EP1480969A1 - Dithiolane derivatives for immobilizing biomolecules on noble metals and semiconductors - Google Patents

Abstract

The invention relates to dithiolane derivatives of the formula (A), and to conjugates from said dithiolane derivatives with organochemical or biological-chemical molecules. The invention further relates to a method for producing said dithiolane derivatives and conjugates, to a coated noble metal or semiconductor structure that comprises a noble metal-coated substrate or a semiconductor substrate and a diothiolane derivative or inventive conjugate immobilized thereon, to a biochip that contains the inventive noble metal or semiconductor structure and to the use of the inventive dithiolane derivatives for immobilizing organochemical or biological-chemical molecules on noble metals, noble metal alloys or semiconductors.

Description

 Dithiolane derivatives for immobilizing biomolecules on precious metals and semiconductors

The present invention relates to dithiolane derivatives, conjugates of the dithiolane derivatives with organic chemical or biochemical molecules, processes for producing the dithiolane derivatives and the conjugates, a coated noble metal or semiconductor structure which comprises a noble metal coated substrate or a semiconductor carrier and a dithiolan derivative immobilized thereon or a conjugate according to the invention, a biochip which contains the noble metal or semiconductor structure according to the invention and the use of the dithiolan derivatives according to the invention for immobilizing organochemical or biochemical Molecules on precious metals, precious metal alloys or semiconductors.

The chemistry of anchoring biological-chemical molecules, especially oligonucleotides, to surfaces plays a crucial role in many analytical and biomedical applications. This applies, for example, to the production of oligonucleotide libraries on flat material carriers ("oligonucleotide chips" or "DNA microarrays" or "oligonucleotide microarrays"), which have become indispensable tools in modern biomedical diagnostics ( Niemeyer et al. (1999) Angew. Chem. 19: 3039-3043), but also for biomolecules that are immobilized on different types of, for example, particulate, in particular spherical, carriers. It is not relevant whether the biomolecules such as oligonucleotides from monomeric or oligomeric synthons 1968

are assembled on the surface or whether they are applied to the surface after the synthesis as completed chains of the monomeric units, for example as oligonucleotide chains. It is crucial, however, that the anchoring on the surface is stable and functional, i.e. It is ensured, for example, that the immobilized organic-chemical or biological-chemical molecules, such as oligonucleotides, peptides, etc., do not detach even under repeated or multiple use under the conditions of use. On the other hand, however, in some applications it is desirable that the grafted (immobilized) molecule e.g. can also be split off for the purpose of analysis under certain conditions.

A large number of possibilities for producing biomolecules, in particular oligonucleotides, applied to glass, plastics and other non-metallic supports have already been described in the prior art (Wölfl (2000) BioChip Technologies, Laboratory World, Transcript 3: 12-20). Of particular interest are Oligonucleotide chips on precious metal, preferably gold, surfaces due to their simple handling and the possibility of producing so-called "self-assembled monolayers" (SAM), which enable efficient control of the density of the immobilized molecules, which results in a high signal intensity. can be achieved. These SAMs of organic molecules on noble metal surfaces are usually generated by reaction with thiols (Bain et al. (1988) J. Am. Chem. Soc. 111: 321-335), which in turn, as anchor groups, immobilize the desired molecules such as, for example, Mediate oligonucleotides (Herne et al., J. Am. Chem. Soc. 119: 8916-8920).

The binding energy of the interaction of elemental gold with (alkane) thiols is of the order of about 30 to about 40 kcal / mol (Nuzzo et al. (1986) J. Am. Chem. Soc. 109: 2358-2368; Nuzzo et al. (1990) J. Am. Chem. Soc. 112: 558-569). However, the interaction of gold and other noble metals with vicinal dithiol compounds should be considerably more stable, since the binding energy per molecule increases here (Wang et al. (1998) J. Phys. Chem. B, 102: 9922-9927; Cheng et al. (1995) Anal. Chem. 67: 2767-2775). Letsinger et al. (Bioconjugate Chem. 11: 289-291, 2000) describe a vicinal dithiol compound and its interaction with gold surfaces. However, the anchor group is a synthetic steroid derivative that is difficult to access. When such sterically problematic groups are used, it is hardly possible to produce SAM on the noble metal surface, which is why the compounds described in this prior art were not used to produce biochips.

Some lipoic acid constructs are known in the prior art which are used in particular for immobilizing biomolecules on gold surfaces; see. e.g. Madoz et al. (1997) J. Am. Chem. Soc. 119: 1043-1051; Blonder et al. (1998) J. Am. Chem. Soc. 120: 9335-9341; Stevenson et al. (2002) J. Nanosci. Nanotech. 2; 397-404; Kim et al. (2002) Langmuir 18: 1460-1462. However, these lipoic acid conjugates have significant disadvantages. Et al the (bio) molecule to be conjugated with the lipoic acid derivative must first be derivatized accordingly in order to make it accessible for reaction with the lipoylation reagent. In addition, it is not possible with the known lipoylation reagents to introduce lipoic acid residues into any amine or hydroxyl groups using standard chemical methods.

Holupirek (dissertation, University of Ulm, section Polymers, 1976) describes the possibility of attaching lipoic acid as a substituent to oligonucleotides and using these substituents to purify the target oligonucleotide from the product mixture of solid-phase synthesis via affinity chromatography on organic mercury columns. However, the method proved to be impractical, which was due in particular to the very inefficient introduction of the lipoic acid residue into oligonucleotides. In addition, regeneration of the lipoylated oligonucleotide was difficult after affinity purification.

It is therefore the object of the present invention to provide compounds which are particularly suitable for the formation of SAM on precious metal or semiconductor surfaces. to provide gene that can be easily and efficiently introduced into the organic chemical or biological chemical molecules to be immobilized.

