AU2011253622A1

AU2011253622A1 - Oligonucleotide synthesis using photocleavable linkers

Info

Publication number: AU2011253622A1
Application number: AU2011253622A
Authority: AU
Inventors: Robert Haner; Jurg Hunziker; Sirnon Matthias Langenegger; Francois Jean-Charles Natt
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2006-01-18
Filing date: 2011-11-23
Publication date: 2011-12-15

Abstract

The present invention relates to a process for the preparation of an oligomeric 5 compound made up of two or more individual oligomers, in which said oligomeric compound the individual oligomers are separated by a photocleavable linker, comprising the step of photoactively cleaving said linker.

Description

Australian Patents Act 1990 - Regulation 3.2A ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title "Oligonucleotide synthesis using photocleavable linkers" The following statement is a full description of this invention, including the best method of performing it known to us:- OLIGONUCLEOTIDE SYNTHESIS USING PHOTOCLEAVABLE LINKERS This application is a divisional of Australian Patent Application No. 2007207131, the entire content of which is incorporated herein by reference. 5 Background of the invention Preparation of a double stranded DNA or RNA usually involves two independent multi-step processes (i.e. synthesis, deprotection. purification and quality assurance). While not an issue for most applications, this becomes rate-limiting for scaling up the technology, e.g. for high-throughput applications or for therapeutic applications which 10 require large amount of oligonucleotides. One approach, described by Pon et al [1], termed tandem synthesis, is based on the principle that one (long) oligonucleotide containing a post-synthetically cleavable linker is prepared. Subsequent cleavage then yields the two complementary strands (illustrated in Scheme 1). According to Pon, Richard T.; Yu, Shuyuan. Nucleic Acids Research 2005, 33(6), 1940-1948 15 and Pon, Richard T.; Yu, Shuyuan. PCT Int. Apple. 2002), WO 2002020537 A2 and Ferreira, Fernando; Meryer, Albert; Vasseur, Jean-Jacques; Morvan, Francois. J. Org. Chem. Online publication, 2005., two or more oligonucleotides separated with a base-labile linker are synthesized sequentially. The linker is then cleaved under the conditions used for the support cleavage and the base/phosphodiester deprotection 20 of the oligonucleotides. One drawback with this procedure is that it does not allow the purification of the oligonucleotides by the trityl-on approach since only the 5-terminal oligonucleotide will bear this residue. Incorporation of photocleavable residues in oligonucleotides has been described for reversible labeling or immobilization of oligonucleotides and in applications such as SNP genotyping, WO 9967619, or as protecting group in RNA synthesis Stutz, Alfred; Pitsch, Stefan. Synlett 1999, 25 (Spec.), 930-934. . Recently, photoactivatable siRNAs or "caged interfering RNAs" have been reported.. In these cases, the siRNA antisense strand was modified either on its 5'-end by the introduction of a photocleavable moiety bearing a label group, W02004045547, or internally by the covalent attachment of 4,5-dimethoxy-2 nitrophenyl groups to the oligoribonucleotide phosphodiester backbone Shah, Samit; 30 Rangarajan, Subhashree : Friedman, Simon, H. Angew. Chem. Int. Ed. 2005,44, -1328-1332. As such, the oligonucleotide could be photo-activated at a desired time point of the biological experiment, e.g. after its transfection in a cell.

WO 2007/082713 PCT/EP2007/000337 -2 The inventors have developed a compound which can be used to simplify the process of synthetically preparing double stranded ribonucleic acids, and provides a method which has several advantages over existing methods. Especially, the use of the compounds of the present invention simplifies the process of the synthetic preparation of double-stranded ribonucleic acids such as siRNAs. By performing the method according to the invention, both strands of a double stranded ribonucleic acid can be obtained from a single synthesis without compromising the quality of the reagent, since it is possible to purify the photocleavable oligonucleotide before release of both strands through irradiation. This feature can be of particular importance in high-throughput applications (e.g. siRNA libraries) or in large scale applications (e.g. siRNA therapeutics). The photocleavable nucleic acids can also be used as such in enzymatic applications (e.g. the incorporation in plasmids), or in biological experiments (e.g. in cellular assay or in animal model assay) and released at any stage of the experiment. Lastly, the inter-oligonucleotide photocleavable linker can be designed to integrate additional functionalities such as label residues or cargo residues which may allow its detection of enhance its pharmacological properties The inventors have developed a new synthesis strategy using a novel photocleavable linker for the one-step synthesis of multiple compounds. The linker and the use thereof is applicable to the preparation of multiple biopolymers such as for instance polypeptides, polysaccharides or polynucleotides or combinations thereof. It can be especially useful in applications where a controlled ratio of two or more reagents is required. It is particularly suited, but not limited, to the preparation of short interfering RNAs (siRNAs) since it allows the synthesis of both strands as one long self complementary oligonucleotide with a photocleavable linker resulting after oligonucleotide deprotection and purification in one long oligonucleotide which can also be called photocleavable short hairpin RNA (photo-shRNA). With respect to siRNA synthesis, this strategy offers the following advantages over standard siRNA preparation; only one molecule is synthesized, purified, and analyzed; light irradiation can be performed on a purified photo-shRNA which consequently ensures the annealing of the siRNA duplex with a perfect stoichiometry; sample tracking of individual strands is not required since non-annealed strands WO 2007/082713 PCT/EP2007/000337 never exist; light irradiation of photo-shRNA to release siRNA can be done at any time, even in biological experiments (e.g. in situ irradiation of photo-shRNA post transfection or post-injection); and the linker may be derived to bear functional groups which may enhance cellular uptake or tissue-specific delivery. The results disclosed herein show that the proposed ortho-nitrobenzyl based linkers are perfectly compatible to standard RNA or DNA oligonucleotide synthesis using phosphoramidite chemistry. The linkers are stable under cleavage and deprotection conditions required to release crude oligonucleotides as well as the aqueous acidic conditions required removing the terminal 5'-dimethoxytrityl group. The present invention provides a compound and the use of the compound which allows the synthesis of multiple purified oligonucleotides in a single synthesis process. In its current form, cleavage of the linker by light irradiation releases oligonucleotides bearing a terminal phosphate residue at the linker anchoring terminus. While this may be a disadvantage for some applications requiring terminal hydroxyl groups, it turns to be an advantage for the preparation of siRNAs which require a phosphate group at the 5'-terminus of the guide strand for biological function Meister, Gunter; Tuschl, Thomas. Nature 2004, 431(7006), 343-349. The simplicity of the method according to the invention is shown in Figure 1. In a first aspect the invention relates to a process for the preparation of an oligomeric compound made up of two or more individual oligomers, in which said oligomeric compound the individual oligomers are separated by a photocleavable linker, comprising the step of photoactively cleaving said linker. The individual oligomers may be independently chosen from the group consisting of oligonucleotides, oligosaccharides, oligopeptides. In one embodiment, the individual oligomers are oligonucleotides which may or may not be complementary. Preferably, the oligomers are fully or partially complementary. Partial complementarity means that 50%-99% of the nucleotides in the oligonucleotides are complementary.

