HK1126183B - Polynucleotide labelling reagent - Google Patents

Description

Polynucleotide labeling reagent

The present invention relates to a novel substance in the field of nucleotide chemistry and a process for its preparation. These substances are so-called phosphate mimetics, in which the hydroxyl groups are replaced by the corresponding mimetics.

In particular, the invention relates to labelling reagents for the preparation of a novel class of modified oligonucleotides.

State of the art

Various methods of synthesizing nucleotides or oligonucleotides having modified phosphate moieties are known in the art.

Typically, synthetic (deoxy) oligonucleotides are prepared on solid phases using phosphoramidite chemistry. Glass beads having pores of a certain size (hereinafter abbreviated as CPG ═ pore-fixing glass) are generally used as the solid phase. The first monomer is attached to the support via a cleavable group so that free oligonucleotides can be cleaved off after completion of solid phase synthesis. In addition, the first monomer contains a protected hydroxyl group, in which case Dimethoxytrityl (DMT) is generally used as a protecting group. The protecting group may be removed by acid treatment. Then, at the 5 'end, 3' phosphoramidite derivatives of (deoxy) ribonucleosides, also bearing a DMT protecting group, are coupled successively to the reactive groups released in the DMT protecting group in each case during the cycling. Alternatively, 3 'dimethoxytrityl-protected 5' phosphoramidites are used for reverse phase oligonucleotide synthesis. The H-phosphonate approach is also particularly useful for introducing modifications on the phosphate backbone, for example, to prepare radioisotope-labeled phosphorothioates. Various protocols for preparing modified or labeled oligonucleotides are also known: oligonucleotides labeled at the 3' end were prepared according to the prior art using trifunctional carrier materials (US5,290,925, US5,401,837). Generally, an oligonucleotide labeled at the 5' end is prepared using a labeled phosphoramidite in which a labeling group is bound to the phosphoramidite via a C3-12 linker (US 4,997,928, US5,231,191). Furthermore, modifications can be introduced at each base into the oligonucleotide (US5,241,060, US5,260,433, US5,668,266), or by introducing internal non-nucleoside linkers (US5,656,744, US 6,130,323).

Alternatively, internucleoside phosphates can be labelled by post-synthesis labelling of phosphorothioates (Hodges, r.r. et al, Biochemistry 28(1989)261-7) or by post-labelling functionalised phosphoramidites (Agrawal, s., Methods in mol. biology 26(1994) Protocols for oligonucleotide Conjugates, chapter 3, Humana Press, Totowa, NJ). However, these methods have not been accepted due to the instability of phosphoramidites and thioesters phosphates.

It is also known from the prior art that modifications can be introduced at the internucleoside phosphate residue of an oligonucleotide. The most prominent examples are phosphorothioate (Burgers, P.M., and Eckstein, F., Biochemistry 18, (1979)592-6), methylphosphonate (Miller, P.S., et al, Biochemistry 18(1979)5134-43) or borano (borano) phosphate (WO 91/08213). To prepare methylphosphonate oligonucleotides, specific monomers must be synthesized. In contrast, conventional phosphoramidites or H-phosphonates can be used for the synthesis of phosphorothioates and boranophosphates, in which case the borato or thio modification can be introduced directly during or after oligonucleotide synthesis by using special reagents which react with H-phosphonates or phosphotriesters. Although all of these methods produce modified oligonucleotides, the conditions of the synthetic chemistry used for this do not allow for the introduction of labels or functional groups detectable in the methods directly onto the phosphate backbone of the oligonucleotide chain during oligonucleotide synthesis.

Baschang, G.and Kvita, V., "Angewandte Chemie" 85(1) (1973)43-44 describes the preparation of trialkyl (aryl) iminophosphates by reacting nucleotide phosphotriesters with azides, such as methanesulfonyl azide, which are, however, unstable and decompose.

Nielsen, J. and Caruthers, M.H., J.am.chem.Soc.110(1988)6275-6276 describe the reaction of deoxynucleoside phosphites which are provided with a 2-cyano-1, 1-dimethylethyl protecting group in the presence of an alkyl azide. Furthermore, the authors propose that this principle is suitable for the preparation of nucleotides modified on phosphate residues, but do not state what type of modification prepared using the disclosed method may have particular advantages. In particular, the authors propose the introduction of alkyl residues.

WO 89/09221 discloses N-alkyl phosphoramidites, more precisely oligonucleotides substituted with N-alkyl on at least one phosphate residue, which are prepared by oxidation of a nucleoside phosphite (with a protecting group) with iodine in the presence of a suitable alkylamine.

WO 03/02587 discloses the preparation of modified oligonucleotides in which H-phosphates are changed to phosphoramidates by amination.

Thus, all of these publications describe the preparation of molecules containing phosphoramidate rather than phosphate residues. However, phosphoramidate-containing molecules are susceptible to hydrolysis because the amine groups are protonated in an acidic environment and then replaced by water.

