WO2004092174A1 - Pyrrolopyrroles utilises comme marqueurs fluorescents pour des biomolecules et des particules spheriques - Google Patents

Pyrrolopyrroles utilises comme marqueurs fluorescents pour des biomolecules et des particules spheriques Download PDF

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Publication number
WO2004092174A1
WO2004092174A1 PCT/EP2004/003990 EP2004003990W WO2004092174A1 WO 2004092174 A1 WO2004092174 A1 WO 2004092174A1 EP 2004003990 W EP2004003990 W EP 2004003990W WO 2004092174 A1 WO2004092174 A1 WO 2004092174A1
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group
marker
biomolecule
particle
amino
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PCT/EP2004/003990
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German (de)
English (en)
Inventor
Otto S. Wolfbeis
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Chromeon Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/004—Diketopyrrolopyrrole dyes

Definitions

  • Pyrrolopyrrole as a fluorescent marker for biomolecules and spherical particles
  • the present invention relates to fluorescent marker dyes which are particularly suitable for labeling biomolecules and / or particles.
  • fluorescence labeling of biomolecules plays an important role in bioanalytics and biological research.
  • fluorophores that are non-covalent to biomolecules such as. B. bind proteins or DNA (z. B. intercalators), and those that can be covalently (chemically) bound to biomolecules.
  • One of the most commonly used fluorophores is that of fluorescein. It has the advantage that it can be excited very effectively with the 488 nm line of the argon ion laser, since its absorption maximum in aqueous solution is around 490 nm. Conversely, the availability of markers from the group of fluoresceins has contributed to the fact that the argon ion laser has become a standard tool in fluorescent bioanalytics.
  • fluoresceins have two disadvantages that have not yet been remedied: on the one hand, their fluorescence is strongly pH-dependent, which leads to incorrect measurement results (and thus analyzes) in the case of unexpected pH changes in a test solution, and on the other hand, fluoresceins are relatively photolabile and bleach under longer exposure, so that the fluorescence intensity slowly decreases. This also leads to incorrect results.
  • fluorescent markers A large number of fluorescent markers have already been described. In general, these are fluorescent chromophores that carry a reactive group that forms a chemical (covalent) bond with another functional group of a biomolecule.
  • the general reaction scheme of a covalent label can be shown as follows become:
  • Finen means fluorophore, on which there is a reactive group Jf, which enters into a fine chemical reaction with a second reactive group H1 on a biomolecule or particle.
  • a reactive group Jf which enters into a fine chemical reaction with a second reactive group H1 on a biomolecule or particle.
  • a (fluorescence) dye can be provided with a biotin group. Biotin binds to the proteins avidin or streptavidin (SA) with high affinity (Kd approx. 10 13 mol / L).
  • the biotin (strept) avidin bond can be used to achieve a non-covalent attachment of dyes to biomolecules.
  • Typical examples of the groups X and Y are given in Table 1.
  • a fluorophore F can be introduced into a biomolecule and thus made accessible to a fluorescence-analytical method. These primarily include fluorescence immunoassays, ELISAs, hybridization assays, ligand-receptor interactions, pharmaceutical screening studies and enzyme inhibition tests.
  • Polymeric particles can also be stained via such conjugation reactions if they carry corresponding groups (Y). Alternatively, they can simply be stained with lipophilic dyes that dissolve in the corresponding polymer.
  • Fluorochromes are - preferably in bioanalytics - fluorescence systems that are suitable for the covalent staining of biomolecules.
  • Numerous fluorochromes (with absorption maxima between 300 and 900 nm) are known. In the short-wave spectral range, these are primarily the coumarins, fluoresceins and Bodipy dyes.
  • the chemical structures of each representative of the classes mentioned, namely the Alexa 430 (A), the Oregon Green (B) and a Bodipy dye (C) are shown below.
  • the fluoresceins and many coumarins have the disadvantage of pH-dependent fluorescence.
  • the Bodipy dyes A (described in US Pat. Nos. 6,005,113, 5,433,896, 5,338,854, 5,274,113) are photolabile in aqueous solution and are often hydrolytically unstable.
  • the rhodamines form another group of fluorochromes. They are largely free from the disadvantage of pH sensitivity, but they are compatible with that Argon ion lasers cannot be excited efficiently and they always carry a positive charge on the ring, which causes undesirable electrostatic effects.
