Multifunctional reagent for stable attachment of molecules or oligomers to surfaces

Abstract

The invention relates to a compound having the chemical structural formula (I) where R1 = H or an alkyl radical, R2 = CPG-LCAA-succinyl- where LCAA-CPG = long chain alkylamine controlled pore glass and succinyl = -(C=O)-CH2-CH2-(C=O)-OH, or R2 = DMTr (dimethoxytrityl) radical or another protecting group, such as Fmoc or 2-cyanoethoxy-N,N-diisopropylphosphonous ester amide, R3 = CPG-LCAA-succinyl- where LCAA-CPG = long chain alkylamine controlled pore glass; succinyl = -(C=O)-CH2-CH2-(C=O)-OH or R3 = DMTr (dimethoxytrityl) radical or another protecting group, such as Fmoc or 2-cyanoethoxy-N,N-diisopropylphosphonous ester amide, and R4 = H or an alkyl radical. The invention also relates to derivatives of these compounds and to a use of these compounds.

Description

[0001] Multifunctional reagent for stable binding of molecules or oligomers to surfaces

[0002] The invention relates to a multifunctional reagent for the stable binding of molecules or oligomers to surfaces. The invention also relates to a surface with molecules or oligomers bound via such a reagent, as well as to the use of such a reagent.

[0003] Numerous applications, such as molecular diagnostics, require stable binding of molecules or oligomers to a specific target surface, such as a gold surface.

[0004] Oligonucleotides, and oligomers in general, can be immobilized on a surface using well-known methods such as non-covalent adsorption or covalent coupling. Known methods for immobilizing oligonucleotides on a glass surface (SiO2) are based on well-established silicone chemistry.

[0005] When bound to gold surfaces, it is known that thiols and disulfides are adsorbed on gold in ordered monolayers. However, oligonucleotides bound to a gold surface via only a single thiol anchor are unstable under mechanical stress, such as during washing steps, so the stability of the oligonucleotides on the surface is often increased by forming multiple Au-S bonds. EP 1 626 952 B1 proposes a multifunctional reagent for the synthesis of thiol-modified oligomers.

[0006] Despite these advances, there is still a need in molecular diagnostics and DNA chip technology for reagents for stable binding of various molecules and oligomers to desired target surfaces or carriers.

[0007] The object of the present invention is therefore to provide reagents particularly suitable for this purpose. This object is achieved by the features of the independent claims. Further developments of the invention are the subject of the dependent claims.

[0008] The invention relates to a compound having the stereochemical structural formula I: where

[0009] RI = H or an alkyl radical,

[0010] R2 = CPG-LCAA-succinyl-, where LCAA-CPG = long chain alkylamine controlled pore glass and succinyl = -(C=O)-CH2-CH2-(C=O)-OH, or

[0011] R2 = DMTr- (dimethoxytrityl-) residue or another protecting group, such as

[0012] Fmoc or 2-cyanoethoxy-N,Ndiisopropyl -phosphonous acid ester amide, is

[0013] R3 = CPG-LCAA-succinyl-, where LCAA-CPG = long chain alkylamine controlled pore glass; succinyl = -(C=O)-CH2-CH2-(C=O)-OH or R3 = DMTr- (dimethoxytrityl-)-residue or another protecting group, such as Fmoc or 2-cyanoethoxy-N,Ndiisopropyl-phosphonous acid ester-amide, and

[0014] R4 = H or an alkyl radical.

[0015] The functional group of the described compound with the stereochemical structural formula I without the residues R3 and R4 is referred to as the DTPA group in this description.

[0016] The invention also includes, in particular, a derivative of said compound 1, in particular a derivative with an oligonucleotide or with a structure derived from an oligonucleotide, such as a bridged nucleic acid (locked nucleic acids, LNA), phosphothioate, 2'0-methyl RNA, methylphosphonate, or 2'fluoro RNA and L-DNA, with one or more DTPA groups, i.e. one or more functional groups of the described compound with the stereochemical structural formula I without the radicals R3 and R4.

[0017] Locked nucleic acids (LNA) are xenonucleic acids and consist of modified nucleotides. In an LNA, the ribose unit of the RNA building blocks is linked with an additional bridge between the 2' oxygen and 4' carbon atom. This locks the ribose in the 3'-endo (north) conformation and makes it structurally less flexible than its unmodified analogue. This significantly increases its hybridization properties (melting point) and its affinity for base stacking.

[0018] In a preferred embodiment, the said derivative is an oligonucleotide derivative, in particular with one, two or three DTPA group(s) at the terminal position of the oligonucleotide (3 Z or 5'). Two or three DTPA groups can advantageously be directly linked to each other.

[0019] In another, equally advantageous embodiment, the said derivative is an oligonucleotide derivative with one, two or three DTPA group(s) in the internal position of the oligonucleotide.

[0020] In this embodiment, two or three DTPA groups can also be advantageously coupled to one another. Alternatively, the DTPA groups of the oligonucleotide derivative can all be spaced apart from one another.