WO 2007/082713 PCT/EP2007/000337 -4 In a preferred embodiment, the individual oligomers are oligoribonucleotides which may be fully or partially complementary. In a preferred embodiment, the linker is stable under the deprotection conditions of each individual oligorner. Preferably, the linker group is cleavable by UV or visible light irradiation. In a preferred embodiment, said oligonucleotides are two oligoribonucleotides In an additional embodiment, the linker is a compound of formula I, PG R6 X R5 R1 R4 R2 R3 wherein; PG is (Ar1)(Ar2)(Ar3)-C-, wherein Ar1, Ar2, Ar3 are independently chosen from the group consisting of;

CH

3 0C6H 4 - and C 6

H

5 -, or PG is a substituted silyl group (RI')(R2')(R3')Si-, wherein R1', R2', R3' is independently chosen from the group consisting of lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkyloxy, or aryloxy; X is 0, N, or S; R1, R2, R3, R4, and R5 is independently chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, halogen. CN. COOH. C(O)O lower alkvl/arvi. CONR'R". CHO. C(0) lower alkvl/arvi.

WO 2007/082713 PCT/EP2007/000337 OH, 0- lower alkyl/aryl, OC(O) lower alkyl/aryl, SH, S- lower alkyl/aryl, SO 3 H, S020 lower alkyl/aryl, SO 2 NIR'R", NH 2 , N- lower alkyl/aryl, NHC(O) lower alkyl/aryl, and at least one of the substituents R1-R5 is a nitro, a nitrosyl, or a diazo group; two or more of the substituents RI, R2, R3, R4, and R5 can form one or several rings which may be further substituted with groups defined as for R1, R2, R3, R4, or R5; at least one of the substituents R1, R2, R3, R4, or R5 is a phosphoramidite, a phosphonate, or a phosphotriester bearing group able to form a phosphodiester or phosphorothioate linkage to the growing oligonucleotide chain or an amine, an activated carboxylic ester, an isocyanate or an isothiocyanate, able to form an amide, a urea or a thiourea linkage to the growing oligonucleotide chain; R6 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, CN, COOH, C(0)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, 0-lower alkyl/aryl, OC(O)lower alkyl/aryl, S-lower alkyl/aryl, SO 3 H, SO 2 0-lower alkyl/aryl, SO 2 NR'R", N- lower alkyl/aryl, NHC(O) lower alkyl/aryl. This linker is preferably cleavable by light, such as UV light or visible light, or a laser beam. Even more preferred is a process as described above, wherein the linker is a compound of formula 11, WO 2007/082713 PCT/EP2007/000337 -6 PG R6 X R5 R1 R4 R2 'U R3 RIO R11 R9 R12 R8 R8 .Y R7 z Formula I wherein, PG is (Ar1)(Ar2)(Ar3)C-, wherein Ar1, Ar2, Ar3 are independently chosen from the group consisting of;

CH

3 0C 6

H

4 -, C 6

H

5 -, or PG is a substituted silyl group (R1')(R2')(R3')Si-, wherein R1', R2', R3' is independently chosen from the group consisting of lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkyloxy, or aryloxy; X is O, N, or S; R1, R2, R3, R4, and R5 is independently chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, halogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, OH, 0- lower alkyl/aryl, OC(O) lower alkyVaryl, SH, S- lower alkyl/aryl, SO 3 H, S0 2 0 lower alkyl/aryl, SO 2 NR'R", NH 2 , N- lower alkyl/aryl, NHC(O) lower alkyl/aryl, and at least one of the substituaents R1 -R5 is a nitro, a nitrosyl, or a diazo group; two or more of the substituents R1, R2, R3, R4, and R5 can form one or several rings which may be further substituted with groups defined as for R1, R2, R3, R4, or R5; WO 2007/082713 PCT/EP2007/000337 -7 R6 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, CN, COOH, C(0)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alky;l/aryl, 0- lower alkyllaryl, OC(O) lower alkyl/aryl, S- lower alkyl/aryl, SO 3 H, S020- lower alkyl/aryl, SO 2 NR'R", N- lower alkyl/aryl, NHC(O) lower alkyl/aryl; U, V, W are forming a chain which replaces one of the substituents R1 - R5 on one end and one of the substituents R7 - R1 1 on the other end; U, V, W can independently be absent, or be an alkylene (-R-), cycloalkylene (-R-), or arylene (-Ar-) group, -0-, -S-, -NR'-, -C(O)-, -C(0)0-, -C(O)NR'-, -OC(0)0-, OC(O)NR'-, -NR'C(O)NR"-, -OC(S)NR'-, -NR'C(S)NR"-, -S(O)-, -S(0 2 )-, -S(0 2 )NR'-, OP(0 2 )0-, and may contain a label or fluorophore or a group which serves to improve the pharmacological profile of the oligonucleotide. R7, R8, R9, R10, and R1 I are independently chosen form the group consisting of, hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, halogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, OH, 0- lower alkyl/aryl, OC(0) lower alkyl/aryl, SH, S- lower alkyl/aryl, SO 3 H, S0 2 0 lower alkyl/aryl, SO 2 NR"R", NH2, N- lower alkyl/aryl, NHC(O) lower alkyl/aryl and at least one of the substituents R-RI 1 is a nitro, a nitrosyl, or a diazo group; RI 2 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) Iower alkyl/aryl, 0- lower alkyl/aryl, OC(O) lower alkyl/aryl, S lower alkyl/aryl, SO 3 H, S0 2 0- lower alkyl/aryl, SO 2 NR'R", N- lower alkyVaryl, NHC(O) lower alkyl/aryl; Y is 0, N, or S; Z is a phosphoramidite, a phosphonate, or a phosphotriester group able to form a phosphodiester or phosphorothioate linkage to the growing oligonucleotide chain or an amine, an activated carboxylic ester, an isocyanate or an isothiocyanate which is WO 2007/082713 PCT/EP2007/000337 able to form an amide, a urea or a thiourea linkage to the growing oligonucleotide chain. Even more preferred is a process according to the above, wherein the linker is a compound of formula 1, PG R6 X R5 R1 R4 R2 R3 Formula I wherein; PG is dimethoxytriphenylmethyl; X is 0; R1 is a nitro group; R3 is -CH 2 -0-P(N[iPr]2)-0-CH 2