  • M stands for O, S, NR or C (CH 3 ) 2
  • n can assume the values 0 to 3, absorb at well over 500 nm, mostly even over 600 nm, and are also positively charged.
  • the new markers should have pH-independent fluorescence and high photostability.
  • the dyes have the basic chemical structure of a pyrrolo (3,4-c) pyrrole, on which there are reactive groups that enable a reaction with biomolecules or with functional surfaces.
  • Biomarkers on the other hand, must be readily soluble, ideally in water, but at least in water-miscible solvents, since biomarkers are almost always carried out in aqueous solution. In addition (and in contrast to color pigments) they must have a high reactivity (even at room temperature), since reactions with biomolecules can practically only be carried out in the temperature range between 0 and 50 ° C. On the one hand, the dyes according to the invention are preferred
  • the pyrrolopyrroles according to the invention form a class of substances which has the following advantages:
  • fluorophores of general type 1 or II are suitable as biomarkers if they are activated by introducing reactive chemical groups in such a way that they are mixed with biomocules in predominantly aqueous solution at room temperature (or only a slightly elevated temperature, for example ⁇ 50 ° C) voluntarily enter into a chemical binding reaction.
  • the compounds are preferably made more soluble by suitable chemical modification, in particular the solubility in water or aqueous solutions is increased. Solubility can e.g. can be increased by alkyl groups or hydroxyalkyl groups on nitrogen.
  • the new biomarkers have absorption maxima between 400 nm and 650 nm, in particular between 400 and 580 nm, and can be produced and used relatively easily as monoreactive fluorochromes for staining biomolecules.
  • Monoreactive means that the dye molecules contain exactly one group which enables a covalent bond or an affinity bond to a biomolecule or polymer particle. Compared to fluoresceins, they have significantly improved photostability and quantum yields, and their absorption and fluorescence spectra are largely pH-independent.
  • the reactive dyes according to the invention have the general chemical structure I,
  • R 1 to R 4 can represent H or any organic substituent, but in particular a linear, branched, cyclic one saturated or unsaturated alkyl radical, a (hetero) aryl radical or a heterocyclic radical, each of which individually with one or more halogen, carboxy, cyano, hydroxy, alkoxy, amino (or substituted amino), aryl, Heteroaryl, sulfo or phospho groups can be substituted, wherein
  • R 1 and R 3 can also represent halogen, CN, COOH, CONR 1 , or COO-alkyl,
  • R 1 and R 2 or R 3 and R 4 can be connected to one another via aliphatic, aromatic or heterocyclic rings, X stands for O, S, C (CN) 2 or NR 5 , where R 5 is for R 1 - R 4 has the meaning indicated, and in which at least one radical R 1 to R 5 represents or contains any group (including a biotin group) which enables covalent labeling or affinity labeling of a biomolecule or a polymer particle.
  • R 1 to R 3 can represent H or any other organic substituent, but in particular a linear, branched, cyclic, saturated or unsaturated alkyl radical, a (hetero) aryl radical, or a heterocyclic radical, each of which is may be substituted with one or more halogen, carboxy, cyano, hydroxy, alkoxy, amino (or substituted amino), aryl, heteroaryl, sulfo or phospho groups, where
  • R 1 and R 2 via aliphatic, aromatic or heterocyclic rings with can be linked to one another, R 1 and R 3 can also represent halogen, CN, COOH, CON (R 1 ) 2 or COO-alkyl, X represents O, S, C (CN) 2 , or NR 5 , where R 5 has the meaning given for R 1 - R 3 ,
  • Y stands for OR 6 , SR 6 , N (R 6 ) 2 , OPOCI 2 , where R 6 is the one given for R 1 - R 3
  • the radicals R 1 to R 4 in compounds of type I and the radicals R 1 to R 3 in compounds of type II can be hydrogen or an organic substituent.
  • the organic substituent preferably has 1 to 30 C atoms, in particular 1 to 20 C atoms.
  • the organic substituent can furthermore have heteroatoms, in particular selected from O, S, N, P and halogens, in particular F, Cl, Br or J.
  • the organic substituent is particularly preferably a linear branched or cyclic, saturated or unsaturated alkyl radical , in particular a Ci to C12, more preferably a C. to C 6 and even more preferably a Ci to C 4 alkyl radical.
  • the organic substituent is furthermore preferably an aryl radical which can contain heteroatoms, in particular O, N, S or P or a heterocyclic radical which can contain heteroatoms, in particular selected from O, N, S and P.
  • the organic substituent in particular in its preferred embodiment as an alkyl radical, (hetero -) aryl radical or heterocyclic radical can be substituted one or more times, for example with halogen, carboxy, cyano, hydroxyl, alkoxy, amino, aryl, heteroaryl, sulfo - or phospho groups.