This object is achieved by the embodiments of the present invention characterized in the claims.

In particular, a dithiolane derivative of the formula A

Formula A

provided.

In the dithiolane derivative of the present invention, a dithiolane ring is covalently linked via a bridge member R ¹ to a carbonyl (X = 0) or sulfonyl (X = S) function derived from an acid residue. The bridge link R ¹ does not have to be present, but can consist of an optionally substituted or unsubstituted aliphatic or heteroaliphatic chain, which can be saturated or unsaturated, straight-chain or branched, but can also be an aromatic radical or contain aromatic radicals and will typically include between 0 and 20 carbon atoms. Preferred bridge members are, for example, corresponding single or multiple methylene groups such as methylene, di-, tri-, tetra-, penta- and hexamethylene. A particularly preferred bridge member R ¹ is a tetramethylene group. The carbonyl or sulfonyl group belonging to the acid residue is connected to the amino group or hydroxyl group of a diol spacer via an amide (Y = NH) or ester bond (Y = 0).

The radicals R ² and R ^{3 of} the diol spacer can be the same or different and are in principle not particularly restricted. They can be selected independently of one another from aliphatic and heteroaliphatic groups, which can be saturated or unsaturated, straight-chain or branched and / or optionally substituted and usually comprise 1 to 10 C atoms. However, R ² and R ³ can also be corresponding substituted or unsubstituted aromatic groups or contain them. From the point of view of the formation of SAM by the dithiolane derivative according to the invention or conjugates produced therefrom on corresponding surfaces, such as noble metals, R ¹ , R ² and R ^{3 are} preferably selected such that a number of C atoms differ from the dithiolane Group up to the group -OR ⁴ or -OR ⁵ results from about 5 to about 20, with saturated aliphatic or heteroaliphatic groups being preferred as sterically less problematic groups. The radical R ^{2 of} the diol spacer is therefore preferably selected from (CH ₂ ) _n groups, where n is an integer from 1 to 10, more preferably 1 to 5. Spacers in which R ^{2 is} methylene (CH ₂ ) are particularly preferred. Accordingly, the radical R ³ is preferably selected from (CH ₂ ) _m groups, where m is an integer from 1 to 10, more preferably 1 to 5. Spacers with R ³ = methylene are particularly preferred. Particularly preferred diol spacer is 3-aminopropane-1,2-diol or generally a spacer in which R ² and R ^{3 are} methylene.

The hydroxyl groups of the diol spacer in the dithiolane derivative of the above formula A according to the invention are identical or differently substituted or unsubstituted. According to the invention, the radicals R ⁴ and R ⁵ can therefore both be, for example, both an H atom or an acid-labile protective group, for example dimethoxytrityl or a related group. The radicals R ⁴ and R ⁵ are preferably different. In a particularly preferred embodiment, which can be used, for example, as a starting point for a further derivatization to obtain particularly advantageous lipoylation reagents, R ^{4 is} H and R ⁵ is an acid-labile protective group or vice versa (cf. in this connection the specific examples of the reaction schemes shown in FIGS. 1 to 3). Suitable acid-labile protective groups, such as dimethoxytrityl and similar groups, are known to a person skilled in the art and are described, for example, by Seliger in Beaucage et al. (Ed.) Current Protocols in Nucleic Acid and Chemistry, Vol. 1: 2.3.1-2.3.34, Wiley & Sons, New York, 2000. The relevant disclosure content of this document is hereby incorporated in full by reference into the present invention. Protective groups of this type are particularly suitable for coupling reactions with nucleic acids such as oligonucleotides. Further substituents of the hydroxyl groups of the diol spacer are amino and / or hydroxy coupling groups. Coupling groups of this type are known in the prior art and are correspondingly activatable residues, for example those residues which are used to anchor biomolecules, in particular oligonucleotides, but also low molecular weight organic chemical molecules, on supports containing amino or hydroxyl groups, for example appropriate polymer carriers, for example long-chain amino-CPG, in particular aminopropyl-CPG, can be used. Such coupling groups are in particular dicarbonyl groups such as a succinyl group. Other amino and / or hydroxy coupling groups which are preferably used in accordance with the present invention are phosporamidite groups, for example a beta-cyanoethoxy-diisopropylaminophosphane radical. Thus, the coupling group, as an activatable radical, enables the dithiolane bridge-spacer unit to be linked to a hydroxyl or amino group.

Particularly preferred dithiolane derivatives of the present invention are those in which R ^{4 is} H or an acid-labile protective group, for example a dimethoxytrityl group, and R ^{5 is} a dicarbonyl group, in particular a succinyl group. Conversely, it can also be R ⁵ H or an acid-labile protective group and R ^{4 can be} a dicarbonyl group. Particularly preferred embodiments of such a dithiolane derivative are designed such that the dicarbonyl group, for example a succinyl group, is covered with a polymer carrier an amide or ester bond is connected. Such a particularly preferred dithiolane derivative of the present invention is a dithiolane modifier shown in the following formula I, in this case a lipoic acid modifier:

Formula I (DMT = Dimethoxytrityl)

In another specific embodiment of the dithiolane derivative of the present invention, R ⁴ in the above formula AH or again an acid-labile protective group (for example a dimethoxytrityl group) and R ⁵ in the above formula A is a phosphoramidite group, where the substituents R ⁴ and R ^{5 may} also be reversed. An N, N-diisopropyl-beta-cyanoethoxyphosphoramidite as R ⁵ or R ^{4 is} very particularly preferred.

