Reactive monomers for the oligonucleotide and polynucleotide synthesis , modified oligonucleotides and polynucleotides, and a method for producing the same
OwnerMarkus Schweitzerinventor
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Abstract
The invention relates to the production of modified oligonucleotides and to their use for conjugation reactions. The invention further relates to reagents and to methods for producing aldehyde-modified oligonucleotides that contain aldehydes that are protected (masked) as acetals. Once said acetals are incorporated into the oligonucleotides the oligonucleotides are converted to aldehydes and are used for conjugation. The conjugation reaction can be carried out with the free oligonucleotide or with the oligonucleotide that is still immobilized on the substrate.
Claims23
A reactive monomer of the formula (I), wherein l, v independently of one another are 0 or 1 and a is an integer between 1 and 5
- X—Ll—Vv—(A)a (I)
- where
- X is a phosphoramidite (II),
- 8
- wherein R2 and R3 independently of one another are a branched or unbranched C1 to C5 alkyl radical and R1 is methyl, allyl or β-cyanoethyl,
- V is a branching unit composed of an atom or of a molecule having at least three binding partners,
- A is an acetal of the formula (IV),
- 9
- where the radicals Y and Z independently of one another are identical or different branched, unbranched or cyclic, saturated or unsaturated hydrocarbons having from one to 18 carbon atoms, it also being possible for the radicals Y and Z to be linked to one another,
- and wherein
- L are linkers which are suitable for linking X to A or X to V and V to A.
A reactive monomer as claimed in any of the preceding claims, wherein the radicals Y and Z independently of one another are methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, tert-butyl.
A reactive monomer as claimed in any of the preceding claims, wherein the linkers L are selected from the group consisting of branched, unbranched or cyclic, saturated or unsaturated C1 to C18 hydrocarbons or the group which is a polyether —(CH2)k—[O—(CH2)m]o—O—(CH2)p— where k, m, p independently of one another are an integer from 0 to 4, and o is an integer from 0 to 8, or the group which is an amine —(CH2)w—NH—(CH2)u— where w and u independently of one another are an integer from 0 to 18, or the group which is an amides —(CH2)q—C(O)—N—(CH2)r— or —(CH2)q—N—C(O)—(CH2)r— where q and r independently of one another are an integer from 0 to 18, it also being possible for the linker L to be linked to V via an oxygen bridge.
A reactive monomer as claimed in any of the preceding claims, wherein the linkers L are selected from the group consisting of the (CnH2n)-alkyl radicals where n is an integer from 0 to 18 or the group which is a polyether —(CH2)k-[O—(CH2)m]o—O—(CH2)p- where k, m, p independently of one another are 2 and o is an integer from 2 to 4 or the group which is an amine —(CH2)w—NH—(CH2)u— where w and u independently of one another are an integer from 3 to 6 or the group which is an amide —(CH2)q—C(O)—N—(CH2)r— or —(CH2)q—N—C(O)—(CH2)r— where q and r independently of one another are an integer from 1 to 5, it being possible for the linkers L to be linked to V also via an oxygen bridge.
A reactive monomer as claimed in one or more of the preceding claims, which has the following structure
- 12 13
A mono-, oligo- or polynucleotide, obtainable by linking the mono-, oligo- or polynucleotide terminally to at least one reactive monomer of the formula (I).
- X—Ll—Vv—(A)a (I)
- wherein l, v independently of one another are 0 or 1 and a is an integer between 1 and 5, where
- X is a phosphoramidite (II) or a phosphonate (III),
- 14
- wherein R2 and R3 independently of one another are a branched or unbranched C1 to C5 alkyl radical and R1 is methyl, allyl or β-cyanoethyl,
- V is a branching unit composed of an atom or of a molecule having at least three binding partners,
- A is an acetal of the formula (IV),
- 15
- where the radicals Y and Z independently of one another are identical or different branched, unbranched or cyclic, saturated or unsaturated hydrocarbons having from one to 18 carbon atoms, it also being possible for the radicals Y and Z to be linked to one another,
- and wherein
- L are linkers which are suitable to link X to A or X to V and V to A. M. A mono-, oligo- or polynucleotide as claimed in claim 9, which corresponds to the formula VIII
- (M)s[—X′—LlVv(A)a]z (VIII)
- where (M)s are from s monomeric units of any sequence, where s is 1 or greater and (M)s can be branched or unbranched, and X′ is a phosphorus-containing group of the formula (IX), which is terminally linked to the mono-, oligo- or polynucleotide,
- 16
- where U is O or S, W is OH, SH or H and Q is O or NH, and in which z is 1 or greater and l, v, a, L, V and A have the above-mentioned meaning.
A mono-, oligo- and polynucleotide, which have been modified with at least one aldehyde group and in which the nucleotide chain comprises p-RNA, homo DNA, p-DNA or CNA.
Dependencies are inferred from the claim text.