding biomolecule labeling reactants. The key steps of the syntheses of their chromophores are shown in Scheme 3. The preparation of 2,2′-(1H-pyrazole-1,3-diyl)bispyridine chromophore 15 involves treatment of 6-methyl-2acetylpyridine 12 with N,N-dimethylformamide dimethyl acetal to give rise to (E)-3-(dimethylamino)-1-(6-methylpyridin-2yl)prop-2-en-1-one 13, which is cyclized to 2-methyl-6-(1Hpyrazol-3-yl)pyridine 14 with hydrazine hydrate followed by

406 Bioconjugate Chem., Vol. 20, No. 3, 2009

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Scheme 2. Derivatization of Terpyridine

Figure 1. A ligand with two 2,6-bis(N-pyrazol-11-yl)pyridine subunits.

Scheme 3. Preparation of Chromophores Comprising Pyridine Conjugated to Five-Membered Heteroaromatic Ring

hydrogen sulfide in ammonia followed by cyclization with 2-bromoacetylpyridine (43). Lanthanide(III) chelates based on 2,6-bis(N-pyrazolyl)pyridine 17 and 6,6′-(1H-pyrazole-1,3-diyl)bis(pyridine) 15 are among the best and most widely used luminescent terbium(III) chelates synthesized: they have excellent photophysical properties including high luminescent quantum yield and relatively high energy of their lowest triplet state. The chromophore of the lanthanide chelate comprising 1,2,4-triazol-3,5-ylene subunit 20 is negatively charged, a property that stabilizes the chelate. Its suitability in immunoassays has been demonstrated (43). No applications for the chelate comprising the chromophore 21 have been published. Also, chelates derived from 2,6-bis(N-pyrazol11-yl)pyridine motif attached to aceto/benzophenone and iminodiatetic subunits 22 (Figure 1) have been synthesized (51); however, their quantum yields were significantly lower than those of simpler complexes.

THE CHELATING PART

reaction with 2-bromo-6-methylpyridine (29). The key reaction of the most recent synthesis of 2,6-bis(3-methyl-1-pyrazolyl)pyridine 17 starts from nucleophilic substitution of the bromine atoms of 2,6-dibromopyridine 16 with potassium 3-ethoxycarbonylpyrazole in diglyme (50). The triazole derivative 20 has been prepared using 2-pyridinecarboxylate 18 as the starting material that was initially treated with hydrazine hydrate to give 2-pyridylhydrazide 19, the reaction of which with picolinonitrile at 160 °C gave the desired heterocycle (43). Synthesis of the thiazole derivative 21 includes treatment of picolinonitrile with

The lanthanide chelate has to be stable in the presence of external chelators such as EDTA and relatively low pH conditions sometimes used in biochemical assays. Tolerance to high temperatures is required in nucleic acid based assays employing thermal cycling. Because the chromophoric moiety binds weakly to the lanthanide ion, the chelate requires additional metal coordination sites. Enhancement in stability has been obtained by adding chelating groups such as carboxylic acid and phosphonate moieties to the chromophore. Alternatively, incorporating several fluorogenic ligands into one structure (52) or encapsulating the metal ion to the chromophoric structure forming polycyclic cage-type compounds such as cryptates and macrocyclic Schiff’s bases have been used to enhance stability. Carboxylic Acid Derivatives. Methods for attaching chelating carboxylic and phosphonic acid moieties to chromophores are shown in Scheme 4. A pyridine ring 23 has been converted to the N-oxide 24 using an oxidizer such as m-chloroperbenzoic acid or magnesium perphthalate (29, 35, 40, 53). There are enormous differences in the reactivities of the pyridine rings: while the terminal pyridine rings of terpyridine are readily oxidized with m-chloroperbenzoic acid in dichloromethane in a few hours at ambient temperature, a prolonged reaction time (perphthalate in acetic acid; 8 days at 80 °C) was required for the preparation of the di-N-oxide of 15 (29). The N-oxides have been converted to the corresponding carbonitriles 25 by a modified Reissert-Henze reaction, the reduction of which with borane yields the amines 26 that have been carboxymethylated with alkyl bromoacetate followed by hydrolysis to give the iminoacetic acid derivatives 27. The nitrile 25 has also been hydrolyzed to carboxylic acid 28, which was esterified followed by reduction to the alcohol 29 (29, 53-57). The cyanide 25 has also been obtained by nucleophilic substitution of 2-bro-

Reviews


Scheme 4a

a

X ) Br or Cl; some substituents are omitted for clarity.