A specific embodiment of the dithiolane derivative is a lipoic acid-substituted phosphoramidite reagent of the following formula:

Formula II (DMT = Dimethoxytrityl) The dithiolane derivative according to the invention is excellently suitable for introducing dithiolane groups (with an attached bridge member) into (low molecular weight) organochemical compounds and biochemical molecules which have amino and / or hydroxyl groups. Another object of the present invention therefore forms a conjugate of the dithiolane derivative as defined above and an organic chemical or biochemical molecule containing at least one amino or hydroxyl group, which conjugate the group via the amino or hydroxyl group -R ⁴ or the group -R ⁵ in formula A above.

The corresponding lipoic acid derivative is particularly suitable for this inventive use of the dithiolane derivative, since this substituent, when applied to a noble metal surface, such as a gold surface, provides high binding energy. In addition, derivatives of the present invention derived from lipoic acid form particularly easily controllable SAMs. In addition, lipoic acid is easily available as a starting material and at an affordable price. Furthermore, the lipoic acid substituent is a naturally occurring biological molecule, which is why this substituent is used, for example, in medical-therapeutic or medical-diagnostic applications of immobilized or non-immobilized corresponding conjugates, for example oligonucleotides, especially one Antisense or antigen therapy poses less or no risk of toxicity. Of course, however, other compounds which have the characteristic dithiolane radical can also be used as a substituent in the dithiolane derivative according to the invention and thus also in the conjugate according to the invention.

The organic-chemical or biological-chemical molecule in the conjugate according to the invention is generally understood to mean any such compound which has at least one amino or hydroxyl group. According to the invention, the organic-chemical molecule is, for example, a low-molecular organic-chemical compound with a molecular weight of, for example, <1500 Da to understand. Biochemical molecules in the conjugate according to the invention are, in particular, those which have at least a portion which comes from a naturally occurring compound. Preferred biochemical molecules are accordingly peptides, oligopeptides, polypeptides, in particular proteins, monosaccharides, oligosaccharides, polysaccharides, lipids and their constituents, in particular fatty acids, and also nucleic acids and their constituents, that is to say nucleosides, nucleotides, oligonucleotides and polynucleotides. Mixed forms of the above-mentioned biochemical molecules can of course also be used in general, for example lipoproteins, glycoproteins etc. Particularly preferred biochemical molecules in the conjugate according to the invention are nucleic acids and their building blocks, that is to say nucleosides, nucleotides, oligonucleotides and polynucleotides. These can be deoxyribonucleotide species as well as ribonucleotide species and their mixed forms. Furthermore, the nucleic acid species according to the invention can be single-stranded or double-stranded or both single-stranded and double-stranded. In addition, the term oligonucleotide or polynucleotide also encompasses analog structures, such as, for example, peptide nucleic acids. Thus, all possible oligonucleotide-analog structures can be used in the conjugate according to the invention, which have at least one chemical modification compared to naturally occurring molecules. The term “chemical modification” means that the nucleic acid species containing the conjugate according to the invention is changed by replacing, adding or removing individual or more atoms or groups of atoms in comparison to naturally occurring nucleic acid species.

The chemical modification is preferably designed such that the nucleic acid contains at least one analog of naturally occurring nucleotides.

In a list that is by no means exhaustive, examples of nucleotide analogs that can be used according to the invention are phosphoramidates, phosphorothioa- te, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine and 2-alkoxy-substituted ribonucleotide species.

In the case of the conjugate of the dithiolane derivative and the above nucleoside / nucleotide species (ie mononucleosides, mononucleotides, oligonucleotides and polynucleotides) according to the invention, the dithiolane derivative, in particular a lipoic acid derivative, can be via the 5-C atom of the corresponding nucleo - Sids / nucleotides or conjugated via the terminal 5'-C atom of the oligo- or polynucleotide (where the nucleoside, nucleotide, oligo- or polynucleotide replaces the group -OR ⁵ in the above formula A) or it can be conjugated via the 3'-C atom of the nucleoside / nucleotide or via the terminal 3'-C atom of the oligo- or polynucleotide (the nucleoside, nucleotide, oligo- or polynucleotide being the group -OR ⁴ in the above formula A replaced).

For the 3'-terminal dithiolane conjugation of nucleic acid species, a dithiolane derivative is particularly suitable, such as a corresponding lipoic acid derivative, which is bonded to a suitable polymer support via a corresponding coupling group according to the invention. A particularly preferred example of a dithiolane derivative which is particularly suitable for the 3'-terminal conjugation is shown in the formula I above.

A dithiolane-substituted, preferably lipoic acid-substituted, phosphorous ester amide reagent is particularly suitable for conjugating a dithiolane derivative as defined above with biomolecules, in particular oligonucleotides and polynucleotides, which enables the dithiolane residue, for example a lipoic acid residue, to be attached terminally and / or internally at any desired position of oligonucleotide chains. Corresponding molecules, in particular oligonucleotides, can thus be dithiolanylated, in particular lipoylated, one or more times with the aid of the present invention. A specific example of a particularly suitable dithiolane derivative of the present invention is shown in Formula II above. The conjugates according to the invention, for example lipoylated oligonucleotides, are advantageous, for example, over known thiol conjugates in that they can easily be stored for a long time, while the conventional thiol conjugates are stable only under inert conditions.

The present invention further relates to a process for the preparation of the dithiolane derivative defined above, comprising the steps: (a) providing a dithiolane compound of the formula B.

Formula B

where R ¹ and X are as defined in formula A above and Z = NH or O,

(b) activating the carbonyl or sulfonyl group of the dithiolane compound and (c) reacting the activated dithiolane compound with a compound of formula C.

Formula C.

wherein R ² , R ³ and Y are as defined in Formula A above.