  • the amino groups can be substituted, for example with one or two alkyl or hydroxyalkyl groups, preferably each with 1 to 6 carbon atoms.
  • the radicals R 2 and R 4 in compounds of type I and the radical R 2 in compounds of type 2 AI are particularly preferred. alkyl radicals or hydroxyalkyl radicals, in particular with 1 to 6 carbon atoms.
  • R 1 and R 2 can also be connected to one another via aliphatic, aromatic or heterocyclic rings.
  • R 1 and R 3 can also be halogen, in particular F, CI, Br or J, for CN, COOH; CON (R 1 ) 2 or COO-alkyl are available.
  • At least one of the radicals R 1 to R 5 in compounds of the formula I or at least one and preferably exactly one of the radicals R 1 to R 3 , R 5 to R 6 or Y of the compounds of the formula II contains a group which is covalent Labeling or affinity labeling of a biomolecule or a polymer particle enables.
  • groups are, for example, carboxylic acid esters, in particular reactive or activated carboxylic acid ester derivatives, maleimides, haloacetyls, sulfochlorides, chlorotriazines, -SCN or -OCN groups, pyrrlium groups, phosphoramidite or biotin or streptavidin.
  • These groups mediate the binding of the dyes according to the invention to the molecules to be labeled, that is to say in particular biomocules or polymer particles, and accordingly contain a unit which can bond with the grouping of the bio-cooler or the polymer particle.
  • the marker dyes according to the invention preferably contain at least one group which increases the solubility of the dyes in aqueous media or water, in particular in media with a water content> 5% by weight, more preferably> 50% by weight ,
  • groups which increase solubility are preferably ionic groups, for example groups which are negatively or positively charged.
  • the dyes preferably contain a sulfo, sulfate, phosphate, phosphonate or quaternized ammonium group.
  • the invention relates to pyrrolo (3,4-c) pyrroles of the typical structure I or 11. They have absorption maxima between 400 and 650 nm and are suitable in reactive form for fluorescent staining of proteins, polynucleic acids and biological receptors, but also of polymer particles. Their fluorescence quantum yields are consistently higher than 0.5. Similarly, fluorescence-labeled proteins, oligonucleotides and particles are also described.
  • the materials stained in this way are preferably excited with violet, blue or blue-green light-emitting diodes or diode lasers for fluorescence and are preferably used in fluorescence-analytical determination methods, eg. B. in immunoassays, in hybridization, in the cytometry, and in pharmaceutical screening.
  • the basic bodies of the fluorochromes according to the invention can be synthesized in various ways. Corresponding general regulations can be found in the above-mentioned patents and in IP Lorenz et al. in Chemistry Eur. J. 8 (2002) 4047-4055 (where metal complexes of diketopyrrolopyrroles are described), and in CJH Morton et al. in Tetrahedron 58 (2002) 5547-5565 (where new and different heterocycles are prepared from diketo-pyrrolopyrroles). Of general importance is the reaction of succinic acid di-tert-butyl ester with a nitrile and subsequent reaction with an amine of the general structure R 4 -NH 2, the products of the general structure 1 are formed. The compounds of type 1 (and derivatives derived from them) serve as the starting material for further syntheses:
  • the red (and green fluorescent) compound 3 can be activated with amino groups directly after activation to the N-hydroxy-succinimide ester (NHS ester) Response.
  • the deep red sulfochloride 4 is an alternative in which the sulfo group causes reactivity to amino groups.
  • the yellow (and blue-green fluorescent) quaternized compound 5 can also be conjugated to amines via an NHS ester.
  • the red isothiocyanate 6 reacts with amines at room temperature to form a thiourea bridge in the sense of
  • the red-violet alcohol 7 shown below can, as will be shown below, be converted into a phosphoramidite ester, which can then be conjugated to the ribose residues of polynucleotides and thus enables their fluorescence labeling.
  • the violet dicyanomethylene derivative 8 all those biomolecules which have an avididn, streptavidin or neutravidine group can be stained via its biotin group, since these have an extraordinarily high affinity for biotin.
  • keto groups in the pyrrolo (3,4-c) pyrroles according to the invention can be converted with Lawesson's reagent into the corresponding thio-pyrrolo (3,4-c) pyrroles of type 9a:
  • any of the above carboxylic acids e.g. 3 or 5
  • DCC dicyclohexylcarbodiimide
  • F stands for the pyrrolo (3,4-c) pyrrolo fluorophore.