mopyridine derivative with cyanide ion (54). Treatment of 29 with phosphorus tribromide in DMF or chloroform gives the bromide 30 that has been allowed to react with iminoacetic acid ester followed by hydrolysis to give 27. Several alternative approaches have been used when 2-methylpyridine or its derivatives 31 have been used as the key intermediates. One route involves free radical halogenation (24, 48, 53, 55) to 30 followed by substitution with iminoacetic acid ester and ester hydrolysis. However, the yield of the free radical reaction may be low due to the formation of unsymmetrical byproduct and the products of different halogenation steps. The yield is highly related to the structure of the alkylpyridine derivative (57). Another route comprises oxidation of 31 to the corresponding N-oxide 32. Treatment of this with acetic anhydride followed by base-catalyzed hydrolysis gives the methanol 29 that was converted to the bromide 30 (29). The picolinate 31 has also been oxidized to carboxylic acid 28 using selenium(IV) oxide in refluxing pyridine (53). When 2-alkyl picolinates are used as key intermediates, the ester is either hydrolyzed to acid (23) or it is reduced to alcohol with NaBH4, followed by bromination with PBr3, substitution with iminoacetic acid ester, and hydrolysis (23). A method disclosed by Hale and Solas (52) includes basecatalyzed condensation of 2-acetylfuran and benzaldehyde to give the pentanedione 33, which was further converted to the

furylpyridine 34 by treatment with hydroxylamine that was finally oxidized to the desired acid 35. Phosphonate ligands (36, 37) have been synthesized by an Arbuzov reaction between the bromide 30 and triethyl phosphate followed by ester hydrolysis and by treatment of the amine 26 with H3PO3 and formaldehyde in the presence of HCl (53). Because there are fewer water molecules coordinated to the lanthanide ion than in the corresponding carboxylate chelate, the phosphonate group has a positive effect on the luminescence. The phosphonate chelates have a high negative charge, and therefore, their coupling to bioactive molecules could change the physical properties of the conjugate. No biomolecule labeling reactants containing phosphonates as chelating units have been synthesized. Azamacrocycles. Macrocyclic lanthanide chelates comprising an intracyclic chromophoric unit (pyridine, bipyridine, terpyridine, bis(pyrazol-1-yl)pyridine) and pendant carboxylate groups have been shown to exhibit high chelate stability and promising photophysical properties (23, 40, 58-63). Also, macrocycles in which the chromphore comprises two nonconjugated pyridine subunits tethered to each other via a nitrogen-containing hydrocarbonchainhasbeendescribedandclaimedinpatents(28,32). The crucial step in the ligand synthesis is the macrocyclization reaction. Azamacrocyles have been traditionally synthesized via the method of Richman and Atkins (64) by condensing disodium


Hovinen and Guy

Scheme 5. Preparation of Azamacrocyclesa

a

Z ) an aromatic group.

salts of pertosylated amines with dihalogeno fragments of the chromophore (Scheme 5). According to the latest modification of this reaction, the synthesis involves a reaction between a bifunctional electrophile (e.g., a dibromide or ditosylate) and a pernosylated amine in dry DMF at an elevated temperature in the presence of cesium or sodium carbonate (65-67). Finally, the tosyl or nosyl groups are removed with acid or thiol in the presence of base, respectively, giving the desired azamacrocycle. Carboxyalkylation of the secondary amines gives the desired ligands. Also, a cyclization reaction utilizing the Mitsunobu reaction between a dihydroxy fragment of the chromophore and a pernosylated amine has been used (68). Galaup et al. have reported an elegant direct method to 18-membered hexaazamacrocyles comprising a functionalized 2,2′:6′,2′′-terpyridine moiety 41, exploiting a metal template ion effect on the macrocylization step (60). The acetate pedant arm 40 was synthesized in five steps starting from diethylenetriamine. Brunet et al. allowed a diamino fragment of the chromophore to react with DTPA anhydride in the presence of base (63) to give 43. The reaction was performed in high dilution, and the yield of the desired ligand was relatively low. Lanthanide chelates based on 1,4,7-triazacyclononane 45 and 1,4,7-triazacyclodecane also exhibit high kinetic and thermodynamic stabilities (28, 32). Their synthesis is straightforward and involves alkylation of the azacycloalkane with the bromomethyl derivatives of the desired chromophore 44.

Cryptates. Macropolycyclic inclusion complexes, cryptates, have been designed to provide lanthanide chelates of high kinetic and thermodynamic stability. Practically, all lanthanide chelate cryptates containing bivalent heterocyclic unsaturated fivemembered rings and/or one or more pyridine derivatives as a chromophore have been claimed in patents (42, 43, 62, 69, 70). However, only few of these compounds have been synthesized and converted to the corresponding biomolecule labeling reactants. Preparation of cryptates comprising the 4,13-diaza18-crown 6 ether moiety involves alkylation of the secondary amines with a chromophore having two bromoalkyl groups (Scheme 6). The cryptand formation is assisted by using a metal ion as a directing template. When the metal ion used is a lanthanide (42, 71), the desired lanthanide chelate is formed; otherwise, an additional prolonged metal exchange reaction is needed. The usefulness of cryptates containing a 4,13-diaza18-crown 6 ether subunit for biomolecule derivatization has not been demonstrated. The only commercialized lanthanide cryptant is the europium chelate based on three bipyridine moieties 46. It has been synthesized by treatment of 2,6′-aminomethylbipyridine and 2 equiv of the corresponding dibromide in the presence of base. Cryptates are kinetically inert and have good stability, because the lanthanide ion is tightly closed inside the molecular cavity. However, the positive net charge may have an adverse effect on antibodies, which are widely used bioactive components in