The radicals R ⁴ and R ^{5 are} further introduced into the dithiolane-diol compound formed by processes known to a person skilled in the art. According to a preferred embodiment of the process according to the invention, the activation of the carbonyl or sulfonyl group of the dithiolane compound according to formula B above takes place by converting it to an activated ester, in particular to a dicarboximide ester, such as an NHS ester. By activating the compound according to formula B, for example lipoic acid, using a suitable dicarboximide, such as hydroxysuccinimide, to give the corresponding ester, the yield in the synthesis of the central compound according to formula C above can be increased significantly. Alternatively, the activation of the dithiolane compound according to the above formula B, for example also with a carbodiimide compound, such as dicyclohexylcarbodiimide (DCC). According to a further embodiment of the production process according to the invention, the dithiolane compound according to formula B above can be converted to a corresponding acid halide, for example by reacting the dithiolanoic acid compound with oxalyl chloride to give the corresponding acid chloride. Since the acid chloride formed is often not very stable, steps (b) and (c) above are advantageously carried out simultaneously by adding the appropriate agent for the preparation of the acid halide and the corresponding diol spacer compound to the compound of the formula B.

The preparation of the dithiolane derivative according to the invention by activating the starting dithiolane compound by a carbodiimide is shown below using the example of the preparation of the dithiolane derivative of the above formula I from lipoic acid, aminopropane-1,2-diol and amino-CPG (Scheme 1 acc. Fig. 1). Another scheme shows the corresponding preparation of the above phosphoramidite derivative according to formula II (scheme 2 according to FIG. 2). Scheme 3 below (FIG. 3) shows an example of the process according to the invention for the preparation of the dithiolane derivative with the aid of the conversion of lipoic acid to an NHS ester, the further synthesis steps to a 3'-lipoic acid modifier CPG or one Lipoic acid phosphoramidites are shown. The preparation of lipoic acid aminopropanediol is an example of the preparation of the dithiolane derivative according to the invention from the dithiolanoic acid, in this case Lipoic acid, using oxalyl chloride and reaction with aminopropane-1, 2-diol in scheme 4 (Fig. 4).

Furthermore, the present invention comprises a method for producing the conjugate according to the invention, which comprises the steps:

(i) providing the dithiolane derivative according to the invention and (ii) reacting the dithiolane derivative with an organic chemical or biological chemical molecule containing at least one amino or hydroxyl group.

The dithiolane derivative is preferably provided by the production process defined above. The organochemical or biochemical compound containing at least one amino or hydroxyl group is preferably one of the compounds specified above.

The process according to the invention for producing the conjugate is particularly preferred if a dithiolane derivative is used which contains a dicarbonyl group which connects the dithiolane bridge-spacer construct to a polymer support via an amide or ester bond. A specific example of such a compound is shown in Formula I above.

According to this particularly preferred embodiment of the present invention, the lipoic acid residue is connected via an amide bond to the amino group of a 3-aminopropane-1,2-diol spacer. One of the hydroxyl groups of the spacer is substituted with a protective group, here dimethoxytrityl, which is split off selectively before the construction of an oligonucleotide chain. The further hydroxyl group of the 3-aminopropane-1, 2-diol spacer is, for example, a succinyl residue with a group known from the prior art which is used for anchoring oligonucleotides to polymer supports containing amino or hydroxyl groups. After the 4,4'-dimethoxytrityl group has been cleaved off using a process known in the prior art, the structure of the oligonucleotide chain is generated from this exposed hydroxyl group in accordance with the prior art, it being irrelevant whether this structure of 3'- or 5 - End of ago. After the construction of a corresponding nucleic acid chain, for example an oligonucleotide chain, with the cleavage from the polymer support known in the prior art, for example with ammonia or amines, the residue containing the lipoic acid or dithiolane group remains bound to the oligonucleotide chain via the spacer.

Another preferred method for producing a conjugate according to the invention uses the dithiolane derivative in which R ^{4 is} H or an acid-labile protective group, for example a dimethoxytrityl group, and R ^{5 is} a phosphoramidite group, in particular N, N-diisopropyl-beta-cyanoethoxyphosphoramidite , is. A specific embodiment of a dithiolane derivative according to the invention, in this case a lipoic acid derivative, is shown in Formula II above. Here, too, a hydroxyl group of the spacer is protected with a corresponding protective group, for example dimethoxytrityl. The second hydroxyl group is connected to an activatable radical known from the prior art, for example a phosphoramidite radical, in formula II a (beta-cyanoethoxy) diisopropylaminophosphane radical. According to the invention, this activatable radical enables attachment to a hydroxyl or amino group. Following this reaction step, the dimethoxytrityl protective group is split off in accordance with known processes. The hydroxyl function obtained in this way can then optionally be used to attach a further nucleotide unit. The dithiolane derivatives according to the invention are therefore very particularly suitable for producing corresponding nucleic acid conjugates, oligonucleotides being particularly preferred. The dithiolane derivative of the present invention, comprising the specific bridging member diol spacer structure, has the advantage over the reagents known in the prior art that it can easily be used in standard nucleic acid synthesis processes, such as, for example, in the phosphoramidite method and anchoring to polymer supports etc., can be set. Such methods are described, for example, by Gait (Oligonucleotide synthesis: a practical approach, IRL Press, Oxford, 1987), and the relevant disclosure content of this document is fully incorporated into the present invention. The course of a synthesis cycle in the phosphoramidite method is shown by way of example in FIG. 6.

The dithiolane derivative according to the invention is particularly suitable for immobilizing organic chemical or biological chemical molecules on noble metals or noble metal alloys and on semiconductors.

Thus, according to the invention, a coated noble metal structure is also provided, which

(a) a substrate made of a carrier material,

(b) at least one layer of one or more noble metals and / or noble metal alloys and at least partially formed on the substrate

(c) comprises at least one dithiolane derivative according to the invention immobilized at least in regions on the noble metal layer and / or at least one conjugate according to the invention immobilized at least in regions on the noble metal layer as defined above.