Furthermore, WO 01/14401 proposes a method in which the phosphate residue is substituted by N-ClO₃、N-NO₂Or N-SO₂R substituted nucleotide building blocks or oligonucleotides. According to the teachings of WO 01/14401, the material can be prepared by reacting the free hydroxyl group of a deoxynucleoside with amidophosphonyl chloride in the presence of pyridine. However, this type of preparation is complicated, time-consuming and not suitable for the conventional synthesis of nucleotides or oligonucleotides.

The preparation of Acc azides such as acyl and sulfonyl azides is simple and well known (reviewed: Brase, S. et al, Angewandte Chemie 117(2005)5320-5374, 3.4 and 3.5.2). They are preferably prepared from acid chlorides or sulfonyl chlorides using sodium azide or from hydrazides using nitrous acid.

For example, the dye sulfonyl azide is also used in dyeing processes (e.g. DE 19650252). Cyanogen azide can be easily prepared by reacting sodium azide with cyanogen bromide in acetonitrile (McMurry, J.E., et al, J.organic Chemistry 38(16) (1973) 2821-7). Heteroaryl azides can be prepared by nucleophilic substitution of a halogen with an azide, and can also be prepared from heteroaryl hydrazines. Provided that the electron-withdrawing nitrogen is in the para or ortho position relative to the azide group, as this will only form a resonance stable phosphate mimic. In this regard, ortho-and para-N-alkylpyridinium azides are particularly suitable. Some acyl, sulfonyl and pyridine azides are also commercially available.

It is therefore a technical object forming the basis of the present invention to provide improved labelled oligonucleotides and to provide labelling reagents which can be used in a simple process for their preparation.

Brief description of the invention

Accordingly, the present invention relates to a compound having the chemical structure N₃-SO₂-a reagent of benzene-L-M-X, characterized in that L is a linker, preferably-NH-CO-polyethyleneglycol or-NH-CO- (CH)₂) n and n are natural numbers of 1-18.

M is selected from the group consisting of-NH-, -O-, and-S-,

x is a protecting group or a detectable unit.

In one embodiment, X is selected from DMT, TFA, Fmoc and S-alkyl, wherein alkyl is a chain of 1-6 carbon atoms. Alternatively, X is a labeled compound, such as a fluorescent compound.

In a second aspect, the present invention relates to the use of a compound having X as a protecting group as disclosed above for modifying a nucleic acid, preferably a single stranded oligonucleotide. In a preferred embodiment, the modification comprises the steps of:

-reacting the 3 'phosphoramidite with the 5' OH terminus of the nascent oligonucleotide chain, and

-reacting the intermediate product with a reagent as disclosed above.

In a third aspect, the invention relates to a method for preparing a reagent as disclosed above, characterized in that

Has a chemical formula N₃-SO₂-benzene-NH₂With an activated carbonic acid having the formula a-CO-L-M-X,

wherein

L is a linker, preferably- (CH)₂) n- (n is a natural number of 1 to 18) or polyethylene glycol,

m is selected from the group consisting of-NH-, -O-, and-S-,

x is a protecting group or a detectable unit,

and A is selected from the group consisting of chloride, anhydride and N-hydroxy-succinimide.

Alternatively, according to the invention, the agent may be prepared by reacting a compound of formula N₃-SO₂-benzene- (CH)₂) A compound of n-COCl (n ═ 0-6) and a compound of formula NH₂-(CH₂) M-M-X compound, characterized in that

m is 0 or a natural number from 1 to 10,

m is selected from the group consisting of-NH-, -O-, and-S-,

x is a protecting group or a detectable unit.

Detailed Description

Basic idea of the invention

It is an object of the present invention to provide labeling reagents which can be used to prepare oligonucleotides containing modified phosphate residues, and thus preferably also detectably labeled, in a simple manner.

The central idea of the present invention is to start with a trivalent phosphorus atom and react it with a reagent to form a stable phosphate mimic. According to the invention, for this purpose, the phosphorus atom containing at least one hydroxyl residue with a protecting group is reacted with an azide having the structure N ═ N-Acc (where Acc is an electron acceptor or an electron acceptor substituted with a residue R, R being any organic substituent). This forms a pentavalent phosphorus atom to which a strong electron-withdrawing electron acceptor group is covalently attached via the N atom. This group ensures that the compounds prepared in this way, unlike phosphoramidate compounds known in the prior art, are resonance stable and are not susceptible to hydrolysis.

This idea of the invention can be used in all processes where trivalent phosphorus is formed as an intermediate product.

In conventional oligonucleotide synthesis using phosphoramidites, a phosphotriester having a trivalent phosphorus atom is formed as an intermediate product. The first and second ester bonds represent internucleoside linkages. The phosphorus atom is attached to the protected hydroxyl group via a third ester linkage, for example, to a β -cyanoethoxy group. Instead of oxidation with iodine, according to the invention, the nascent oligonucleotide may be reacted with an appropriate azide during which the trivalent phosphorus atom is oxidized to the pentavalent atom by covalently linking-N-Acc to the phosphorus atom while cleaving nitrogen.