Reviews Scheme 6. Synthesis of Cryptands and Schiff’S Basesa


vibrations are decreased, giving rise to enhanced luminescence. Chelate stability has been enhanced by linking several chelates together to give rise to multidentate structures. However, the chelate has to be saturated using an excess of lanthanide ion if the single subunit is tridentate (75). Since lanthanide chelates do not suffer from concentration quenching, development of chelates bearing several lightabsorbing moieties is possible. In particular, chelates comprising a triazacyclononane 56 backbone have been shown to be highly luminescent (28, 30, 32). The corresponding triazacyclododecane 57 is practically nonluminescent (30).

THE LINKER

a

L ) a linker.

common assay formats. In addition, water and negatively charged quenchers present in samples must be removed by treatment with fluoride ion. These disadvantages are not present in 47, since the molecule has a net charge of -1. Also, its quantum yield is relatively high (70). Macrocyclic Schiff Bases. Lanthanide chelates having a macrocyclic Schiff base 48 as a ligand can be prepared from 2,6-diacetylpyridine and diaminoalkane by using a lanthanide ion as a template (Scheme 6). Although Schiff bases are normally labile, the lanthanide ion stabilizes the macrocyclic moiety if the structure formed is an 18-membered macrocycle (72). Due to limited chelate stability, assays have been performed in the presence of excess lanthanide ion. Detection sensitivity has been enhanced by using the chelate in association with dissociative enhancement (73) or as a mixed ligand chelate with thenoyltrifluoroacetone (74). Figure 2 shows different ligands containing the pyridinyl-4phenylethynyl chromophore. The corresponding chelates differ from each other with regard to lanthanide/ligand ratio and stability constants of the formed complex. The tridentate pyridine dicarboxylic acid 49 forms 1/1, 1/2, and 1/3 complexes with lanthanide(III) ions, but only the 1/3 complex is highly luminescent. Thus, this ligand is unsuitable for biomolecule derivatization. Ligands with seven or more chelating groups 50-56 form luminescent 1/1 chelates with lanthanide(III) ions and thus are suitable for bioconjugation. The stability of the chelate is usually increased as the number of ligating atoms of the chelate are increased. Simultaneously, the number of coordinating water molecules in the first coordinating sphere of the lanthanide ion and the quenching effect of O-H

Although in many applications a reactive group for biomolecule conjugation can be attached directly to the chromophoric group or to the chelating part, it is often desirable, for steric reasons, to have a linker between the reactive group and the chromophoric group or chelating part, respectively. The linker is especially important in case the chelate should be used in solid-phase syntheses of oligopeptides and oligonucleotides, but it is often desirable also when labeling biomolecules in solution. In one strategy for attaching a linker, an aromatic bromide or iodide was allowed to react with a linker tethered to a terminal alkynyl group using Sonogashira reaction (28). Elongation of an amine function was performed using reactions of peptide bond formation. Aromatic amino groups of low reactivity require the use of reactive activators such as HATU (76) and 1-(2mesitylenesulfonyl)-3-nitro-1,2,4-triazole (24). For the preparation of terpyridyl ethers with an ω-substituent, three methods have been reported. The method of Newkome and He (77) involves nucleophilic substitution of 4′-chloro-2,2′: 6′,2′′-terpyridine with various ω-substituted alcohols or thiols in the presence of an excess of KOH in DMSO at elevated temperature (Scheme 7, route A). Although the reactions have to be performed in rather drastic reaction conditions, a wide variety of linkers have been introduced in high yield (78, 79). Another tethering strategy, developed by Constable and coworkers (80) involves an SN2 reaction between the nucleophilic 2,6-di(pyridin-2-yl)pyridin-4(1H)-one and a tether molecule bearing a good leaving group in its structure (Scheme 7, route B). The third method is based a reaction 2,6-di(pyridin-2yl)pyridin-4(1H)-one and an alcohol under Mitsunobu reaction (Scheme 7, route C). Since the reaction conditions are mild, a wide range of linkers tethered to various substituents can be synthesized (81). The reaction has shown to be applicable to pyridine derivatization also. The addition of a binding arm to energetically isolated positions, such as to the chelating imidoacetate group, allows development of structures without changing the energetic profile of the aromatic part on conjugation. However, synthesis of these structures is more challenging unless the presence of two linker arms for biomolecule derivatization is accepted. Attachment of a single linker arm to the chelating imidoacetate group involves two SN2 reactions with different amines of similar reactivities reducing the yield of the desired product (82). The method also calls for preparation of a modified iminoacetic acid ester (Scheme 8). When the chromophores are linked to each other via a nitrogen-containing hydrocarbon chain, the linker has been attached to this unit (Scheme 9). Several nine dentate derivatives have been synthesized by a SN2 reaction of a primary amine in the presence of an excess of a bromide (28, 32, 83). The corresponding azamacrocycles have been synthesized using a carbon-substituted cyclic amine (28, 32, 84) (Scheme 10). Sitespecific derivatization of azacycloalkane bearing a single tether