[0021] It is also advantageously possible for the oligonucleotide derivative to have several spaced blocks of two or three DTPA groups.

[0022] The compound represented stereochemically below shows an advantageous embodiment of an oligonucleotide derivative according to the invention with three DTPA groups at the 3' terminal end of an oligonucleotide:

[0023] The preparation of the compound according to the invention preferably starts from the starting substance (S;R)-cyclodithiothreitol, (R,S)-cyclodithiothreitol. Alternatively, although currently less preferred, (ss,rr)-cyclodithiothreitol can also be used as the starting substance. The basic synthesis route, particularly starting from these starting substances, can follow the procedure described in the article by P. Liepold et al., Anal Bioanal Chem (2008): 391:1759-1772.

[0024] The invention also includes a surface with molecules or oligomers attached via one or more DTPA groups of the type described.

[0025] In particular, the invention contains a surface with at least one derivative of the type described above, which is bound to the surface via the derivative's DTPA groups. The at least one derivative is preferably an oligonucleotide derivative of the type described.

[0026] The surface is preferably metallic, but non-metallic surfaces with absorption mediated by metal ions are also possible. The surface does not have to be flat; it can also be the surface of a particle, particularly a nanoparticle, for example.

[0027] With particular advantage, the surface is a gold surface, in particular a gold contact pad, a gold test area or a gold particle, in particular a gold nanoparticle.

[0028] In advantageous designs, the gold surface is a surface of sputtered gold with a rough surface texture.

[0029] The invention also encompasses the use of the above-mentioned compound having the stereochemical structural formula I for modifying oligomers, in particular for immobilizing modified oligomers on surfaces.

[0030] The invention further comprises the use of the above-mentioned compound having the stereochemical structural formula I for binding enzymatic, chromogenic, fluorogenic, radioactive or chemiluminescent labels, substances intercalating in nucleic acid oligomers, metals, metal ions, hormones, proteins, peptides, nucleolytic and proteolytic agents, biotin, antigens, haptens, antibodies or receptors to molecules or oligomers.

Claims

Patent claims 1. Compound with the stereochemical structural formula I: RI = H or an alkyl radical, R2 = CPG-LCAA-succinyl-, where LCAA-CPG = long chain alkylamine controlled pore glass and succinyl = -(C=O)-CH2-CH2-(C=O)-OH, or R2 = DMTr- (dimethoxytrityl-)-rest or another protecting group, such as Fmoc or 2-cyanoethoxy-N,Ndiisopropyl -phosphonous acid ester-amide, is R3 = CPG-LCAA-succinyl-, where LCAA-CPG = long chain alkyamine controlled pore glass; succinyl = -(C=O)-CH2-CH2-(C=O)-OH or R3 = DMTr- (dimethoxytrityl-) radical or another protecting group, such as Fmoc or 2-cyanoethoxy-N,Ndiisopropyl -phosphonous acid ester amide, and R4 = H or an alkyl radical.

2. A derivative of the compound of claim 1, in particular a derivative with an oligonucleotide or with a structure derived from an oligonucleotide, such as a bridged nucleic acid (locked nucleic acids, LN A), phosphothioate, 2'O-methyl RNA, methylphosphonate, or 2'fluoro RNA and L-DNA, with one or more DTPA groups according to claim 1.

3. Oligonucleotide derivative according to claim 2, having one, two or three DTPA group(s) at the terminal position of the oligonucleotide (3' or 5').

4. Oligonucleotide derivative according to claim 3 with two or three DTPA groups directly coupled to one another.

5. Oligonucleotide derivative according to claim 2, having one, two or three DTPA group(s) in the internal position of the oligonucleotide.

6. Oligonucleotide derivative according to claim 5, with two or three DTPA groups directly coupled to one another.

7. Oligonucleotide derivative according to claim 5, with exclusively spaced-apart DTPA groups.

8. Oligonucleotide derivative according to claim 2, having several spaced blocks of two or three DTPA groups.

9. Surface with molecules or oligomers attached via one or more DTPA groups according to claim 1.

10. Surface with at least one derivative according to claim 2, which is bound to the surface via the DTPA groups of the derivative, wherein the at least one derivative is preferably an oligonucleotide derivative according to one of claims 3 to 8.

11. Surface according to claim 9 or 10, characterized in that the surface is a gold surface, in particular a gold contact pad, a gold test surface or a gold particle, in particular a gold nanoparticle.

12. Surface according to claim 11, characterized in that the gold surface is a surface of sputtered gold with a rough surface texture.

13. Use of the compound of claim 1 for modifying oligomers, in particular for immobilizing modified oligomers on surfaces.

14. Use of the compound of claim 1 for binding enzymatic, chromogenic, fluorogenic, radioactive or chemiluminescent labels, substances intercalating in nucleic acid oligomers, metals, metal ions, hormones, proteins, peptides, nucleolytic and proteolytic agents, biotin, antigens, haptens, antibodies or receptors to molecules or oligomers.

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