Subsequently, the synthesis of oligonucleotides can be continued as known in the art. Stable oligonucleotides are obtained as end products which are modified in almost any way at one or more internucleotide phosphate residues.

Definition of

Within the scope of the present invention, some of the terms used are defined as follows:

a reactive group denotes a group of a molecule capable of reacting with another molecule under suitable conditions while forming a covalent bond. Examples of reactive groups are hydroxyl, amino, thiol, hydrazine, hydroxyamino, diene, alkyne and carboxylic acid groups.

A protecting group refers to a molecule that reacts with one or more reactive groups of the molecule, such that, as part of a multi-step synthetic reaction, only one particular, unprotected reactive group may react with a target reaction partner. Examples of commonly used protecting groups for protecting hydroxyl groups are beta-cyano-ethyl, beta-cyanomethyl, trialkylsilyl and allyl. Protecting groups used to protect the amino group are trifluoroacetyl and Fmoc. Other possible protecting groups are outlined in standard textbooks (Greene, T.W., Protective groups in organic synthesis, Wiley Interscience publishers, John Wiley & Sons (1981) New York, Chichester, Brisbane, Toronto; Souveaux, E.G., Methods in mol. biology 26(1994) Protocols for oligonucleotide Conjugates, Humana Press, Totowa, NJ, Chapter 1, ed.S. Agrawal).

Linker is defined as a carbon chain of 0-40 carbon atoms in length. The linker chain may also have one or more internal nitrogen, oxygen, sulfur and/or phosphorus atoms. The linker may also be branched, for example also dendritic. The linker connects the nucleotide or nucleotide chain to the detectable unit or a reactive group optionally protected by a protecting group.

In the present invention, the linker preferably has at least 6 atoms. Also preferably, the chain consists of C atoms, which may contain up to 20 heteroatoms. In particular embodiments, the linker may comprise one or more of the following structures:

-NR-(C＝O)-，

-C(＝O)-NR-，

-S(＝O)₂-NR-，

-NR-S(＝O)₂-

(R ═ H or C)₁-C₆Alkyl radical)

Or O-CH₂CH₂-O. Detectable units are understood to mean substances which can be detected by means of analytical methods. For example, they may beTo units for detection by mass spectrometry, immunology or by means of NMR. In particular, the detectable unit is also a substance detectable by optical methods (e.g. fluorescence and UV/VIS spectroscopy), such as fluorescein, rhodamine and gold particles. They also include intercalants and minor groove binders, which also have an effect on the melting behavior and whose fluorescence is altered by hybridization.

Phosphoramidite means a molecule containing a trivalent phosphorus atom that can be coupled to the 5' end of a nucleoside or nucleoside derivative. Thus, phosphoramidites are useful in oligonucleotide synthesis. In addition to the (deoxy) ribonucleotide phosphoramidites used for chain extension, phosphoramidites derivatized with labels are also present, which labels the oligonucleotides in a similar way during or at the end of oligonucleotide synthesis (Beaucage, S.L, Methods in Molecular Biology 20(1993)33-61, ed.S.Agrawal; Wojczewski, C, et al, Synlett 10(1999) 1667-1678).

In the present invention, the term "oligonucleotide" includes not only (deoxy) oligoribonucleotides but also oligonucleotides containing one or more nucleotide analogues with modifications on the phosphate backbone (e.g. methylphosphonate, phosphorothioate), sugars (e.g. 2 ' -O-alkyl derivatives, 3 ' and/or 5 ' aminoribose, LNA, HNA, TCA) or modified bases (e.g. 7-deazapurine). In this regard, the invention also includes conjugates and chimeras comprising non-nucleoside analogs (e.g., PNA) or other biopolymers such as peptides. Furthermore, the oligonucleotides of the invention also contain one or more non-nucleoside units such as spacers, for example hexaethylene glycol or Cn (n ═ 3.6) spacers, at each position.

The term "electron acceptor" includes atomic structures that tend to attract free electron pairs. One measure is the Hammett constant. The Hammett constant σ p involved in the specific embodiment of the invention exceeds a certain value of 0.30, preferably 0.45, particularly preferably 0.60.

In addition, the electron acceptor must be compatible with all chemical reactions in oligonucleotide synthesis, i.e.

It should not be oxidized by iodine

It must be inert to dichloroacetic acid and trichloroacetic acid, and

it must be inert to bases, in particular to ammonia

It should not react with trivalent phosphoramidates.

Examples of electron acceptors that satisfy these conditions are:

-NO₂、SO₂-R, -CN, -CO-R, pyrinidinyl, pyridyl, pyridazinyl, hexafluorophenyl, benzotriazolyl (Hansch, C, et al, chem. reviews 91(1991) 165-. In addition, these acceptors can also be bound to the nitrogen atom in the form of a vinyl or phenyl group.