Hovinen and Guy

Figure 2. Ligands containing 4-(phenylethynyl)pyridine chromophore. Scheme 7

L ) a Linker; Lv ) a leaving group.

arm at one of the chromophores was performed using a bisprotected triazacyclononane (28, 32).

THE REACTIVE GROUP In several applications, covalent conjugation of the chelate to bioactive molecules is required. Most commonly, this is performed in solution by allowing a chelate with a reactive group to react with a functional group of a bioactive molecule (85). Although derivatization of synthetic biomolecules can be conveniently performed on solid phase using standard oligopep-

tide and oligonucleotide synthesizers, solution-phase labeling of large biomolecules, such as proteins, is also important. In these cases, the labeling reaction must be as selective and effective as possible. Preparation of the most common activations is depicted in Scheme 11. Labeling in Solution. Synthesis of the activated chelate often involves reaction of an amino chelate with the desired activator. Treatment of an amine with thiourea gives rise to the corresponding isothiocyanate (86). An aromatic isothiocyanate reacts smoothly with primary amino groups forming thiourea bonds (optimal pH of the reaction is 9.0-9.5). Aromatic isothiocyanate is commonly employed for the coupling of lanthanide chelates to proteins, oligopeptides, and oligonucleotides tethered to primary amino groups. Since the aliphatic isothiocyanate is considerably less reactive, this group does not have preparative value. Amino groups of biomolecules can be selectively labeled also with arenesulfonyl activated chelates (10). Due to quite drastic reaction conditions, the activation has to be performed before chelate formation. Although the activated chelate contains two reactive arenesulfonyl groups in its structure, no formation of protein polymers during labeling (protein-protein cross-links)

Reviews


Scheme 8. Introduction of the Linker Arm to an Energetically Isolated Position of Terpyridinea

a

L ) a linker; R ) an alkyl group.

Scheme 9. Introduction of the Linker between the Chromophoresa

a

L ) a linker; R ) an alkyl group; Z ) an aromatic group.

have been observed. The same has been later observed for an europium chelate tethered to two aromatic isothiocyanate groups (87). When high reactivity is required, the 4,6-dichloro-1,3,5triazin-3-yl group (DTA) is the activation of choice. The activated chelate has been prepared by treating the amino chelate with cyanuric acid. DTA allows labeling of all functional groups of proteins (87, 88). This function has been used even in the labeling of hydroxy groups of sugars. Due to the high reactivity, the selectivity is lost. A study has been conducted to evaluate various less reactive DTA derivatives and their effects on chelate luminescence and antibody functionality by replacing one of the chlorine atoms in the triazine ring with a deactivating substituent (89). Histidine and tyrosine residues can be selectively labeled using diazonium coupling. Although no luminescent lanthanide chelates tethered to diazo groups have been synthesized (90), they have been claimed in several patents. Thiols are know to react with haloacetamides, maleimides, disulfides and their analogues, mercurials, vinyl sulfones, aryl halides, aziridines, and oxiranes (91). For coupling purposes, the reactions of haloacetamides, maleimides, and disulfides are most commonly employed. From them, the haloacetyl derivatives display a wide range of other reactivities, while disulfides are practically completely specific to thiols. The reactivity of maleimides is intermediate between them. The problem with using disulfides is lability of the bioconjugate in the presence of reducing agents, which are often present in the conjugation reaction. The haloacetamide chelates have been synthesized by allowing the amino chelates or ligands to react with the haloacetic anhydride or halide (87). Because of the high reactivity of haloacetamides, the pH of the conjugation reaction has to be carefully controlled. At too low pH, the reaction is slow, while at too high pH, amino functions, often abundantly present in the bioactive molecule to be labeled, compete with the target mercapto function for the label. Accordingly, the haloacetamides are most specific toward thiols at neutral pH. Maleimides have been synthesized by allowing amino chelates or ligands to react with carboxylic acid derivatives bearing an ω-maleimide substituent in the presence of appropriate activating agents (76). Disulfides have been prepared by allowing amino chelates to react with the NHS ester of 3-(2-(pyridin-2-yl)disulfanyl)propanoic acid (92). Ge and Selvin allowed a modified DTPA anhydride to react with primary maleimidopropionic acid hydrazide and aminoethylmethanethiosulfonate to give the corresponding mercapto selective labeling reactants (93).