Furthermore, a coated semiconductor structure is provided according to the invention, comprising a thiol-binding semiconductor carrier and at least one dithiolane derivative according to the invention immobilized at least in regions on the semiconductor and / or at least one conjugate according to the invention immobilized at least in regions on the semiconductor as defined above. Preferred semiconductor structures according to the invention are those in which gallium arsenide or indium phosphide is coated with dithiolane derivatives or conjugates according to the invention; with regard to the binding of thiols on semiconductors cf. eg Lunt et al. (1991) J. Appl. Phys. 70: 7449; Gu et al. (1995) Langmuir 11: 1849-1851; Zerulla et al. (1999) Langmuir 15, 5285-5294. Layer structures in which the immobilized dithiolane derivative and / or the immobilized conjugate form / form a self-assembled monolayer (SAM) are preferred according to the invention. This embodiment of the coated noble metal or semiconductor structure according to the invention provides a controlled layer of the immobilized molecules, for example biomolecules such as oligonucleotides. By forming a SAM, it is possible to control the density of the immobilized molecules in such a way that the best possible balance between a high density of, for example, probe molecules used in an array (i.e. the greatest possible number of binding sites for potential binding partners (target molecules) per Area unit) and their accessibility for the target molecules to be examined, which results in a high signal intensity when used in a biochip (for example a corresponding microarray). In particular, to increase the hybridization efficiency in immobilized probe oligonucleotides, the layer structure can be formed according to the invention in such a way that, in addition to the dithiolane derivative according to the invention or the conjugate, for example, a spacer (cf. Southern et al. (1999) The Chiping Forecast 21: 5- 9; Southern et al. (1997) Nucl. Acid Res. 25: 1155-1161). Furthermore, the hybridization efficiency can be increased, for example, by using probe oligos loosely packed on the surface (Southern et al. (1997), supra).

In comparison to other immobilization strategies, a so-called mixed oligo-SAM can be formed in the conjugated oligonucleotides according to the invention, in addition to the dithiolane derivative according to the invention or the conjugate thereof with an oligonucleotide, a customary alkanethiol for controlling the density of the desired oligo-SAM is added. This also means that the oligonucleotide used is easily accessible for efficient hybridization. These measures can increase the hybridization efficiency up to 100% (Levicky (1998) JACS. 120: 9787-9792). Another great advantage of the dithiolane-modified molecules immobilized on a noble metal, in particular of oligonucleotides modified with lipoic acid, is the high binding capacity on the noble metal. For example. Gold has a binding capacity of lipoic acid of 7.1 x 10 ^"10 mol / cm ² (Pirrung (2002) Angew. Chem. 114: 1326-1341). Furthermore, the layer structures according to the invention are those from the prior art, particularly conventional ones thiol-modified oligonucleotide layers, in that, due to the presence of the vicinal dithiol group, the dithiolane derivative of the present invention has a higher binding energy on the carrier material, which is why a more stable connection can be expected from the double anchoring (see Wang et al. (1998 ), supra; Cheng et al. (1995), supra).

The coated noble metal or semiconductor structures according to the invention are further distinguished by the fact that the constituents and the structure itself can be produced by standard synthesis processes. In contrast, conventional thiol-modified oligonucleotides from the prior art require elaborate treatment or workup so that the -SH group desired for chemisorption of the corresponding conjugate is released.

Numerous noble metals such as gold, silver, copper, platinum, palladium, ruthenium and iridium and alloys of noble metals are suitable for the coated noble metal structure according to the invention. A particularly preferred noble metal according to the invention is gold. The coated noble metal structure according to the invention can be designed in such a way that the substrate is at least partially flat. In particular, glass, plastics, semiconductors (in particular silicon) and metals, preferably those which differ from the noble metal of the coating, are used as carrier materials. If necessary, the surface of the carrier material can be treated or coated in accordance with a method known to a person skilled in the art in order to bind the noble metal or the noble metal alloy to be applied. Glass carriers are currently used as standard for biochips, in particular microarrays. On the other hand, there are also particles made of precious metals or Precious metal alloys or particulate carrier materials coated therewith in question, so that the substrate can also be designed in particulate form according to the invention. In the case of a particulate configuration of the coated noble metal structure according to the invention, materials such as polymers, for example polystyrene, glass, silica, etc., are particularly suitable.

In the case of flat structures, the noble metal or semiconductor structure is particularly suitable for use as a nucleic acid array, in particular as a DNA microarray, on biochips.

Therefore, a biochip is also provided according to the invention, which comprises the coated noble metal / semiconductor structure according to the invention.

Further advantages of coating precious metals, in particular gold, with dithiolane derivatives and conjugates derived therefrom, in particular of oligonucleotides, according to the invention are particularly good binding capacity (Wölfl (2000) transcript laboratory value 3), the possibility of developing SAM (Bamdad ( 1998) Biophys. J. 75: 1997-2003), ie the controlled connection of analysis molecules, in particular DNA, for use in diagnostics, and the possibility of rapid production of a biochip, since the immobilization of the desired analysis molecules, such as DNA oligonucleotides, and the blocking of the chip surface within about 3 hours is possible.

The figures show:

1 shows in a synthesis scheme the preparation of a dithiolane derivative according to the invention, in the present case a CPG modifier reagent according to formula I (scheme 1). Lipoic acid is reacted with aminopropane-1,2-diol with activation by DCC, giving the corresponding propane-2,3-diol-1,2-dithiolane-3-pentanoic acid amide. In a further reaction one of the hydroxyl groups is protected with 4,4'-dimethoxytrityl chloride. By implementing with Succinic anhydride is added to the free hydroxyl group, a succinyl residue and then a p-nitrophenyl ester is produced by activation with DCC. Finally, the ester activated in this way is reacted with amino-CPG to give the dithiolane derivative of the formula I coupled to the polymer support.