-CH

2 -CN); R2, R4, R5, and R6 are hydrogen; More preferred is a process according to the above, wherein the linker is a compound of formula 1i WO 2007/082713 PCT/EP2007/000337 -9 PG R6 X R5 R1 R4 R2 'U R3 RIO W R11 R9 R12 R R8 .'Y R7 z Formula i wherein; PG is dimethoxytrphenylmethyl; X and Y are 0; RI and R7 are nitro groups: R2, R4, R5, R6, R8, RIO, R11, and R12 are hydrogen; U is oxygen and replaces R3; V is -CH 2

-CH

2

-CH

2 -; W is oxygen and replaces R9; Z is -P(N[iPr] 2 )-0-CH-CH 2 -CN). In yet a further embodiment, the present invention provides a compound according to formula I, WO 2007/082713 PCT/EP2007/000337 -10 PG R6 X R5 RI

I

R4 R2 R3 formula I wherein; PG is (ArI)(Ar2)(Ar3)C-, wherein Ar1, Ar2, Ar3 are independently chosen from the group consisting of;

CH

3 0C 6

H

4 - and C 6

H

5 -, or PG is a substituted silyl group (R1')(R2')(R3')Si-, wherein R1', R2', R3' is independently chosen from the group consisting of lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkyloxy, and aryloxy; X is 0, N, or S; R1, R2, R3, R4, and R.5 is independently chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, halogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, OH, 0- lower alkyl/aryl, OC(O) lower alkyl/aryl, SH, S- lower alkyl/aryl, SO 3 H, S0 2 0 lower alkyl/aryl, SO 2 NR'R", NH 2 , N- lower alkyl/aryl, and NHC(O) lower alkyl/aryl; and at least one of the substituents RI-R5 is a nitro, a nitrosyl, or a diazo group; two or more of the substituents RI, R2, R3, R4, and R5 can form one or several rings which may be further substituted with groups defined as for RI, R2, R3, R4, or R5; at least one of the substituents RI, R2, R3, R4, or R5 is a phosphoramidite, a phosphonate, or a phosphotriester bearing group able to form a phosphodiester or phosphorothioate linkage to the growing oligonucleotide chain or an amine, an activated carboxylic ester, an isocyanate or an isothiocyanate, able to form an amide, a urea or a thiourea linkage to the growing oligonucleotide chain; WO 2007/082713 PCT/EP2007/000337 - 11 R6 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, CN, COOH, C(0)0 lower alkyl/aryl, CONR'R", CHO, C(0) lower alkyl/aryl, 0-lower alkyl/aryl, OC(0)lower alkyl/aryl, S-lower alkyl/aryl, SO 3 H, S0 2 0-lower alkyl/aryl, SO 2 NR'R", N- lower alkyl/aryl, and NHC(0) lower alkyl/aryl. More preferred is a compound of formula Il PG R6 X R5 RI R4 R2 R3 R10W R11 R9 R12 I R #R8 zY R7 z Formula |1 wherein, PG is (Ar1)(Ar2)(Ar3)C-, wherein Ar1, Ar2, Ar3 are independently chosen from the group consisting of;

CH

3 0C 6

H

4 -, C 6

H

5 -, or PG is a substituted -silyl group (R1')(R2'XR3')Si-, wherein R1', R2', R3' is independently chosen from the group consisting of lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkyloxy, or aryloxy; X is 0, N, or S; R1, R2, R3, R4, and R5 is independently chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, WO 2007/082713 PCT/EP2007/000337 - 12 halogen, CN, COOH, C(0)O lower alkyl/aryl, CONR'R", CHO, C(0) lower alkyl/aryl, OH, 0- lower alkyl/aryl, OC(O) lower alkyl/aryl, SH, S- lower alkyl/aryl, SO 3 H, S0 2 0 lower alkyl/aryl, S0 2 NR'R", NH 2 , N- lower alkyl/aryl, and NHC(O) lower alkyl/aryl, and at least one of the substituents R1-R5 is a nitro, a nitrosyl, or a diazo group; two or more of the substituents R1, R2, R3, R4, and R5 can form one or several rings which may be further substituted with groups defined as for RI, R2, R3, R4, or R5; R6 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkyihalogen, CN, COOH, C(0)O lower alkyl/aryl, CONR'R". CHO, C(0) lower alky/aryl, 0- lower alkyl/aryl, OC(0) lower alkyl/aryl, S- lower alkyl/aryl, SO 3 H, SO 2 0- lower alkyl/aryl, SO 2 NR'R", N- lower alkyl/aryl, and NHC(O) lower alkyl/aryl; U, V, W are forming a chain which replaces one of the substituents RI - R5 on one end and one of the substituents R7 - RI 1 on the other end; U, V, W can independently be absent, or be an alkylene (-R-), cycloalkylene (-R-), or arylene (-Ar-) group, -0-, -S-, -NR'-, -C(O)-, -C(0)0-, -C(O)NR'-, -OC(0)0-, OC(O)NR'-, -NR'C(O)NR-, -OC(S)NR'-, -NR'C(S)NR"-, -S(O)-, -S(0 2 )-, -S(0 2 )NR'-, OP(0 2 )0-, and may contain a label or fluorophore or a group which serves to improve the pharmacological profile of the oligonucleotide. R7, R8, R9, R10, and RI 1 are independently chosen form the group consisting of, hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, halogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, OH, 0- lower alkyl/aryl, OC(O) lower alkyl/aryl, SH, S- lower alkyl/aryl, SO 3 H, S0 2 0 lower alkyl/aryl, SO 2 NR'R", NH2, N- lower alkyl/aryl, NHC(O) lower alkyl/aryl and at least one of the substituents R7-RI 1 is a nitro, a nitrosyl, or a diazo group; R12 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyVaryl, 0- lower alkyl/aryl, OC(O) lower alkyl/aryl, S- WO 2007/082713 PCT/IEP2007/000337 - 13 lower alkyVaryl, SO 3 H, S0 2 0- lower alkyl/aryl, SO 2 NR'R", N- lower alkyl/aryl, NHC(O) lower alkyl/aryl; Y is 0, N, or S; Z is a phosphoramidite, a phosphonate, or a phosphotriester group able to form a phosphodiester or phosphorothioate linkage to the growing oligonucleotide chain or an amine, an activated carboxylic ester, an isocyanate or an isothiocyanate which is able to form an amide, a urea or a thiourea linkage to the growing oligonucleotide chain. Even more preferred is a compound of formula I, PG R6 X R5 R1 R4 R2 R3 Formula I wherein; PG is dimethoxytripheriylmethyl; X is 0; R1 is a nitro group; R3 is -CH-0-P(NFiPr] 2 0-CH 2