Although N-hydroxysuccinimidyl (NHS) ester is one of the most common functional groups used in bioanalytical chemistry for covalent coupling of labels to primary amino groups of biomolecules, this activation has not been widely applied to lanthanide chelate conjugation. The coupling reaction with NHS esters is performed under mild alkaline conditions under which the ester reacts specifically with primary amino groups. NHS coupling is not harmful to biologically active compounds, and high reaction yields can be achieved, minimizing protein denaturation. Wieder et al. allowed a chelate comprising an aromatic amino group to react with bis NHS ester of dicarboxylic acid to give rise to the chelate tethered to NHS group (92). NHS ester 58 was prepared by allowing the corresponding carboxylic acid to react with N-hydroxysuccinimide in the presence of DCC (46) (Scheme 12). In this case, the reactive group is attached to the energetically isolated position, but the net charge of the chelate is changed. The NHS ester was not isolated but used in situ for bioconjugation. Weibel et al. (94) prepared a europium chelate based on a glutamic acid skeleton bis-functionalized on its nitrogen atoms by 6-methylene-6′carboxy-2,2′-bipyridine chromophoric units 59. Since the propionate function on the glutamic residue remained uncoordinated to the central ion, its esterification into an activated Nhydroxysuccinimidyl ester could be performed. Amino groups can be conjugated directly to carboxylic acid groups in the presence of water soluble carbodiimides such as EDAC. This reaction has been utilized especially for antibody and steroid conjugation (92, 95, 96). O-Alkyl hydroxylamines are known to react chemoselectively with ketones and aldehydes under mild conditions, giving stabile oximes in excellent yield. Although aminooxy chelates have been claimed in numerous patents and patent applications, only one stable luminescent europium chelate tethered to a aminooxy group 60 (Figure 3) has been prepared and used for carbonyl group conjugation (97). The same chelate structure tethered to a mercapto group 61 has been synthesized and exploited in the detection of phosphopeptides by phosphate elimination and subsequent Michael addition (98). Poupart et al. synthesized the aldehyde 62 and used it in oligonucleotide conjugation (99). The Schiff base formed was further stabilized by reduction with borohydride to the corresponding amine. The thioester-mediated ligation of unprotected peptide segments, called natural chemical ligation, has been demonstrated to be extremely effective for the rapid and efficient chemical synthesis of proteins and long oligopeptides (100). The method is based on the reaction of a peptide containing a thioester at the R-carboxy group with the N-terminal Cys residue of another peptide. The initial thioester ligation product then undergoes rapid intramolecular reaction giving rise to a native peptide bond at the ligation site. In addition to the synthesis of proteins and long oligopeptides, natural chemical ligation has been successfully used in the introduction of affinity groups and organic fluorophores to proteins. A lanthanide chelate tethered to thioester bond 63 has been prepared by allowing a chelate bearing a primary amine to react with pentafluorophenyl S-benzyl thiosuccinate (99).


Hovinen and Guy

Scheme 10. Derivatization of Ligands Comprising Azacycloalkane Backbonea

a

L ) a linker; R ) an alkyl group; Z ) an aromatic group.

Scheme 11. Activation of an Aromatic Amino Groupa

a

X ) Br or I.

The copper(I) catalyzed Huisgen’s dipolar [2 + 3] cycloaddition of azide and alkynes, called “click chemistry”, is a powerful direct method to prepare 1,4-disubstituted 1,2,3triazoles (101). This reaction has been utilized increasingly also in the conjugation of various label molecules to nanomaterials and biomolecules. Recently, this reaction has been utilized with lanthanide chelates (99). For example, a protected ligand tethered to an azido group 64 has been reacted with a steroid tethered to an ethynyl group in the presence of copper(I). Removal of the protecting groups followed by treatment with europium ion gave the desired steroid conjugate. Statistical labeling of oligonucleotides, DNA, RNA and proteins was done using labels tethered to derivatives of cisplatinum (102). In nucleic acids, the platinum predominantly coordinates to N7 of guanine residues. Although no luminescent