FIG. 2 shows in synthesis scheme 2 the preparation of a dithiolane derivative according to the invention which has a phosphoramidite group on a hydroxyl oxygen of the diol spacer, propane-2,3-diol-1,2- dithiolan-3-pentanoic acid amide is assumed, which can be shown according to Scheme 1 of FIG. 1. The lipoic acid amide-spacer conjugate is reacted with chlorine-N, N-diisopropyl-beta-cyanophosphine and N, N-diisopropylamine in dry dichloromethane to give the compound of formula II.

3 shows in a further synthesis scheme the coupling of lipoic acid with aminopropane-1,2-diol in an alternative production method for S'-lipoic acid modifier-CPG or lipoic acid phosphoramidite (Scheme 3). First, lipoic acid is used

Hydroxysuccinimide to lipoic acid N HS ester activated. The NHS ester is then reacted with aminopropane-1,2-diol in dichloromethane / triethylamine to give the lipoamide amide spacer derivative again. Then follows the mounting of a ^4,4 -Dimethoxytrityl-protecting group at the terminal hydroxyl group.

Starting from this protected derivative, the corresponding S'-lipoic acid modifier-CPG or a lipoic acid phosphoramidite, for example, can then be produced, as shown in FIG. 1 (Scheme 1) or FIG. 2 (Scheme 2).

4 shows another embodiment of the production of lipoic acid aminopropanethiol in a further synthesis scheme (Scheme 4). In this case, lipoic acid is reacted with oxalyl chloride to give the corresponding acid chloride, thus obtaining an activated compound which reacts with aminopropane-1,2-diol to give the desired product.

5A shows a schematic representation of the immobilization of oligonucleotide-lipoic acid conjugates according to the invention, the lipoic acid derivative being linked to the 3'-C atom of the terminal nucleotide.

FIG. 5B shows, in a representation corresponding to FIG. 5A, the conjugation of the lipoic acid derivative to the terminal 5'-C atom of the conjugated oligonucleotide.

6 shows in a synthesis scheme the oligonucleotide synthesis

Cycle that is run through in the phosphoramidite method (cf. Gait (1987), supra).

The following exemplary embodiments illustrate the present invention without restricting it.

1st embodiment

Production of 3'-lipoic acid modifier CPG

Literature:

Nelson et al. (1989) Nucl. Acids Res. 17: 7179-7186; Nelson et al. (1989) Nucl. Acids Res. 17: 7187-7194 Chemicals used:

D, L-alpha-lipoic acid, 4-pyrrolidinopyridine, 3-aminopropane-1, 2-diol, dicyclohexylcarbodiimide, dichloromethane, 4,4 ^, -dimethoxytriphenylmethyl chloride, pyridine (dry), methanol, dimethylaminopyridine, succinic anhydride, ethyl acetate, di oxane, p-nitrophenol, triethylamine, aminopropyl-CPG (for production see Amino-CPG, http://www.interactiva.de), dimethylformamide, acetic anhydride, diethyl ether.

Preparation of propane-2,3-diol-1,2-dithiolane-3-pentanoic acid amide

To a mixture consisting of 3-aminopropane-1,2-diol (5 mmol, 0.5 g), 4-pyrrolydinopyridine (5 mmol, 0.7 g) and dicyclohexylcarbodiimide (10 mmol, 1.9 g) in 20 ml of dichloromethane, D, L-alpha-lipoic acid (5 mmol, 1 g) was added and the mixture was stirred at room temperature overnight. The precipitated dicyclohexylurea was filtered off and the solvent was evaporated on a rotary evaporator.

Preparation of propane-2,3-diol-3-0-4,4'-dimethoxydiphenylmethyl-1, 2-dithiolane-3-pentanoic acid amide

The viscous, impure product was dissolved in 10 ml of dry pyridine and reacted with 4,4 ^, -dimethoxytriphenylmethyl chloride (5 mmol, 1.69 g) and stirred at room temperature overnight.

10 ml of methanol were then added, the mixture was stirred for a further 10 min and dried under vacuum.

The product was purified using a silica gel column (5 × 20 cm) (yield 61%, elution mixture: dichloromethane / hexane / triethylamine (50/50/1%)) and checked using thin layer chromatography (TLC) (mobile solvent: dichloromethane / Hexane / triethylamine, 50/50/1%), detection: UV light (the trityl group can be made visible under HCl vapor). Preparation of 3'-ubonic acid modifier CPG

To a solution of propane-2,3-diol-3-0-4,4'-dimethoxytriphenylmethyl-1,2-dithioian-3-pentanoic acid amide (1 mmol, 0.61 g) and dimethylaminopyridine (0.9 mmol, 200 mg) in dry pyridine (12 ml), succinic anhydride (3 mmol, 300 mg) was added and the mixture was stirred overnight at room temperature. The mixture was dissolved in ethyl acetate (100 ml), washed with a 0.5 M sodium chloride solution (3 x 100 ml) and a saturated sodium chloride solution (1 x 100 ml) and dried over sodium sulfate. The solvent was removed and the product dried under vacuum.

The dry succinic acid derivative, p-nitrophenol (2.5 mmol, 350 mg) and 0.5 ml of dry pyridine were dissolved in 10 ml of dry dioxane. Dicyclohexylcarbodiimide (4.8 mmol, 1.0 g) was then added to the mixture and the mixture was stirred under mild conditions for 3 h. The resulting dicyclohexylurea was filtered off.

1 g of aminopropane CPG was suspended in the filtrate, 1 ml of triethylamine was added and shaken at room temperature overnight.