-CH

2 -CN); R2, R4, R5, and R6 are hydrogen; More oreferred is a cnmnnuind arcordinn tn formula 11 WO 2007/082713 PCT/EP2007/000337 -14 PG R6 X

R

5 RI R4 R2 U R3 RIOw R11 R9 R12 N I R R8 z.Y R7 z Formula i wherein; PG is dimethoxytriphenylmethyl; X and Y are 0; R1 and R7 are nitro groups; R2, R4, R5, R6, R8, R10, R11, and R12 are hydrogen; U is oxygen and replaces R3; V is -CH 2

-CH

2

-CH

2 -; W is oxygen and replaces R9; Z is -P(N[iPr]2)-O-CHz-CH-CN).

WO 2007/082713 PCTIEP2007/000337 -15 The term "lower" in connection with organic radicals or compounds means a compound or radical which may be branched or unbranched with up to and including 8 carbon atoms, preferably 1-6 or more preferably 1-4, or 2-6 carbon atoms. Lower alkyl represents, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and branched pentyl, n-hexyl and branched hexyl, n-heptyl, branched heptyl, n-octyl and branched octyl. iPr means isopropyl. MATERIALS AND METHODS Synthesis of Photo-Cleavable Phosphoramidites Scheme 2 OH NO 2 1;RO: OIO H DMTOM O'OY OH 1 b 2 R=H 4 CN 3 R=DMT 3-Hydroxymethyl-4-nitro-phenol (1) Compound I was synthesized according to the literature R. Reinhard, B.F. Schmidt, J. Org. Chem., 1998, 63, 2434-2441. (5-[3-(3-Hydroxymethyl-4-nitro-phenoxy)-propoxy]-2-nitro-phenyl}-methanol (2) Compound 1 (2.02 g, 12 mmol) was dissolved in DMF (26 ml). 1,3-Dibromopropane (560 pl, 5,4 mmol), K 2

CO

3 (2.0 g, 14.4 mmol) and potassium iodide (0.2 g, 1.2 mmol) were added and the orange suspension was stirred at 900 C for 3h. The reaction solution was then cooled to room temperature and poured into 140 ml of water. The precipitate was filtered off, washed with water, sat. aq. NaHCO 3 solution and then again twice with water, dried to give 1.82 g of slightly yellow crystals. Yield 89%. TLC (AcOEt/hexane 1:1): Rr 0.21. 'H-NMR (300 MHz, DMSO-d 6 ): 2.27 (q, J = 6.2.

CH

2

CH

2

CH

2 ); 4.30 (t, J = 6.2, CH 2

CH

2

CH

2 ); 4.84 (s, CH 2 OH, C'H 2 OH); 5.59 (s, WO 2007/082713 PCT/EP2007/000337 -16

CH

2 OH, C'H 2 OH); 7.05 (dd, J = 9.1, 2.8, 2 arom. H); 7.36 (d, J = 2.8, 2 arom. H); 8.12 (d, J = 9.1, 2 arom. H). ' 3 C-NMR (75 MHz, DMSO-d6): 28.2; 60.3; 65.0; 112.8; 113.2; 127.5; 139.4; 142.4; 162.9. HR-ESI-MS (pos. mode): 401.0959 ([M+Na]*; calc. 401.0960). [5-(3-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethylJ-4-nitro-phenoxy} propoxy)-2-nitro-phenyl]-methanol (3) 1.8 g (4.76mmol) 2 was dissolved in 45 ml pyridine under nitrogen. A solution of 1.61g (4.76 mmol) DMTCI in 20 ml dry pyridine was added at room temperature. The reaction mixture was stirred over night, diluted with sat. aq. NaHCO 3 solution and extracted twice with AcOEt. The combined organic phases were washed with water and Brine, dried (K 2

CO

3 ) and evaporated under reduced pressure. The resulting oil was purified by column chromatography (silicagel; AcOEt/hexane 1:3, 2% Et 3 N -+ AcOEt, 2% Et 3 N) to give 1.45 g 3 as a yellow foam. Yield 45%. TLC (AcOEt/hexane 1:1): Rr 0.43. 'H-NMR (300 MHz, CDCl 3 ): 2.40 (q, J = 6.0, CH 2