lanthanide chelates tethered to platinum have been synthesized, the patent (102) has claims directed to platinum tethered to any detectable group. Statistical phosphate labeling using lanthanide titanocene conjugates, such as 65 has also been proposed (103). It has been shown that detection sensitivity can be dramatically enhanced by incorporating lanthanide(III) chelates into particles. Beads containing lanthanide(III) chelates as dye molecules have most commonly been prepared simply by swelling chelates into the bead polymer (104). Few of this type of labeled beads are currently commercially available (23). If the lanthanide(III) chelates are not covalently bound to the polymer particles, the signal obtained from the particles may decrease as a function of the time because of leaking. This problem has been avoided by linking the chelate covalently to a matrix (31, 105-107). In case the chelate should be attached to a microparticle or nanoparticle during the manufacturing process of the particles, a lanthanide chelate tethered to a polymerizable group is required. For this purpose, europium(III) chelates based on thienylpyridine subunits tethered to an acrylamide function 66 have been synthesized (31). Labeling on Solid Phase. In almost all biomolecule labelings, the reaction is performed with an excess of an activated label, and accordingly, laborious purification procedures cannot be avoided. In particular, when attachment of several label molecules, or site-specific labeling in the presence of several functional groups of similar reactivity, is desired the isolation and characterization of the resultant biomolecule conjugate is extremely difficult, and often practically impossible. The biomolecule conjugates used in many applications, such as homogeneous quenching assays, must be extremely pure, because even small amounts of fluorescent impurities considerably increase luminescence background and thereby reduce detection sensitivity. Thus, it is highly desirable to perform conjugation of biomolecules on a solid phase, allowing removal of most impurities by washing while the biomolecule is anchored to the solid support. Once the complex is released into the solution, only a single chromatographic or electrophoretic purification is required. Solid-phase labeling of oligopeptides calls for preparation of the corresponding oligopeptide building blocks (108-110). Most commonly, the block has an activatable carboxylic acid function and an R-amino group bearing a transient protecting group. The chelating part has to be masked with permanent protecting

Reviews


Scheme 12. NHS Activation at the Energetically Isolated Position

groups. Building blocks that allow solid-phase introduction of luminescent europium and terbium chelates to olipeptides using standard Fmoc chemistry are shown in Figure 4. The blocks have been coupled either to the amino or carboxyl terminus or to the internal position of the coding sequence using prolonged

Figure 3. Europium(III) chelates tethered to reactive groups.

coupling time but otherwise standard conditions (Scheme 13). The coupling efficiencies of these blocks are comparable to natural amino acid analogues. After completion of the oligopeptide synthesis, the oligopeptides were deprotected and released from the resin using standard procedures. Treatment of the


Hovinen and Guy

Figure 4. Oligopeptide labeling reactants. Scheme 13. Labeling of Oligopeptides on Solid Phase

deblocked oligomers with lanthanide(III) salt converts the oligopeptide conjugate to the corresponding lanthanide chelate.

These blocks have been shown to be suitable for solid-phase steroid derivatization also (108).

Reviews


Figure 5. Oligonucleotide labeling reactants. R ) COOMe.

Several nucleosidic and non-nucleosidic building blocks useful for solid-phase oligonucleotide derivatization with lanthanide chelates have been prepared. Illustrative examples are shown in Figure 5. The key step in this labeling reaction is the phosphitylation of the corresponding protected ligand tethered to a hydroxy group. Synthesis of the hydroxy group comprising the precursor has been performed as described above. The chelating carboxylic acid functions were protected with base labile groups compatible with standard oligonucleotide synthesis. Originally, Kwiatkowski et al. synthesized a non-nucleosidic bipyridine derivative 71 that can be coupled to oligonucleotide by phosphoramidite chemistry (111). Later, various nonnucleosidic terbium, dysprosium, samarium, and europium blocks in which the linker was attached at the chromophore have been prepared (28, 112). The non-nucleosidic blocks are suitable for introduction of a single label molecule to the 5′terminus of an oligonucleotide. The first nucleosidic blocks including 72 were published by Kwiatkowski et al. (113). These blocks comprise a chromophore conjugated to uracil and cytosine base via ethynyl moieties. Accordingly, the nucleobase is a part of the chelate chromophore. They can be introduced also within the coding sequence, since the chelates do not restrict the formation of Watson-Crick base pairs. Later, a more versatile method for the preparation of nucleosidic and acyclonucleosidic oligonucleotide building blocks was developed in which the linker is attached at the N3 of uracil and thymine

base using a Mitsunobu reaction (112-116). This approach was applied to multilabeling of oligonucleotides and in the preparation of solid supports that allow 3′-derivatization. Since the labels are attached at sites involved in base pairing, incorporation of these blocks within coding sequences has not been recommended. The oligonucleotide building blocks described in the literature have been introduced into oligonucleotide structures with high efficiency using a modified deprotection protocol (Scheme 14). Hydrolysis of the protecting groups of the chelating moieties was performed by treatment with aqueous sodium hydroxide prior to ammonialysis to avoid carboxamide formation. The lanthanide ion was introduced as a lanthanide citrate in which the lanthanide is chelated and in solution even under basic conditions. It is known that trivalent lanthanide ions promote hydrolysis of phosphoesters very effectively (117). The catalytic efficiency can be attributed to the formation of a metal hydroxide gel, and the catalysis was suggested to be a complicated heterogenic process (118). The lanthanide clusters are formed already in slightly alkaline solutions. Accordingly, it is extremely important that the excess lanthanide ion is not precipitated under the basic conditions needed. It is also known that certain europium chelates based on Schiff bases hydrolyze RNA (119). The catalytic activity is considerably reduced with negatively charged