The derivatized carrier was washed carefully with dimethylformamide, methanol and diethyl ether and dried.

Finally, the lipoic acid carrier was capped with a solution of acetanhydride / pyridine / dimethylaminopyridine (10: 90: 1). It was then washed thoroughly with methanol and diethyl ether and thoroughly dried under high vacuum.

The synthesis is shown in Scheme 1 (Fig. 1). 2nd embodiment

Production of lipoic acid phosphoramidite

Literature:

Nelson et al. (1989) Nucl. Acids Res. 17: 7179-7186

Chemicals used:

N, N-diisopropylethylamine, dichloromethane (dry), chlorine-N, N-diisopropyl-beta-cyanoethoxyphosphoramidite, methanol, ethyl acetate, hexane, triethylamine

It is to be assumed that propane-2,3-diol-0-4-4'-dimethoxytriphenylmethyl-1,2-dithiolane-3-pentanoic acid amide, which is produced, for example, according to the first exemplary embodiment.

Preparation of propane-2,3-diol-2-0-rN, N-diisopropyl-beta-cvanoethoxyphosphoramidite 1-3-3-r4,4'-dimethoxydiphenylmethane-1,2-dithiolane-3-pentanoic acid amide

Propane-2,3-diol-3-0-4-4'-dimethoxytriphenylmethyl-1,2-dithiolane-3-pentanoic acid amide (1.25 mmol, 2.2 μg) and dry N, N-diisopropylethylamine ( 6 mmol, 0.78 g) were dissolved in 45 ml of dry dichloromethane under argon. Chloro-N, N-diisopropyl-beta-cyanoethoxyphosphoramidite (2.5 mmol, 0.6 g) was slowly added to this solution within 5 min and the mixture was stirred under argon for 1 h. Then methanol (1 ml) was added and the mixture was stirred for a further 10 min. The reaction mixture was partitioned between ice cold ethyl acetate (170 ml) and 10% ice cold sodium hydrogen carbonate (275 ml). The organic phase was washed with 10% cold sodium hydrogen carbonate solution (275 ml) and dried over sodium sulfate. After the solvent was removed under vacuum, the rubbery residue was purified using a 5 x 10 cm silica gel column. The desired product could be methane / hexane / triethylamine (50: 50: 1%) are eluted isocratically (check by TLC, eluent: dichloromethane / hexane / triethylamine (50: 50: 1%), detection: UV light, with the trityl group under HCI vapor can be made visible). The solvent was spun in and the purified lipoic acid phosphoramidite was dried in vacuo. The synthesis is shown in Scheme 2 (Fig. 2).

3rd embodiment:

Synthesis of 3'-, 5'-lipoylated oligonucleotides

The oligonucleotide syntheses were carried out according to a standard protocol on an Expedite TM Syntheziser (PerSeptive Biosystems). All synthesized oligonucleotides were then purified using HPLC as standard. The syntheses of 3'-, δ'-lipoylated oligonucleotides could be confirmed by MALDI.

4th embodiment:

Immobilization of lipoylated oligonucleotides on gold-coated supports

gold support

Commercial slides were used as the substrate, which were sputtered with 10 nm tungsten / titanium (adhesion layer) and 100 nm gold. The gold carriers were cleaned with Piranha solution (70% H ₂ S0 ₄ : 30% H ₂ 0 ₂ ).

immobilization

Oligonucleotides (lip oligonucleotides) modified with 3'-C and 5'-C were added to 1, 2, 5, 10, 15 and 30 pmol in 3 μl of 1 M NaH ₂ PO ₄ (pH 4.1) with lipoic acid cleaned gold carrier applied. The gold carriers were leave saturated chamber for 2 h. After immobilization, the liquid drops were pipetted off and rinsed thoroughly with double-distilled water.

Passivation of the carrier periphery

After immobilization, the gold carrier was incubated with lip oligonucleotides for the prevention of non-specific binding in a 1 mM solution of thio-polyethylene glycol MW 5000 (Rapp Polymers) for 45 min. It was then rinsed carefully with double-distilled water. Remaining water drops were blown off with nitrogen.

Proof of immobilization

To detect the immobilization, the coated slide was hybridized with radioactive labeled oligonucleotides, which had the complementary sequence to the immobilized nucleotides (hybridization with ³² P, 1 M NaCI-TE buffer, overnight at room temperature). In addition, the immobilization of lipoylated oligonucleotides was confirmed with the help of X-ray photoelectron spectroscopy (XPS) studies.