The term "substituted" means that the substituted structure contains another residue at any position, provided that the position is not defined in more detail. The term "optionally substituted" means that the structure referred to in this manner includes embodiments with or without additional residues.

The term "amino-substituted alkyl" includes C₁-C₃₀A linear or branched alkyl group containing at least one amino group, wherein the amino group is protected or bound to a detectable unit by a linker.

The term "electron deficient six membered N-heterocyclic ring" includes N-heterocyclic rings that are alkylated at the sp2 nitrogen such that the total charge of the heterocyclic ring is positive. Examples thereof are pyridinium, pyrimidinium and quinolinium.

The term "nucleotide chain" is understood to mean a molecule or part of a molecule comprising at least two nucleotide residues which are 5 '-3' interconnected by a phosphate moiety.

The chemical labeling reagent of the present invention

The invention relates to a compound having the chemical structure N₃-SO₂-benzene-L-M-X reagent, characterized in that

L is as aboveA defined linker structure. In one embodiment, L is preferably-NH-CO-polyethylene glycol or-NH-CO- (CH)₂) n and n are natural numbers of 1-18. In another embodiment, L is-CO-NH-polyethylene glycol or-NH-CO- (CH)₂) n and n are natural numbers of 1-18.

M is selected from the group consisting of-NH-, -O-, and-S-,

x is a protecting group or a detectable unit.

The moiety attached to benzene is disposed in either the meta or para configuration. Preferably, they are in the para configuration. Further, the benzene may be substituted at one or more positions with a non-bulky substituent such as a halogen (e.g., chlorine).

In which L is- (CH)₂) In the case of n-, n is a natural number from 1 to 18, preferably from 2 to 12, most preferably from 3 to 6.

In the case where L is polyethylene glycol, the preferred chain length may be 2 (diethylene glycol) -6 (hexaethylene glycol).

Preferably, M is-NH-.

In case X is a protecting group, it is selected from DMT (dimethoxytrityl), TFA (trifluoroacetyl), Fmoc ((fluoren-9-yl) methoxycarbonyl), and-S-alkyl, wherein the chain length of the alkyl group is 1-6 carbon atoms. Depending on the nature of M, different protecting groups are chosen. TFA and Fmoc are highly preferred for-NH-, and are particularly preferred. for-O-and-S-, DMT is suggested. for-S-, an-S-alkyl group may be used.

Where X is a detectable unit, the detectable unit may be a colored labeling dye, such as a fluorescent label. Further examples are mass labels, haptens (such as digoxgenin or Biotin), or small peptides, all of which are detectable by antibodies. Preferably, X is a fluorescent compound, such as fluorescein or any other fluorescent dye used for real-time PCR.

It is noted that the definitions of X, M and L and the specific embodiments disclosed are also used to disclose the following sections: "Synthesis of the chemical labeling reagent of the present invention", "preparation of the oligonucleotide of the present invention", and "oligonucleotide produced with the labeling reagent of the present invention".

Synthesis of the chemical labeling reagent of the present invention

The invention also provides a simple and direct method for synthesizing the compounds of the invention.

Has a chemical formula N₃-SO₂-benzene-NH₂Can be prepared by standard methods in the art from commercially available, reasonably inexpensive N₃-SO₂-benzene-NH acetyl is readily available.

The compound is reacted with an activated carbonic acid having the formula A-CO-L-M-X

Characterized in that A is selected from the group consisting of chloride, anhydride and N-hydroxysuccinimide (NHS ester). Preferably, A is N-hydroxysuccinimide.

L is a linker, preferably- (CH)₂) n-or polyethylene glycol. In which L is- (CH)₂) In the case of n-, n is a natural number from 1 to 18, preferably from 2 to 12, most preferably from 3 to 6. In the case where L is polyethylene glycol, the preferred chain length may be 2 (diethylene glycol) -6 (hexaethylene glycol).

M is selected from the group consisting of-NH-, -O-, and-S-,

x is a protecting group or a detectable unit as disclosed above.

Some of the compounds covered by the general structure A-O-CO-L-M-X are also commercially available as reagents to provide for post-synthetic labeling of oligonucleotides. In particular, fluorescent dyes are often provided in the form of NHS esters, as is the case with LC Red 640 and LC Red 610 (Roche Applied Science Cat. Nos: 12015161001, 03, 03561488001). However, in the present invention, the compound is first reacted with N₃-SO₂-benzene-NH₂Reaction such that the compound is then available as an azide. The azide can then be used to introduce a label already during oligonucleotide synthesis, as described below.

Alternatively, N can be synthesized according to the methods described above₃-SO₂-benzene-L-NH-X (X as protecting group) and then deprotecting X to synthesize the labeling reagent, wherein X must be selected from protecting groups that can be removed under conditions reasonably stable to azide. Suitable protecting groups are trityl, Boc and phenylacetyl. Then, the obtained N₃-SO₂-benzene-L-NH₂Can be reacted with an activated ester of a detectable group, for example, with a NHS ester of a commercially available dye.