  • Compounds 3 and 5 were implemented in this way. From alcohols (eg 8), phosphoramidite II, which is suitable for labeling sugar residues of DNA or RNA, is obtained in turn by reaction with the phosphine I in a manner known per se, as will be described further below.
  • iodoacetyl derivatives which are suitable for dyeing thiol groups can be obtained from alcohols by reaction with iodoacetyl chloride:
  • a special form of activation consists in the reaction of pyrrolo (3,4-c) pyrroles of type 9 (if R 2 is not H) with POCI 3 , which leads to phosphoryl chloride 8c.
  • phosphoric acid esters react directly with amino groups of proteins in the following sense:
  • F / stands for the pyrrolopyrrole fluorophore and Pr for protein.
  • the pyrrolo (3,4-c) pyrroles according to the invention have a significantly improved solubility in all solvents if there are no NH groups.
  • the dyes described in the abovementioned patents also relate primarily to pyrrolopyrroles with free NH groups, since this is particularly advantageous because of their poor solubility with regard to the applications described there.
  • Solubility-improving N-alkyl groups and N-hydroxyalkyl groups can already be introduced during the synthesis of the basic structure, but subsequent alkylation can also take place, e.g. B. with the help of speaking alkyl halides or alkyl tosylates and with the addition of an acid scavenger such. B. potassium carbonate, an amine base or an alcohol.
  • the alkylation brings about a significant improvement in the solubility of the compounds, which makes them only suitable as a biomarker, but can also serve at the same time to introduce functional groups, e.g. B. a carboxy group, a hydroxy group, or another functional or reactive group.
  • the water solubility of the pyrrolo (3,4-c) pyrroles can be further improved if sulfo groups are introduced. This is done either by using sulfonated benzonitriles (or their esters) in the initial synthesis phase, or by reacting aryl-1,4-diketo-pyrrolo (3,4-c) pyrroles by sulfonation with fuming sulfuric acid.
  • a significantly larger Stokes shift ( ⁇ 50 nm) is noticeable in comparison with fluorescein.
  • the molar decadal absorption coefficient is 24,000 (M "1 cm " 1 ).
  • the color of the marker can be varied by other substituents in the para position of the aryl radical, or by replacing the aryl with alkyl or other aryl radicals.
  • the absorption maximum of 1,4-diketo-3,6-bis (4-dimethylaminophenyl) pyrrolo (3,4-c) pyrrole is already at 550 nm. It therefore appears blue-violet in solution. At the same time, the molar absorbance increases to approximately 80,000 M "1 cm " 1 .
  • the dyes according to the invention are outstandingly suitable for bioconjugation.
  • Reactivated carboxylic acids preferably their NHS esters
  • Type 10a thioethers can also be reacted with primary or secondary amines and lead to type 9b products, where R stands for the protein residue.
  • the type II phosphoramidites described above react with a nucleoside in a manner known per se to form the primary conjugate III. After oxidation with iodine, fluorescence-labeled nucleosides of type IV are obtained.
  • ß stands for a nucleobase
  • F for the pyrrolo (3,4-c) pyrrole fluorophore.
  • Unconjugated dye is separated from labeled protein by gel permeation chromatography (Sephadex G25) on a 15 cm column (usually 1 cm) with 22 mM phosphate buffer (pH 7.2) as eluent. The fastest running (orange colored) band contains the labeled protein. The unconjugated excess label is in the slowest moving orange-red zone. 2. Labeling of an amino-modified nucleic acid oligomer with the marker 3c
  • the 15'-amino-modified 15-oligomer 3'-ATA-GTG-TCT-TAG-TAC- (CH 2 ) 6-NH 2 is reacted with the isothiocyanate ⁇ in the following manner:
  • the oligomer (0.2 mg ) is dissolved in 5 mL acetonitrile and heated to 50 ° C. Then a solution of 0.1 mg of the dye in 1 mL acetonitrile is slowly added dropwise. After 1 h, the acetonitrile is evaporated off and the residue is subjected to preparative HPLC (acetonitrile as eluent). Oligomer and residual (excess) dye can be easily detected or separated using the green inherent fluorescence of the conjugate.
  • Porous glass beads (1.0 g; pore size 70 nm; with aminopropyl groups on the surface (40 - 100 ⁇ mol per gram of beads; obtained from Sigma, product no. G-5019) are suspended in bicarbonate buffer solution of pH 8.0. Slowly and A solution of 3 mg of the OSl ester of 5 in DMF is added dropwise with rapid stirring at 50 ° C. After one hour, the yellow-colored glass particles are separated off, washed with copious amounts of distilled water and methanol until no dye is left in the wash water The particles are then dried and stored in a dry state, and the yellow-colored particles have a strong green fluorescence.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

