Rhodamine derivatives and the use thereof

Details Rhodamine derivatives and the use thereof Rhodamine derivatives and the use thereof

1999-06-02

2001-02-06

Dentz, Bernard (Department: 1612)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C435S006120, C435S071100, C436S546000, C436S800000

Reexamination Certificate

active

06184379

ABSTRACT:

The claimed invention concerns new pentacyclic rhodamine derivatives whose fluorescence emission maxima are in the range of ca. 520 to 720 nm, biomolecules labelled with these derivatives and their use in diagnostic systems.
Numerous rhodamine derivatives are known which can be used as markers (labelled substances) in the form of hydrophilic derivatives with appropriate reactive groups. Such compounds are for example obtained by reacting NHS esters of tetramethylcarboxyrhodamine or carboxy-rhodamine 101 (RHODOS) with proteins containing amino groups, which are used in diagnostic systems. Examples of hapten-fluorescent conjugates are described in EP-A-285179 but the described compounds have absorption maxima at wavelengths of less than 600 nm.
EP 0 543 333 discloses long wavelength rhodamine-like derivatives which have an absorption maximum above 600 nm. However, at the time of the invention a conversion of the described compounds into corresponding phosphoramidites was unknown.
EP 0 567 622 also discloses the preparation of long wavelength rhodamine derivatives with absorption maxima above 600 nm. However, the synthesis of corresponding nucleic acid conjugates and the use of the described compounds as a component of fluorescence resonance energy transfer systems like those that have become established in nucleic acid analysis (Bio Techniques Vol. 22, No.1, p. 130-138, 1997) has not been previously described.
The fluorescent dye fluorescein is widely used because of its spectral properties and its general availability and it is used for a wide variety of applications. In so-called FRET systems fluorescein usually acts as a fluorescence resonance energy donor in which various fluorescence resonance energy acceptors can be potentially used depending on the analytical test procedure. However, the use of FRET systems in nucleic acid analysis has previously been limited to the use of cyanine derivatives such as Cy5 (PCR Methods Appl. 4, 357-362, 1995) and the rhodamine derivatives TAMRA and ROX (Anal.Biochem. 252, 78-88, 1997).
The object of the present invention is therefore to provide new rhodamine derivatives with improved spectral properties in the long wavelength range which are especially suitable for forming a fluorescence resonance electron transfer pair together with other dyes to enable the use of biomolecules labelled with the rhodamine derivatives for analytical or diagnostic purposes.
Hence the invention concerns compounds of the general formulae
in which Ca-Cd each denote a C atom, and Ca and Cb as well as Cc and Cd can either be linked together by a single bond or by a double bond; X1 to X16 denote independently of one another either halogen, sulfonic acid, hydrogen or an alkyl residue with 1-20 C atoms in which the alkyl residue can be substituted with one or several halogen or sulfonic acid residues. R1 and R2 are either identical or different and denote either hydrogen, alkyl with 1-20 C atoms, polyoxyhydrocarbyl units, phenyl or phenylalkyl with 1-3 carbon atoms in the alkyl chain in which the alkyl and/or phenyl residues can be substituted by one or several hydroxy, halogen, sulfonic acid, amino, carboxy or alkoxycarbonyl groups where alkoxy can have 1-4 carbon atoms. R1 contains at least one activatable group. R2 and X4 can be optionally linked together by a bridge composed of 0-2 C atoms. In contrast to the previously known state of the art, these compounds are characterized in that A1, A2 and A3 can independently of one another denote halogen, cyano, hydrogen, carboxylic acid, phosphoric acid or sulfonic acid; B1 denotes either halogen, cyano or hydrogen; and B2 is selected from a group comprising hydrogen, amide, halogen and an alkyl residue with 1-20 C atoms.
It has turned out to be advantageous when at least one residue of the group A1, A2, A3 and B1 represents a halogen and in particular fluorine or chlorine. Preferably all residues A1, A2, A3 and B1 are halogens and in particular fluorine or chlorine. The preparation of such compounds is relatively simple when all residues A1, A2, A3 and B1 are either only fluorine or only chlorine. Compounds are also preferred in which B2 is identical with a hydrogen atom. However, B2 can alternatively also denote fluorine or chlorine, such compounds being particularly simple to synthesize when all the residues A1, A2, A3, B1 and B2 are either only fluorine or only chlorine.
The residues X1, X2, X3, X4, X7, X10, X14 and X15 are preferably hydrogen. The residues X5, X6, X8, X9, X11, X12, X13 and X16 are preferably methyl residues. Those compounds are particularly preferred in which all residues X1, X2, X2, X4, X7 and X10 represent hydrogen residues and all residues X5, X6, X8, X9, X11 and X12 represent methyl residues.
The residues R1 and R2 can either be identical or different but at least R1 contains an activatable group such as amino, hydroxyl or carboxyl. R1 preferably contains exactly one activatable group. In a particularly preferred embodiment R1 is a hydroxyethyl residue or a carboxypropyl residue.
If R2 is different from R1, R2 is preferably hydrogen or an alkyl with 1 to 20 C atoms. It is then particularly preferably hydrogen or an ethyl residue.
The compounds according to the invention provide molecules which, due to their spectral properties (absorption maxima in the range of ca. 500 nm and above and emission maxima in the range between ca. 520 and 720 nm), are very suitable as dyes and in particular as fluorescent dyes. In this connection the spectral properties of the molecules can be changed by modifying the identity, number and position of the halogen residues with regard to A1, A2, A3 and B1. In this manner it is possible to produce fluorescent dyes with almost any absorption and emission maxima in the range between ca. 520 nm and 720 nm. Hence a subject matter of the invention is also the use of the rhodamine derivatives according to the invention as fluorescent dyes.
In order to prepare conjugates which contain the rhodamine derivatives according to the invention, activated derivatives can be synthesized which are for example suitable for labelling biomolecules or other analytical reagents. The activated derivatives are prepared by utilizing at least one of the activatable groups on the residues R1 and R2 and the activation can be carried out by known standard protocols that are known to a person skilled in the art. Usually the activation is achieved by means of at least one hydroxyl group, amino or carboxyl group of R1 and R2.
Various reactive groups can be introduced depending on the later application. Phosphoramidites and H phosphonates can for example be derived from a hydroxyl group. Hence rhodamine phosphoramidites or H-phosphonates are usually prepared by previously known protocols (Methods in Mol.Biol.Vol 20, “Protocols for Oligonucleotides and Analogs, Synthesis and Properties”, S. Agrawal publisher Humana Press Totowa, N.J.).
In contrast, N-hydroxy-succinimide (NHS) esters and maleinimide alkylamides are usually derived from a carboxyl group. NHS esters are preferably prepared by the process described in EP 0 543 333 in which the free carboxylic acid is reacted with NHS in the presence of a condensation reagent such as DCC or MEI. Rhodamine isothiocyanates are preferably prepared by reacting amino groups with thiophosgene (Advanced Organic Chemistry, McGraw Hill, 2
nd
edition, p. 383, 1997).
Hence the invention also concerns activated derivatives of the rhodamines according to the invention. The reactive groups of the activated derivatives are preferably phosphoramidite, N-hydroxy-succinimide (NHS) ester, maleinimide alkylamide, H-phosphonate or isothiocyanate.
The invention additionally concerns conjugates which are obtainable by binding rhodamine compounds according to the invention or their activated derivatives. Consequently such conjugates are composed of at least two components, of which one component represents a rhodamine derivative according to the invention. The conjugates are prepared by standard protocols starting from the activated derivatives. The conjugation methods that are

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