In another aspect of the invention, the synthesis may begin with compound N₃-SO₂-benzene- (L) n-C (═ O) Cl, n ═ 0-1. The compounds are readily synthesized from alkylsulfonyl chlorides substituted with carboxylic acids, which may be attached to the aromatic ring either directly or through a linking moiety. Commercially available compounds are for example Cl-S (═ O)2-Ph-COOH or Cl-S (═ O)2-Ph- (CH2)2 COOH. The sulfonyl chloride is transferred to the sulfonyl azide by reaction with sodium azide and the carboxylic acid can then be converted to the acid chloride by standard methods (e.g., FR 1455154). Alternatively, N3-S- (═ O)2-Ph-NH2 can be reacted with a dicarboxylic anhydride to produce N3-S (═ O)2-Ph-NHC (═ O) (CH2) COOH, and the carboxylic acid converted to the acid chloride by standard methods. Then, compound N₃-SO₂-benzene- (L) n-C (═ O) Cl can be reacted with NH2-L-M-X, which is commercially available, or can be easily prepared by reacting NHs esters with excess NH₂-L-NH₂Synthesis of NHS ester reacted from detectable groups.

Preparation of oligonucleotides of the invention

In general, the invention relates to labeling reagents for the preparation of modified oligonucleotides characterized by trivalent phosphorus derivatives of the following chemical structure

Wherein PG represents a protecting group

A represents the 5' end of the nucleotide or nucleotide chain, or it represents a linker bound to a solid phase,

b represents the 3' end of the nucleotide or nucleotide chain, or it represents a linker

And the following structure N₃-SO₂-azide reaction of benzene-L-M-X, characterised in that

L is a linker, preferably-NH-CO-polyethylene glycol or-NH-CO- (CH)₂) n and n are natural numbers of 1-18.

M is selected from the group consisting of-NH-, -O-, and-S-,

x is a protecting group or a detectable unit.

Beta-cyanoethyl, methyl, alkyl or silyl groups are particularly preferred as Protecting Groups (PG). Alternatively, methylphosphonates may be prepared according to the invention, wherein-O-PG is CH₃And (4) replacement.

The method of the invention can also be used in general, in particular, in conventional oligonucleotide synthesis. Accordingly, the present invention also relates to a method comprising the steps of:

a) reaction of the 3 'phosphoramidite with the 5' OH terminus of the nascent oligonucleotide chain

b) And the following structure N₃-SO₂-azide reaction of benzene-L-M-X, characterised in that

L is a linker, preferably-NH-CO-polyethylene glycol or-NH-CO- (CH)₂) n and n are natural numbers of 1-18.

M is selected from the group consisting of-NH-, -O-, and-S-,

x is a protecting group or a detectable unit.

In this case, the 5 ' OH terminus of the nascent oligonucleotide strand may be the 5 ' terminus of the 5 ' terminal nucleotide or the free OH group of the CPG.

Conventional oligonucleotide chemistry starts on a reactive solid support material. Solid support material means a polymer forming a solid phase containing reactive groups on which other molecules can be immobilized. In the case of oligonucleotide synthesis, the support material is usually porous glass beads with a defined pore size, so-called fixed pore glass particles (CPG). Alternatively, polystyrene residues and other organic polymers and copolymers (Ghosh, P.K., et al, J.Indian.chem.Soc.75(1998)206-218) may also be used. Glass and semiconductor chips can be used as solid support materials if the oligonucleotides remain immobilized after synthesis on a substrate. The solid phase support materials are commercially available.

The support may be attached to the terminal reactive hydroxyl residue protected by a protecting group, such as DMT (dimethoxytrityl), via a so-called linker group containing a cleavable bond. Linker groups with cleavable bonds represent groups between the trifunctional spacer and the solid support material and which can be cleaved by simple chemical reactions. They may be succinyl or oxalyl or other linker groups containing a cleavable ester bond. Other linker groups are known to those skilled in the art (Ghosh, P.K., et al, J.Indian.chem.Soc.75(1998)206- & 218).

The linker group is essential for the synthesis of oligonucleotides intended to remain in aqueous solution after completion of the synthesis using a support material. If, in contrast, for the preparation of nucleic acid arrays, the oligonucleotides should remain on the surface of the support material after synthesis (U.S. Pat. No. 5,624,711; Shchepinov, M.S., et al, Nucl. acids. Res.25(1997)1155-1161), a cleavable linker group is not necessary, but preferably a non-cleavable linker group.