Hovinen and Guy

Scheme 14. Labeling of Oligonucleotides on Solid Phase

ligands (120). Accordingly, the choice of ligand structure is highly important. Noncovalent Binding. There exist assay formats in which no covalent binding between biomolecule and label is necessary. The example of preparation of nanobeads by swelling chelates into particles was discussed above. As another example, a cytotoxicity assay in which target cells are labeled with the acetoxymethyl ester of 2,2′:6′,2′′-terpyridine-6,6′-dicarboxylic acid has been developed (121). The hydrophobic ligand 76 penetrates easily through cellular membranes of viable cells. Upon the action of intracellular esterases, the acetoxymethyl esters are hydrolyzed, generating impermeable hydrophilic ligands within cells. The ligand leakage upon cell disintegration is monitored by enhancing the supernatant with an excess of Eu3+. Another example of an assay application in which no reactive group is needed is the separation of eosinophilic and basophilic cells (122). In this application, positively and negatively charged chelates bind with negatively and positively charged cell surfaces, respectively. Reaction of the almost nonluminescent chelate 9d (Figure 6) with singlet oxygen converts it to the luminescent endoperoxide 77. This has been utilized in the quantitation of 1O2 generated in various biological systems (38).

of the used biomolecules are almost unchanged. The same strategy has been used also for terbium chelates (124, 125). Very recently, Romieu et al. reported an elegant method for improvement of water solubility of organic dyes (126). The method involves the addition of 3-amino-2-sulfopropanoic acid to dyes tethered to active esters of carboxylic acids. The new labeling reactant formed contains a carboxylic acid group for biomolecule conjugation. Although the applicability of 3-amino2-sulfopropanoic acid to derivatization of lanthanide chelates has not been published, the molecule is worth trying if enhanced water solubility is needed. Since lanthanide chelates are often extremely hydrophilic, their solubilities in organic solvents are low. This is a serious problem when developing luminescent chelates to be polymerized to produce nanobeads. However, certain europium(III) chelates having thienylpyridine subunits have moderate solubil-

SOLUBILITY In general, lanthanide chelates exhibit good water solubility. However, large aromatic chromophores may cause hydrophophic aggregation. The problem has been solved by introduction of hydrophilic substituents to the chelate structure. For example, the aqueous solubility of compound 50 has been enhanced by the addition of COOH (83) or carbohydrate (123) groups to the chromophore (Figure 7). Also, the nonspecific binding of labeled biomolecules is reduced, whereas the affinity properties

Figure 6. Labels for applications where no covalent binding between biomolecule and label is needed.

Reviews

ity in organic solvents such as THF that allow the preparation of the beads (31).

THE LANTHANIDE(III) ION Lanthanide(III) chelates are most commonly prepared by the treatment of the free ligand with a slight excess of lanthanide chloride at pH 6-7. When the chelation is completed, the excess of lanthanide is conveniently removed by increasing the pH of the reaction mixture to ca. 11 where the uncomplexed lanthanide ion precipitates as lanthanide hydroxide (86). Alternatively, the chelate formation is achieved by passing the ligand through a column of strong cation exchange resin loaded with the appropriate lanthanide ion (76). Eu3+, Tb3+, Sm3+, and Dy3+ are the four lanthanide ions that can be used as central ions of luminescent lanthanide chelates. Since there are larger differences in the vibrational levels of the lowest excited state and the highest ground state of Eu3+ and Tb3+ than of Sm3+ and Dy3+, europium and terbium chelates are more efficient light emitters, and thus they are applied more often. The decay times of Sm3+ and especially Dy3+ chelates are short (39, 127, 128). Due to the similarities in the energy levels of Eu3+ and Sm3+, ligands highly luminescent with europium are useful with samarium (129). This is the case with the terbium and dysprosium pair also. For example, electron releasing substituents in the aromatic moiety of phenyl and naphthyl substituted 2,6-[N,N-di(carboxyalkyl)aminoalkyl]pyridines have advantageous effects on the photophysical properties on their chelates with terbium and dysprosium, while the corresponding europium and samarium chelates are practically nonluminescent (131). However, ligands based on 2,6-bis(Npyrazolyl)pyridine and 6,6′-(1H-pyrazole-1,3-diyl)bis(pyridine) have been shown to form luminescent chelates with Eu3+, Sm3+, Tb3+, and Dy3+ ions. Accordingly, these ligands are highly useful for multiparametric homogeneous assays simplifying considerably the preparation of chelates needed in these applications. Also, dual-label assays based on luminescent Eu3+ and Tb3+ chelates of terpyridine have been proposed (82). It has been observed repeatedly that even small modifications in the structure of labeling reagents may cause significant changes in the luminescence properties of the chelate. In this respect, europium is the most robust metal: it is possible to develop assays in which the labeling reagents coupled to a bioactive molecule have luminescence properties almost identical to their parent compounds (40). The use of terbium chelates is more complex due to their greater sensitivity to the environment. Li and Selvin have shown that the emission intensity of certain luminescent Tb3+ but not the corresponding Eu3+ chelates decreases upon conjugation to single-stranded oligonucleotides, and that the signal increases upon hybridization (132). The emission intensity is at its highest for unconjugated chelate. This