CH

2

CH

2 ); 2.56 (t, J = 6.4, CH 2 OH); 3.78 (s, 2 OMe); 4.33 (t, J = 6.0, CH2CH2CH 2 ); 4.65 (s, CH 2 ODMT); 4.99 (d, J = 6.4, CH 2 OH); 6.8-6.95 (m, 6 arom. H); 7.2-7.4 (m, 8 arom. H); 7.47 (m, 2 arom. H); 7.70 (m, 2 arom. H); 8.12 (d, J = 9.1, 1 arom. H); 8.18 (d, J = 9.1, 1 arom. H). HR-ESI-MS (pos. mode): 703.2264 ([M+Na]*; calc. 703.2267). Diisopropyl-phosphoramidous acid 5-(3-(3-[bis-(4-methoxy-phenyl)-phenyl methoxymethyl]-4-nitro-phenoxy)-propoxy)-2-nitro-benzy ester 2-cyano-ethyl ester (4) 1.0 g (1.47 mmol) 3 was dissolved in 6 ml CH 2

CI

2 under nitrogen. Then 0.6 ml HOnig's base, and 0.38 g (1.62mmol) 2-cyanoethyl diisopropylamidochlorido phosphite were added and the mixture was stirred for 3 h at room temperature. The reaction mixture was directly applied onto silica gel and purified by column chromatography (silica gel (50g); AcOEt/hexane 3:7, 2% Et 3 N -+ AcOEt, 2% Et 3 N). 1.05 g 4 as a yellow foam was obtained. Yield 81%. TLC (AcOEt/hexane 1:1): Rr 0.79. 'H-NMR (300 MHz, CDCl 3 ): 1.21 (d, J = 6.9, 2 MeCHN): 2.40 (q, J = 6.0,

CH

2

CH

2

CH

2 ); 2.60 (t, J = 6.3, CH 2 CN); 3.6-4.0 (m, OCH 2

CH

2 CN, 2 Me 2 CHN); 3.78 (s, 2 OMe); 4.32 (t, J = 6.0, CH 2

CH

2

CH

2 ); 4.65 (s, CH2ODMT); 5.14 (m, CH 2 OP); 6.8 6.95 (m, 6 arom. H); 7.2-7.4 (m, 8 arom. H); 7.47 (m, 2 arom. H); 7.70 (m, 2 arom. H); WO 2007/082713 PCT/EP2007/000337 - 17 8.11 (d, J = 9.0, 1 aroma. H); 8.18 (d, J = 9.1, 1 arom. H). 31 P-NMR (162 MHz, CDC 3 ): 149.12. HR-ESI-MS (pos. mode): 903.3326 ([M+Na]*; calc. 903.3346). Scheme 3 HO DMTO DMTO INO DMTCI NO2

NO

2 HO HO HO 5 6 CNN N NN N~ N DMTO DMTO

NO

2

NO

2 NC,_- 0 N NC O P N' 7 8 {4-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethylJ-3-nitro-phenyl)-methano (5) and {4-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-2-nitro-phenyl} methanol (6) (4-Hydroxymethyl-2-niitro-phenyl)-methano (TCl Tokyo Kasei, 3.0g, 16.4 mmol) was dissolved in pyridine (30ml) under an argon atmosphere. 4,4'-Dimethoxytrityl chloride (5.55g. 16.4mmol) was added in portions over a period of 30 minutes while cooling the solution to O'C. The reaction mixture was stirred over night at room temperature, diluted with sat. aq. NaHCO 3 solution and extracted twice with AcOEt. The combined organic phases were washed with water and brine, dried (NaHCO 3 ) and evaporated under reduced pressure. The resulting oil was purified by column chromatography (silicagel; AcOEt/hexane 1:4, 1% Et 3 N -+ AcOEt, 1%Et 3 N) to give 0.91g of 5 (11%) and 3.18g of 6 (40%) as yellow foams.

WO 2007/082713 PCT/EP2007/000337 - 18 Analytical data for 5: TLC (AcOEt/hexane 1:2): R, 0.11. 'H-NMR (300 MHz, CDCI 3 ): 1.90 (t, J=6.4, CH 2 OH); 3.70 (s, 2 OMe); 4.52 (s, CH 2 0DMT); 4.68 (s, CH 2 OH); 6.72 6.79 (m, 4 arom. H); 7.06-8.03 (m, 12 arom. H). EI-MS: 485 [M+']. Analytical data for 6: TLC (AcOEt/hexane 1:2): R, 0.24. 'H-NMR (300 MHz. CDC 3 ): 1,71 (t, J=6.4, CH 2 OH); 3.71 (s, 20Me); 4,19 (s, CH20DMT); 4,85 (s, CH 2 OH); 6.73 6.78 (m, 4 arom. H); 7..06-8.03 (m,12 arom. H). EI-MS: 485 [M+]. Diisopropyl-phosphoramidous acid 4-[bis-(4-methoxy-phenyl)-phenyl methoxymethyl]-3-nitro-benzyl ester 2-cyano-ethyl ester (7) Alcohol 5 (300mg, 0.62mmol) was dissolved in 2.4ml CH 2

CI

2 under an argon atmosphere. 2-Cyanciethyl-2(diisopropylamido)phosphite (0.28ml, 0.77mmol) and tetrazolide (145mg, 0.846mmol), dissolved in CH 2

CI

2 (2.4ml) were added. The mixture was stirred at room temperature for 3 h, diluted with sat. aq. NaHCO 3 solution and extracted twice with CH 2

CI

2 . The combined organic phases were dried (NaHCO 3 ) and concentrated under reduced pressure. The resulting oil was purified by column chromatography (silicagel;AcOEt/hexane 1:4, 1%N-methyl-morpholine) to give 7 (253mg, 61%) as a yellow foam. TLC (AcOEt/hexane 1:2): Rr 0.50. 'H-NMR (300 MHz, CDCl 3 ): 1-20 (2d, J=6.8, 4MeCHN); 2.65 (t, J=6.4, CH 2 CN); 3.61-3.69 (m,

OCH

2

CH

2 CN); 3.77 (s, OMe); 3.79-3.91 (m, 2Me 2 CHN); 4.57 (s, CH 2 0DMT); 4.76 (m, CH 2 OP); 6.78-6.84 (m, 4 arom. H); 7.20-7.48 (m, 10 arom. H) 7.64 (d, J=8.1, larom. H); 8.01 (s, 1 arom. H); 8.09 (d, J=8.1, 1 arom. H). 31 P-NMR (162 MHz,