The synthesis of oligonucleotides incorporating the structures of the invention is detailed below:

after removal of the DMT protecting group by acid treatment, a reactive hydroxyl group on which chain extension in the 3 '-5' direction can occur is formed. Then, the 3 'phosphoramidite derivative of a (deoxy) ribonucleoside, also bearing a DMT protecting group, is coupled sequentially at the 5' end to the reactive group without the DMT protecting group in the presence of tetrazole. In this process, an intermediate product containing trivalent phosphorus atoms is formed, which forms ester bonds with the individual nucleosides linked together by the reaction and a third ester bond with the protected hydroxyl groups already present in the phosphoramidite used. After completion of the oligonucleotide synthesis, the protecting group, which may be formed, for example, by β -cyanoethyl, methyl, allyl or silyl groups, is then cleaved with ammonia, during which the base protecting group and the linker of the CPG are also cleaved.

Without iodine oxidation, according to the invention, nascent oligonucleotides are of the following structure N₃-SO₂-azide reaction of benzene-L-M-X, characterised in that

L is a linker, preferably-NH-CO-polyethylene glycol or-NH-CO- (CH)₂) n and n are natural numbers of 1-18.

M is selected from the group consisting of-NH-, -O-, and-S-,

x is a protecting group or a detectable unit.

At the position where the phosphate ester mimic will be introduced into the nucleotide chain.

In particular, if X is a protecting group, X may be removed after oligonucleotide synthesis and post-oligonucleotide synthesis labeling with a reactively detectable unit is performed according to methods well known in the art.

However, preferably X is a detectable unit, such as a fluorescent molecule, such that labeling of the nascent oligonucleotide has occurred during phosphoramidite-based oligonucleotide synthesis.

Certain embodiments of the present invention relate to the preparation of dual labeled oligonucleotide probes, wherein one label is preferably incorporated internally into the oligonucleotide according to the methods of the present invention, and another label is incorporated into the oligonucleotide according to methods known in the art, preferably at the 5 'or 3' end.

In the case of 5 ' labeling of the ribose at the 5 ' position of the 5 ' terminal nucleotide, binding is carried out by conventional Methods by using a dye-labeled phosphoramidite at the end of oligonucleotide synthesis (Beaucage, S.L, Methods in Molecular Biology 20(1993)33-61, S.Agrawal Publishers).

The 3' end labeling was performed using a commercially available CPG as a reactive solid support that already contained a detectable label in addition to the tritylated hydroxyl group. After cleavage of the DMT protecting group, standard oligonucleotide synthesis can be started at the now free hydroxyl group.

Alternatively, post-labeling methods known in the art can be used for additional 5 'or 3' labeling (US5,002,885; US5,401,837).

The invention also relates to synthetic intermediates of the invention, which can be prepared prior to standard oligonucleotide synthesis. In this case, preference is given to intermediates which remain bound to the solid phase, have not been deprotected and may contain base-free spacer groups. CPG, which is known to those skilled in the art as phosphate CPG, is preferably used for the preparation, since 3' phosphorylated oligonucleotides are formed after the synthesis of the oligonucleotides. After detritylation, the phosphate CPG is reacted with a spacer phosphoramidite in the presence of an activator: the resulting trivalent phosphorus intermediate is then reacted with N as disclosed above₃-SO₂-benzene-L-M-X reaction, wherein X is a detectable unit. These synthetic intermediates can be stored and used for general 3' labeling as trifunctional CPG.

The phosphorous intermediate is also formed during the synthesis of methylphosphonate, which can be reacted with the azide of the present invention. Methylphosphorous acid amides are also commercially available.

In a reverse phase synthesis scheme (EP 1155027) for standard oligonucleotides and in particular for the synthesis of analogs, for example for N3 '- > P5' oligonucleotides, trivalent phosphorus-containing intermediates are also formed which can be reacted according to the invention with the azides of the invention. The corresponding phosphoramidites are commercially available.

Oligonucleotides produced using the labeling reagents of the invention

The synthetic scheme of the present invention allows for the preparation of a variety of oligonucleotides modified on the phosphate backbone. The degree of modification, the diversity of modification and the charge are determined according to the intended use.

The present invention includes any compound comprising at least once the following structure

Wherein

A represents a nucleotide or the 5' end of a nucleotide chain, or OH

B represents a nucleotide or the 3' end of a nucleotide chain, or OH

Acc represents-SO₂-benzene-L-M-X, characterized in that

L is a linker, preferably-NH-CO-polyethylene glycol or-NH-CO- (CH)₂) n and n are natural numbers of 1-18.

M is selected from the group consisting of-NH-, -O-, and-S-,

x is a protecting group or a detectable unit.

It will also be appreciated by those skilled in the art that the hydroxyl groups of the oligonucleotide are typically present in a deprotonated state.

In addition, the present invention includes methylphosphonates of the structure

Depending on the intended use of the oligonucleotide, the above structure may occur once, twice, multiple times or even on all phosphate residues present on the oligonucleotide. The phosphate residue within the oligonucleotide is a so-called internucleoside phosphate, wherein

A represents the 5' end of the first nucleoside,

b represents the 3' terminus of the second nucleoside within the nucleotide chain.