L'invention concerne des pyrrolo(3,4-c)pyrroles représentés par la structure typique (I) ou (II). Ces composés possèdent des maximums d'absorption compris entre 400 et 650 nm et peuvent être utilisés sous forme réactive pour la coloration fluorescente de protéines, d'acides polynucléiques et de récepteurs biologiques, mais aussi de particules polymères. Leurs rendements quantiques de fluorescence sont sans exception supérieurs à 0,5. L'invention concerne également des particules, des protéines et des oligonucléotides marqués en fluorescence au moyen desdits composés. La fluorescence des matières ainsi colorées est provoquée de préférence par excitation au moyen de diodes lumineuses ou de lasers à diodes violets, bleus ou bleus-verts, ces matières pouvant être utilisées de préférence dans des procédés d'analyse par fluorescence, p. ex. dans des dosages immunologiques, des essais d'hybridation, en cytométrie et dans le criblage de médicaments.
PCT/EP2004/003990 2003-04-16 2004-04-15 Pyrrolopyrroles utilises comme marqueurs fluorescents pour des biomolecules et des particules spheriques WO2004092174A1 (fr)

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DE10317817A DE10317817A1 (de) 2003-04-16 2003-04-16 Pyrrolopyrrole als fluoreszente Marker für Biomoleküle und sphärische Partikel
DE10317817.1 2003-04-16

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Cited By (5)

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CN102146217A (zh) * 2010-01-28 2011-08-10 富士胶片株式会社 颜料微粒分散体、光固化性组合物以及该分散体中使用的新化合物
US10513515B2 (en) 2017-08-25 2019-12-24 Biotheryx, Inc. Ether compounds and uses thereof
WO2021064073A1 (fr) * 2019-10-03 2021-04-08 Syngenta Crop Protection Ag Composés herbicides
US11236103B2 (en) 2018-07-27 2022-02-01 Biotheryx, Inc. Bifunctional compounds
US11897930B2 (en) 2020-04-28 2024-02-13 Anwita Biosciences, Inc. Interleukin-2 polypeptides and fusion proteins thereof, and their pharmaceutical compositions and therapeutic applications

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CN113087692B (zh) * 2021-04-02 2022-05-17 广西师范大学 一种同时双色双靶向型荧光探针及其制备方法和应用

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102146217A (zh) * 2010-01-28 2011-08-10 富士胶片株式会社 颜料微粒分散体、光固化性组合物以及该分散体中使用的新化合物
US10513515B2 (en) 2017-08-25 2019-12-24 Biotheryx, Inc. Ether compounds and uses thereof
US10927104B2 (en) 2017-08-25 2021-02-23 Biotheryx, Inc. Ether compounds and uses thereof
US11236103B2 (en) 2018-07-27 2022-02-01 Biotheryx, Inc. Bifunctional compounds
WO2021064073A1 (fr) * 2019-10-03 2021-04-08 Syngenta Crop Protection Ag Composés herbicides
US11897930B2 (en) 2020-04-28 2024-02-13 Anwita Biosciences, Inc. Interleukin-2 polypeptides and fusion proteins thereof, and their pharmaceutical compositions and therapeutic applications

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