CDC

3 ): 150.84. ESI-MS (pos. mode): 708 ([M+Na]*; calc. 708). Diisopropyl-phosphoramidous acid 4-[bis-(4-methoxy-phenyl)-phenyl methoxymethyl]-2-nitro-benzyl ester 2-cyano-ethyl ester (8) Alcohol 6 (300mg, 0.62mmol) was dissolved in CH 2

CI

2 (2.4ml) under an argon atmosphere. 2-Cyanoethyl-2(diisopropylamido)phosphite (0.28m, 0.77mmol) and tetrazolide (145mg, 0.85mmol), dissolvded in CH 2

CI

2 . The combined organic phases were dried (NaHCO 3 ) and concentrated under reduced pressure. The resulting oil was purified by column chromatography (silicagel; AcOEt/hexane 1:4, 1 %N-methyl-morpholine) to give 8 (352mg, 85%) as a yellow foam. TLC (AcOEt/hexane 1:2): R, 0.42. 'H-NMR (300 MHz, CDCl 3 ): 1.14 (2d, J=6.8, 4 MeCHN); 2.58 (t, J=6.4, CH 2 CN); 3.54-3.66 (m, WO 2007/082713 PCT/EP2007/000337 -19

OCH

2

CH

2 CN); 3.72 (s, OMe); 3.75-3.96 (m, 2Me 2 CHN); 4.18 (s, CH 2 0DMT); 5.00 (m, CH 2 0P); 6.75-6.8) (m, 4 arom. H); 7.12-7.42 (m, 10 arom. H); 7.58 (d, J=8.1, 1 arom. H); 7.71 (d, J:8.1, I arom. H); 7.97 (s, 1 arom. H). 3 1P-NMR (162 MHz, CDC13): 150.35. ESI-MS (pos. mode): 708 ([M+Na]*; calc. 708). Synthesis of OligonLcleotides. Oligodeoxynucleotides were synthesized on a 392 DNA/RNA Synthesizer (Applied Biosystems) according to the phosphoramidite chemistry[6,7]. The deoxynucleoside phosphoramidites were from Transgenomic (Glasgow, UK). Oligodeoxynucleotides were prepared by the standard synthetic procedure ("trityl-off" mode). Detachment from the solid support and final deprotection was achieved by treatment with 30% ammonium hydroxide overnight at 55 0 C. Oligoribonucleotides were synthesized on a Mermade DNA plate synthesizer (Bioautomation Inc.) according to the TOM protected RNA phosphoramidite chemistry [3]. The ribonucleoside phosphoramidites were from Qiagen AG (Hombrechtikon, CH). Oligonucleotides were prepared according to the standard synthetic procedure ("trityl-on" mode). Detachment from the solid support and base/phosphodiester backbone deprotection was achieved by treatment with aqueous Ammonia/Methylamine solution (1:1) for 30 minutes at 65"C. 2'-TOM deprotection was achieved by treatment with TEA-HF solution for I h at 65 0 C. Purification of oligonucleotides Where specified, oligonucleotides were purified with OASIS cartridges (Waters AG). First, the cartridge was conditioned with 1 ml acetonitrile followed by 1 ml of 0.1M of triethylammonium acetate solution (TEAA). The crude oligonucleotides was loaded on the cartridge which was washed with a 15% acetonitrile solution in 0.1 M TEAA to remove all trityl-off truncated sequences. On-cartridge detritylation was performed with 1 ml of an aqueous 3% dichloroacetic acid solution. Before elution of the purified trityl-off oligonucleotide with a 1:1 acetonitrile/water solution, the cartridge was washed with 1-2 ml of 0.1 M TEAA or water. Scheme 3: Oligonucleotides connected via a photocleavable linker.

WO 2007/082713 PCTIEP2007/000337 -20 igonudeoede-0 ,0 O 0 NO, PAM PAMO standard ofigonudeotide synthesis 0, o N020 0 OUTO~~ oO o 7 0 'I+ .0' O-cagonudeatde Photocleavage of Olicionucleotides. Cleavage of oligonucleotides was performed by irradiation of a solution of the oligonucleotide (0.1 to 10 optical densities) in water (10-100 microliters in a conventional plastic cuvette) with light (352 nm wavelength; two 8Watt tubes) for 15 to 180 minutes. The treatment resulted in the formation of two individual oligonucleotides (Scheme 4 and Figure 1). Scheme 4: Example of the generation of two oligodeoxynucleotides by post-synthetic irradiation. $!.TTTY-0.A h 0 0 o hv (352 nm) TTTTT-p (MW:1539) 0 o hv T A A A A - p (MW: 1575) S- T A A A A-O, A 0 0 (MW: 3506) Table 1: MS analysis of oligonucleotides before and after irradiation. Mcalc. Mmeas. Before irradiation 3506 3506.00 After irradiation 1575 1574.63 1539 1538.63 WO 2007/082713 PCT/EP2007/000337 -21 Scheme 5: Example of the generation of two oligonucleotides by post-synthetic irradiation of a of DNA-RNA oligonucleotide chimera. UUU GGA GGG AUC UCG CUC C TdG 0 0- -m m m m 00 '' 0 H 0 0 McalclOA 754.9 UUU GGA GGG AUC UCG CUC C TdGL- OH + HO O--TmTmTm m Mcalc=6744.2 Ucal=3668.2 Table 2: MS analysis of oligonucleotides before and after irradiation. Mcalc. Mmeas. Before irradiation 10754.9 10757.87 After irradiation 6744.2 6745.56 3668.2 3668.82 A first photocleavable oligodeoxynucleotide was prepared using standard phosphoramidite chemistry by concomitant incorporation of phosphoramidites 8 and 7 on the 5' end of a pentadeoxynucleotide (sequence 5'-AAAAT-3') and further extension by a pentalthymidylate. Upon cleavage/deprotection and desalting, the photocleavable oligodeoxynucleotide was irradiated at 352 nm for 2h on (a 16W UV lamp). The irradiated solution, directly measured by Electrospray Mass Spectrometry (ES-MS), displayed two peaks corresponding to both pentadeoxynucleotides (bearing a terminal phosphate either at 5' or 3'-end) resulting from the cleavage of both orthophenyl moieties (scheme 4). Using the phosphorarnidite 4, a photocleavable chimeric DNA/RNA was synthesized using standard phosphoramidite chemistry on a 96-well Mermade synthesizer. The oligonucleotide consisted of a dodecathymilydate followed by the bis-ortho nitrobenzyl linker and further extended with two deoxynucleotides followed by a 1 9nt Inna nlinnrihnnueilantidp ThA chimprn wa- nrannrpd in thp "tritv-nn" mnnrI ni irifipid - 22 by reverse-phase cartridge and analyzed by Mass Spectrometry before and after light irradiation (366 nm for 15 min. at room temperature). Two peaks were detected corresponding to the dodecathymidylate bearing a phosphate residue on its 5' terminus and the 21 nt long DNA/RNA chimera with a 3'-phosphate residue. 5 We then synthesized on a 96-well Mermade synthesizer one long DNA/RNA chimera composed of two complementary strands separated by the bis-ortho-nitrobenzyl linker. Each strand was formed of a deoxynucleotide dimer on its 3'-end and a 19-nt long oligoribonucleotide. The chimera was prepared in the "trityl-on" mode, purified by reverse-phase cartridge and analyzed by Mass Spectrometry before and after light 10 irradiation (366 nm for 15 min. at room temperature). Before irradiation we observed a unique peak corresponding to the full-length material. After irradiation, the masses corresponding to both strands were observed with a complete disappearance of starting material. 15 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 20 The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general 25 knowledge in the field of endeavour to which this specification relates.