Furthermore, the structures of the invention may be located at the 3 'end or the 5' end of the oligonucleotide. If they are present at the 5' end of the oligonucleotide, then

A represents the 5' end of the nucleotide chain,

b is a hydroxyl group or a linker that may optionally contain a detectable group or another reactive group, and may be used to synthesize a detectable group on the oligonucleotide.

If the electron acceptor contains a substituent which also represents a detectable unit, the oligonucleotide of the invention is present with a double label at the 5' end. If the structure of the present invention is located at the 3' end of the nucleotide chain, then

B represents the 3' end of the oligonucleotide

A is a hydroxyl group or a linker bound to a solid phase, preferably a fixed pore glass particle, e.g.used as a starting material for conventional oligonucleotide synthesis.

Each nucleoside in the oligonucleotide of the invention may comprise any type of nucleoside or modified nucleoside or nucleoside derivative. The sugar units are typically deoxyribose of a DNA oligonucleotide or ribose of an RNA oligonucleotide. The nucleobases comprised in the oligonucleotides of the invention may be naturally occurring bases such as adenine, guanine, thymine, cytidine, uridine, derivatives thereof or so-called universal bases such as nitroindole.

Oligonucleotides labeled with the labeling reagents of the invention can be advantageously used in many different applications in molecular biology, such as real-time PCR. The detectable label is preferably a fluorescent dye or a fluorescence quenching molecule. Corresponding dyes and molecules which can be used as detectable units of oligonucleotides are well known to the person skilled in the art. Examples of dyes and molecules which do not limit the scope of the present invention are: fluorescein, rhodamine, cyanine, merocyanine, carbocyanine, and azo and polyazo compounds.

The labeling reagent of the present invention can be used for synthesizing a real-time PCR probe having the structure as described above, wherein at least one fluorescent label is bound to the phosphate atom of the oligonucleotide chain through an amide/electron acceptor group. Examples of such probes are FRET hybridization probes (WO97/46707) or so-called single-labeled probes (WO 02/14555). In this regard, particularly preferred are oligonucleotide probes in which the internal modification of the present invention is present on the internucleoside phosphate residue.

In this respect, the labeling reagents of the present invention are particularly useful for preparing ditag oligonucleotides having two detectable units. Examples of such probes are TaqMan probes (U.S. Pat. No. 5,804,375), molecular beacons (U.S. Pat. No. 5,118,801). In this aspect, the invention relates to the preparation of a ditag oligonucleotide, wherein a first fluorescent label is bound to the internucleoside phosphate atom of the oligonucleotide chain via an amide/electron acceptor group and a second detectable unit is present at the 5 'terminus or the 3' terminus of the oligonucleotide. Molecules with such labels and methods for their preparation are well known to the expert.

The invention is illustrated in more detail by the following examples, the scope of protection of which derives from the patent claims. The method is to be understood as an example that describes the object of the invention even after changes.

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 to be understood that variations may be made in the methods listed without departing from the spirit of the invention.

Examples

Example 1: 4-Aminobenzenesulfonyl azide (according to DE 2919823)

2,0g (8mmol) of p-acetamidophenylsulfonyl azide was stirred with 4,0g (3.4mL) of 32% hydrochloric acid and heated to 95 ℃ over 20 minutes. A clear solution was formed. Upon cooling to room temperature, the hydrochloride precipitated and was isolated by filtration. The crystals were dissolved in 20ml of water, saturated sodium carbonate solution was added, and then the aqueous phase was extracted three times with dichloromethane. The combined organic phases were washed with water and dried over sodium sulfate. After removal of the solvent, the resulting pale red oil was dried over calcium chloride in vacuo overnight to yield 1.04g (66%) of crude product.

TLC silica (toluene: ethyl acetate: methanol 4: 1) on a plastic slide Rf (product) 0.63,

example 2: (2, 2, 2-trifluoro-acetylamino) -hexanoyl chloride

735mg (3mmol) (2, 2, 2-trifluoro-acetylamino) -hexanoic acid was dissolved in 5mL dry dichloromethane and stirred under a flow of argon at 0 ℃. Then, 0.6mL (6.6mmol) of oxalyl chloride was added dropwise at 0 deg.C, and also several drops of dry dimethylformamide were added dropwise, gas was generated, and the white mixture turned yellow before. The mixture was stirred at room temperature for 70 minutes. The mixture was then evaporated repeatedly with dichloromethane. The crude product was used directly in the next step.

Example 3: 4- [6- (2, 2, 2-trifluoro-acetylamino) -hexanoylamino ] -benzenesulfonyl azide

To 0.57g (3mmol) of sulfanyl azide in 5mL of dimethylformamide was added 0.9mL (6mmol) of triethylamine, followed by dropwise addition of (2, 2, 2-trifluoro-acetylamino) -hexanoyl chloride in 10mL of dimethylformamide. The mixture turned brown and was stirred under argon for 24 hours. The solvent was evaporated, the remaining oil was dissolved in 10mL of dichloromethane and left at room temperature for 1 hour. The precipitate was filtered and the solution was evaporated, yielding 1.10g of crude oil. The obtained compound was used directly for oligonucleotide synthesis.