Figure 7. Europium(III) chelates with enhanced water solubility.


unique property of Tb3+ chelates has been exploited in the development of homogeneous DNA hybridization assays (133-137). Traditionally, the lanthanide-based assay chemistries have allowed only limited multiplexing since the number of usable chelates is limited to four (chelates of Eu3+, Tb3+, Sm3+, and Dy3+) (138). However, when luminescent lanthanide chelates are combined with different homogeneous FRET techniques, more multiplexed detections can be performed, since the same donor chelate can be exploited for different analytes in the reaction. With appropriate selection of acceptor labels, one can use either the emission of donor itself (TR-FQA) (139) or wavelengths in the Red/NIR region (TR-FRET) (140), or wavelengths shorter than the donor emission spectrum (anti Stokes’ shift TR-FRET) (141) to detect the analyte. In addition, extra signal resolution can be obtained by adjusting the decay time of the acceptor signal (142). Accordingly, by using multiple assay chemistries, a single lanthanide chelate can be utilized for measuring at least five analytes simultaneously.

FREEDOM TO OPERATE Patent literature provides a rich source of information about luminescent lanthanide chelates. Although there are numerous excellent scientific publications, only a small fraction of luminescent lanthanide chelates described in the patent literature have also been described in the non-patent literature. Moreover, many chelates published in the non-patent literature were first described in the patent literature. Thus, a search of chemical literature concerning luminescent lanthanide chelates, as well as many other classes of structures, would be incomplete without a review of published patent applications and patents. Fortunately, there are many public resources available for patent literature searching, and the patent laws of most countries require publication of a patent application as a condition of granting a patent. Typically, patent applications are published about 18 months from the date of filing in a patent office, for the public benefit of describing the invention in a manner sufficient for others to practice it after the term of protection granted with the patent has expired. Useful patent information is available free of charge, for example, from www.espacenet.com. This worldwide database was designed especially for nonprofessionals and offers efficient contextual help and help files. In addition to providing millions of uploadable patents and patent application publications, this resource provides information about the legal status of specific patents, as well as the countries in which patent applications are pending and patents are granted. More detailed information on patents and pending patent application is available at many governmental patent offices, such as WIPO (www.wipo.int), EPO (www.epoline.org). and USPTO (www.uspto.gov), academic Internet sites, professional societies’ sites, and fee-based Internet sources, such as Delphion (www.delphion.com), Micropatent (www.micopat.com), and Nerac, Inc. (www.nerac.com). Briefly turning to the value of patents to their owners, a patent is a form of intellectual property that grants to the owner the right to exclude others from making, using, importing, and selling the claimed invention. Notably, a patent does not give the owner the right to practice the invention. Patent rights are based on laws of the granting country and most often provide 20 years of protection from the first filing date of the application. In most countries, a patent can protect any subject matter that falls into one of the following categories: a process, machine, article of manufacture, or composition of matter, or any new and useful improvement thereof. From a chemistry perspective, in most countries a patent can protect a new and useful compound, a composition containing the compound, an article


containing or made from the compound, a process for synthesizing the compound, and a method that involves using the compound. Certain subject matter is not patentable in various countries due to specific prohibitions in patent laws or as a result of court decisions ruling on these laws. In particular, laws of nature, physical phenomena, scientific principles, abstract ideas, purely mental processes, and mathematical algorithms are typically nonpatentable subject matter. Given the inherent complexities of patents, from selecting a type of patent application, meeting patentability requirements, and complying with rules in patent offices throughout the world to more commercial aspects such as exercising provisional rights, licensing, and enforcing patent rights, this review article mentions a minute fraction of patent-related issues of interest to chemists. Readers are encouraged to pursue additional information, which fortunately is increasingly available as professional societies and publishers of chemical research literature more frequently provide educational information about patents.

CONCLUSION Stable luminescent lanthanide chelates have made homogeneous bioaffinity assays possible. However, the use of stable chelates demands very complicated optimization of the chelate structure. The optimal luminescent lanthanide chelate is stable in the presence of additional chelators even at low pH and high temperature. The ideal chelate has an optimal emission profile, high hydrophilicity, small size, good biocompactibility, little effect on biomolecules, and good energy transfer properties. In addition, the chelate should be compatible with different energy acceptors. The synthesis of the molecule should be simple, affordable, and scalable. Naturally, it is advantageous from a commercial perspective that new luminescent lanthanide chelates are protected by patents in relevant international markets.

ACKNOWLEDGMENT J.H. wish to thank Dr. Ilkka Hemmila¨ (PerkinElmer Life and Analytical Sciences, Turku Site) for valuable discussions.

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Bioconjugation with Stable Luminescent Lanthanide(III) Chelates

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