Claims

1. Process for the preparation of an oligomeric compound made up of two or more individual oligomers, in which said oligomeric compound the individual oligomers are separated by a photocleavable linker, comprising the step of photoactively cleaving said linker.

2. Process according to claim 1 wherein the individual oligomers are independently chosen from the group consisting of oligonucleotides, oligosaccharides and oligopeptides.

3. Process according to claim 1 wherein the individual oligomers are oligonucleotides.

4. Process according to claim 1 wherein the individual oligomers are oligonucleotides which are fully or partially complementary.

5. Process according to claim 1 wherein the individual oligomers are oligoribonucleotides which are fully or partially complementary.

6. Process according to claim I wherein the linker is stable under the deprotection conditions of each individual oligomer.

7. Process according to claim 1 wherein the linker group is cleaved by UV or visible light irradiation.

8. Process according to claim 4 wherein said oligonucleotides are two oligoribonucleotides

9. Process according to any one of claims 1-8, wherein the linker is a compound of formula I, WO 2007/082713 PCTIEP2007/000337 -24 PG R6 X R5 R1 R4 R2 R3 Formula I wherein; PG is (Ar1)(Ar2)(Ar3)C-, wherein Ar1, Ar2, Ar3 are independently chosen from the group consisting of; CH 3 0C 6 H 4 - and C6Hj-, or PG is a substituted silyl group (R1')(R2')(R3')Si-, wherein R1', R2', R3" is independently chosen from the group consisting of lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkyloxy, or aryloxy; X is 0, N, or S; RI, R2, R3, R4, and R5 is independently chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, halogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, OH, 0- lower alkyl/aryl, OC(O) lower alkyl/aryl, SH, S- lower alkyl/aryl, SO 3 H, S0 2 0 lower alkyl/aryl, SO;:NR'R", NH 2 , N- lower alkyl/aryl, NHC(O) lower alkyl/aryl, and at least one of the substituents R1-R5 is a nitro, a nitrosyl, or a diazo group; two or more of the substituents R1, R2, R3, R4, and R5 can form one or several rings which may be further substituted with groups defined as for Ri, R2, R3, R4, or R5; at least one of the substituents R1, R2, R3, R4, or R5 is a phosphoramidite, a phosphonate, or a phosphotriester bearing group able to form a phosphodiester or phosphorothioate linkage to the growing oligonucleotide chain or an amine, an activated carboxylic ester, an isocyanate or an isothiocyanate, able to form an amide, a urea or a thiourea linkage to the growing oligonucleotide chain; WO 2007/082713 PCT/EP2007/000337 - 25 R6 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(0) lower alkyl/aryl, 0-lower alkyl/aryl, OC(O)lower alkyl/aryl, S-lower alkyl/aryl, SO 3 H, S0 2 0-lower alkyl/aryl, SO 2 NR'R", N- lower alkyl/aryl, NHC(O) lower alkyl/aryl.