Example 4: synthesis of amino-modified oligonucleotides

5′Ap^*GG GAT CTG CTC TTA CAG ATT AGA AGT AGT CCTATT-p

p^*＝p＝N-SO2-Ph-NH-C(＝O)-(CH2)5-NH₂

Oligonucleotide synthesis was performed on a 1 μmol scale on an ABI 394 synthesizer. Commercially available phosphate ester CPG (Glen Resera) is used as the support material. All other chemicals used for standard synthesis were obtained from Glen Research. Phosphoramidites from Prooligo with a tert-butylphenoxy-acetyl protecting group (known as "tac" or "Expedite" monomers) were used.

Standard protocols were used for the synthesis. Only in the last synthesis cycle, the oxidant was replaced by a 0.1M solution of 4- [6- (2, 2, 2-trifluoro-acetylamino) -hexanoylamino ] -benzenesulfonyl azide in anhydrous acetonitrile and the "oxidation" time was extended to 16 minutes.

The product was cleaved from the support with 33% ammonia at 55 ℃ and purified by reverse phase chromatography on a Poros Oligo R34.6x50mm column. Chromatography: and (3) buffer solution A: triethylammonium acetate in 0.1M water, pH 6.8, buffer B: 0.1M Triethylammonium acetate in water/acetonitrile 1: 1, gradient 2 min, rising from 0% B to 100% B in 45 min. The UV absorption of the eluent was measured at 260 nm. The major portion of the oligonucleotide containing the amino modification was obtained. The solvent was removed on a vacuum centrifuge.

MALDI: calc: 11436.7 found 11435.6

Example 5: post-labelling with LightCycler Red 640

5′Ap^*GG GAT CTG CTC TTA CAG ATT AGA AGT AGT CCTATT-p

p^*＝p＝N-SO2-Ph-NH-C(＝O)-(CH2)5-NH LC Red 640

The residue was taken up in 1ml of 0.1M sodium borate buffer (pH 8.5). A solution of 1mg LightCycler Red 640 NHS ester (Roche Applied Science) in 1000. mu.l DMF was added and the mixture was kept at room temperature overnight. The mixture was evaporated under the same conditions as described above and purified. Detection was performed at 260nm and 625nm using a diode array as the detector. The fractions with double absorption were collected and evaporated in vacuo. The residue was dissolved in double distilled (bidest.) water and evaporated again in vacuo. The residue was then dissolved in double distilled water and lyophilized.

MALDI: calc: 12438.35 found 12435.5

Example 6: n-dansyl 3-aminopropionyl chloride

970mg (3mmol) dansyl beta alanine (alanin) was dissolved in 5mL dry dichloromethane and stirred under a flow of argon at 0 ℃. Then, 0.6mL (6.6mmol) of oxalyl chloride was added dropwise at 0 ℃ followed by addition of a few drops of dried dimethylformamide to generate a gas. The mixture was stirred at room temperature for 100 minutes. The solvent was removed using a rotary evaporator and the residue was then evaporated twice with dichloromethane. The crude product was used directly in the next step.

Example 7: n-dansyl (3-aminopropionyl) benzenesulfonyl azide

To 152mg (0.8mmol) of p-aminobenzenesulfonylazide dissolved in 5mL of dimethylformamide was added 0.11mL (6mmol) of triethylamine, followed by dropwise addition of 0.5mmol of N-dansyl 3-aminopropionyl chloride dissolved in 10mL of dimethylformamide. The mixture was stirred under argon for 24 hours. The solvent was evaporated and the remaining oil was purified by chromatography on silica (eluent toluene/ethyl acetate 1: 1). The product containing fractions were collected and the solution was evaporated.

1H NMR (Bruker DPX 300 MHz): d6 DMSO: 2.53 m 2H, 2.82s 6H, 3.12 m 2H, 7.20 m 2H, 7.59 t 1H, 7.62 t 1H, 7.81 d 2H, 7.93 d 2H, 8.07 t 1H, 8.12 d 1H, 8.27 d 1H, 8.44[1H ], 10.47 s 1H, (protonated form) IR (nujol)2122 cm-1

Claims (4)

1. Has a chemical structure N₃-SO₂-a reagent of-benzene-L-M-X, characterized in that L is-NH-CO-polyethylene glycol or-NH-CO- (CH)₂)_nN is a natural number of 1 to 18,

m is selected from the group consisting of-NH-, -O-, and-S-,

x is selected from DMT, TFA, Fmoc and S-C_1-6-an alkyl group.

2. Use of a compound of claim 1 for modifying nucleic acids.

3. The use of claim 2, wherein the nucleic acid is a single stranded oligonucleotide.

4. The use of claim 2 or 3 for modifying single stranded oligonucleotides comprising the steps of:

-reacting the 3 'phosphoramidite with the 5' OH terminus of the nascent oligonucleotide chain,

-reacting with the reagent of claim 1.