Claims

claims Dithiolane derivative of the formula A Formula A being R ^{1 is} nothing or a straight-chain or branched, saturated or unsaturated, substituted or unsubstituted aliphatic, heteroaliphatic radical with 1 to 20 C atoms or a corresponding substituted or unsubstituted aromatic radical, R ² , R ^{3 are} each independently a straight-chain or branched, saturated or unsaturated, substituted or unsubstituted aliphatic or heteroaliphatic radical having 1 to 10 C atoms or a corresponding substituted or unsubstituted aromatic radical, R ⁴ , R ^{5 are} each independently selected from the group consisting of H, acid-labile protective groups, and amino and / or hydroxy coupling groups, X is O or S and Y is NH or O. The dithiolane derivative according to claim 1, wherein R ^{1 is} tetramethylene. Dithiolane derivative according to claim 1 or 2, wherein X = 0 and Y = N. Dithiolan derivative according to one of claims 1 to 3, wherein R ^{2 is} (CH ₂ ) _n , where n is an integer from 1 to 10, in particular from 1 to 5 A dithiolane derivative according to claim 4, wherein R ^{2 is} methylene. Dithiolan derivative according to one of claims 1 to 5, wherein R ^{3 is} (CH ₂ ) _m , where m is an integer from 1 to 10, in particular from 1 to 5. The dithiolane derivative according to claim 6, wherein R ^{3 is} methylene. Dithiolan derivative according to one of claims 1 to 7, wherein R ^{4 is} H and R ^{5 is} an acid-labile protective group or vice versa. Dithiolan derivative according to any one of claims 1 to 7, wherein R ^{4 is} H or an acid-labile protective group and R ^{5 is} a dicarbonyl group or vice versa. The dithiolane derivative according to claim 9, wherein the dicarbonyl group is connected to a polymer support via an amide or ester bond. A dithiolane derivative according to claim 10, which has the following formula: Formula I wherein DMT is a dimethoxytrityl group. 2. Dithiolan derivative according to one of claims 1 to 7, wherein R ^{4 is} H or an acid-labile protective group and R ^{5 is} a phosphoramidite group or vice versa. 3. dithiolane derivative according to claim 12, wherein the phosphoramidite group is N, N-diisopropyl-ß-cyanoethoxyphosphoramidit. 4. The dithiolane derivative according to claim 13, which has the following formula: Formula II wherein DMT is a dimethoxytrityl group. 15. Conjugate of at least one dithiolane derivative according to one of claims 1 to 14 and an organic chemical or biochemical molecule containing at least one amino or hydroxyl group, the group -OR ⁴ or the via the amino or hydroxyl group Group -OR ^{5 replaced} in formula A according to claim 1. 16. The conjugate according to claim 15, wherein the biochemical molecule is selected from the group consisting of nucleosides, nucleotides, oligonucleotides and polynucleotides. 17. The conjugate of claim 16, wherein the dithiolane derivative via the 5'-C- Atom of the nucleoside / nucleotide or via the terminal δ'-C atom of the oligo- or polynucleotide is conjugated and the group -OR ⁵ in formula A according to claim 1 replaced. 18. The conjugate of claim 16, wherein the dithiolane derivative via the 3'-C- Atom of the nucleoside / nucleotide or via the terminal 3'-C atom of the oligo- or polynucleotide is conjugated and the group -OR ⁴ in formula A is replaced according to claim 1. 19. A method for producing a dithiolane derivative according to any one of claims 1 to 14, comprising the steps: (a) providing a dithiolane compound of formula B. Formula B wherein R ¹ and X are as defined in formula A according to claim 1 and Z = NH or O, (b) activating the carbonyl or sulfonyl group of the dithiolane compound and (c) reacting the activated dithiolane compound with a compound of the formula C. Formula C. wherein R ² , R ³ and Y are as defined in formula A according to claim 1. 20. The method according to claim 19, wherein in step (b) the compound of formula B is converted to an activated ester. 21. The method of claim 20, wherein the activated ester is a dicarboximide ester. 22. The method according to claim 19, wherein in step (b) the compound of formula B is converted to the acid halide. 23. The method according to claim 22, wherein steps (b) and (c) take place simultaneously. 24. A method for producing the conjugate according to any one of claims 15 to 18, comprising the steps: (i) providing the dithiolane derivative according to any one of claims 1 to 14 and (ii) reacting the dithiolane derivative with an organic chemical or biological chemical molecule containing at least one amino or hydroxyl group. W 31 25. The method according to claim 24, wherein the dithiolane derivative is provided by the method according to one of claims 19 to 23. 26. The method according to claim 24 or 25, wherein the dithiolane derivative is defined according to claim 10 or 11. 27. The method according to claim 24 or 25, wherein the dithiolane derivative is defined according to one of claims 12 to 14. 28. The method according to any one of claims 24 to 27, wherein the biochemical molecule is selected from the group consisting of nucleosides, nucleotides, oligonucleotides and polynucleotides. 29. Coated precious metal structure, comprising (A) a substrate made of a carrier material, (B) at least one layer of one or more noble metals and / or noble metal alloys formed at least in regions on the substrate and (C) at least one dithiolane derivative immobilized at least in regions on the noble metal layer according to one of claims 1 to 14 and / or at least one at least in regions the conjugate immobilized on the noble metal layer according to one of claims 15 to 18. 30. Precious metal structure according to claim 29, wherein the immobilized dithiolane derivative and / or the immobilized conjugate form / form a self-assembled monolayer. 31. Precious metal structure according to claim 29 or 30, wherein the noble metal from the group consisting of gold, silver, copper, platinum, palladium, Ruthenium and iridium, as well as alloys of these precious metals, is selected. 32. Precious metal structure according to claim 31, wherein the noble metal is gold. 33. Precious metal structure according to one of claims 29 to 32, wherein the substrate is at least partially flat. 34. Precious metal structure according to one of claims 29 to 32, wherein the substrate is particulate 35. Precious metal structure according to one of claims 29 to _. 34, wherein the carrier material is glass, plastic, a semiconductor or a metal. 36. Coated semiconductor structure comprising a thiol binding Semiconductor carrier and at least one dithiolane derivative immobilized at least in regions on the semiconductor carrier according to one of claims 1 to 14 and / or at least one conjugate immobilized at least in regions on the semiconductor carrier according to one of claims 15 to 18. 37. The semiconductor structure of claim 36, wherein the semiconductor is gallium arsenide or indium phosphide. 38. The semiconductor structure according to claim 36 or 37, wherein the immobilized Dithiolan derivative and / or the immobilized conjugate form / forms a self-assembled monolayer. 39. Biochip comprising the noble metal structure according to one of claims 29 to 35 and / or the semiconductor structure according to one of claims 36 to 38. Use of the dithiolane derivative according to one of claims 1 to 14 for the immobilization of organic chemical or biological chemical molecules on noble metals, noble metal alloys and semiconductors.