10. Process according to any one of claims 1-8, wherein the linker is a compound of formula 11, PG R6 X R5 R1 R4 R2 . VU R3 RIO w R11 R9 R 12 x R R8 zY R7 z Formula i wherein, PG is (Ar1)(Ar2)(Ar3)C-, wherein Ar1, Ar2, Ar3 are independently chosen from the group consisting of; CH 3 0CrH 4 -, C 5 H 5 -, or PG is a substituted silyl group (R1')(R2')(R3')Si-, wherein R1', R2', R3" is independently chosen from the group consisting of lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkyloxy, or aryloxy; X is 0, N, or S; WO 2007/082713 PCTIEP2007/000337 - 26 R1, R2, R3, R4, and R5 is independently chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl. lower alkylhalogen, halogen, CN, COOH, C(0)O lower alkyVaryl, CONR'R", CHO, C(O) lower alkyl/aryl, OH, 0- lower alkyVaryl, OC(O) lower alkyl/aryl, SH, S- lower alkyl/aryl, SO 3 H, SO 2 0 lower alkyl/aryl, SO 2 NR'R", NH 2 , N- lower alkyl/aryl, NHC(O) lower alkyl/aryl, and at least one of the substituents R1 -R5 is a nitro, a nitrosyl, or a diazo group; two or more of the substituents R1, R2, R3, R4, and R5 can form one or several rings which may be further substituted with groups defined as for RI, R2, R3, R4, or R5; R6 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(0) lower alkyl/aryl, 0- lower alkyl/aryl, OC(0) lower alkyl/aryl, S- lower alkyl/aryl, SO 3 H, S0 2 0- lower alkyl/aryl, S0 2 NR'R", N- lower alkyl/aryl, NHC(O) lower alkyl/aryl; U, V, W are forming a chain which replaces one of the substituents R1 - R5 on one end and one of the substituents R7 - R1 I on the other end; U, V. W can independently be absent, or be an alkylene (-R-), cycloalkylene (-R-), or arylene (-Ar-) group, -0-, -S-, -NR'-, -C(O)-, -C(0)0-, -C(O)NR'-, -OC(0)0-, OC(O)NR'-, -NR'C(0)NR"-, -OC(S)NR'-, -NR'C(S)NR"-, -S(O)-, -S(0 2 )-, -S(0 2 )NR'-, OP(O 2 )O-, and may contain a label or fluorophore or a group which serves to improve the pharmacological profile of the oligonucleotide. R7. R8, R9, R10, and R11 are independently chosen form the group consisting of, hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, halogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, OH, 0- lower alkyl/aryl, OC(O) lower alkyVaryl, SH, S- lower alkyl/aryl, SO 3 H, S0 2 0 lower alkyl/aryl, SO 2 NR'R", NH2, N- lower alkyl/aryl, NHC(O) lower alkyl/aryl and at least one of the substituents R7-R1 I is a nitro, a nitrosyl, or a diazo group; R12 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, CN, COOH, C(O)0 lower alkyl/aryl, WO 2007/082713 PCT/IEP2007/000337 -27 CONR'R", CHO, C(O) lower alkyl/aryl, 0- lower alkyl/aryl, OC(O) lower alkyl/aryl, S lower alkyl/aryl, SO 3 H, S0 2 0- lower alkyl/aryl, SO 2 NR'R", N- lower alkyl/aryl, NHC(O) lower alkyl/aryl; Y is O, N, or S; Z is a phosphoramidite, a phosphonate, or a phosphotriester group able to form a phosphodiester or phosphorothioate linkage to the growing oligonucleotide chain or an marine, an activated carboxylic ester, an isocyanate or an isothiocyanate which is able to form an amide, a urea or a thiourea linkage to the growing oligonucleotide chain.

11. Process according to claim 1-8, wherein the linker is a compound according to formula I, PG R6 X R5 R1 R4 R2 R3 Formula I wherein; PG is dimethoxytriphenylmethyl; X is 0; R1 is a nitro group; R3 is -CH 2 -0-P(N[iPr]2)-0-CH 2 -CH 2 -CN); R2, R4, R5, and R6 are hydrogen; WO 2007/082713 PCT/EP2007/000337 -28

12. Process according to claim 1-8, wherein the linker is a compound of formula Il PG R6 X R5 RI R4 R2 U R3 RIO w Ru#R R 12 N R8 R8 zY R7 z Formula i wherein; PG is dimethoxytriphenylmethyl; X and Y are 0; R1 and R7 are nitro groups; R2, R4, R5, R6, R8, RA0, R11, and R12 are hydrogen; U is oxygen and replaces R3; V is -CHrCHrCH 2 -; W is oxygen and replaces R9; Z is -P(N[iPr] 2 )-0-CHz-CHrCN). WO 2007/082713 PCT/EP2007/000337 -29

13. A compound of formula 1, PG R6 X R5 R1 IN R4 R2 R3 formula I wherein; PG is (Ar1)(Ar2)(Ar3)C-, wherein Ar1, Ar2, Ar3 are independently chosen from the group consisting of; CH 3 0C 6 H 4 - and C 6 H 5 -, or PG is a substituted silyl group (R1')(R2')(R3')Si-, wherein R1', R2', R:3' is independently chosen from the group consisting of lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkyloxy, and aryloxy; X is 0, N, or S; RI, R2, R3, R4, and R5 is independently chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, halogen, CN, COOH, C(0)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, OH, 0- lower alkyllaryl, OC(O) lower alkyl/aryl, SH, S- lower alkyl/aryl, SO 3 H, S0 2 0 lower alkyl/aryl, SO 2 NR'R", NH 2 , N- lower alkyl/aryl, and NHC(O) lower alkyl/aryl; and at least one of the substituents R1-R5 is a nitro, a nitrosyl, or a diazo group; two or more of the substituents R1, R2, R3, R4, and R5 can form one or several rings which may be further substituted with groups defined as for R1, R2, R3, R4, or R5; at least one of the substituents RI, R2, R3, R4, or R5 is a phosphoramidite, a phosphonate, or a phosphotriester bearing group able to form a phosphodiester or phosphorothioate linkage to the growing oligonucleotide chain or an amine, an WO 2007/082713 PCTIEP2007/000337 -30 activated carboxylic ester, an isocyanate or an isothiocyanate, able to form an amide, a urea or a thiourea linkage to the growing oligonucleotide chain; R6 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, 0-lower alkyl/aryl, OC(O)lower alkyl/aryl, S-lower alkyl/aryl, SOAH, S0 2 0-lower alkyl/aryl, SO 2 NR'R", N- lower alkyl/aryl, and NHC(O) lower alkyl/aryl.

14. A compound according to claim 13, wherein; PG is dimethoxytriphenylmethyl; X is O; R1 is a nitro group; R3 is -CH 2 -0-P(N[iPr] 2 )-O-CHrCH-CN); R2, R4, R5, and R6 are hydrogen;

15. A compound of formula il PG R6 X R5 RI R4 R2 v' R3 R10 W' R11 R9 R12 N R8 Y R7 z Formula 11 WO 2007/082713 PCT/EP2007/000337 -31 wherein, PG is (Ar1)(Ar2)(Ar3)C-, wherein Ar1, Ar2, Ar3 are independently chosen from the group consisting of; CH 3 0C 6 H 4 -, C 6 H 5 -, or PG is a substituted silyl group (R1')(R2')(R3')Si-, wherein R1', R2', R3' is independently chosen from the group consisting of lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkyloxy, or aryloxy; X is O, N, or S; R1, R2, R3, R4, and R5 is independently chosen from the group consisting of hydrogen, lower alkyl, aryl. aryl lower alkyl, lower alkylaryl, lower alkylhalogen, halogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, OH, 0- lower alkyVairyl, OC(O) lower alkyl/aryl, SH, S- lower alkyl/aryl, SO 3 H, S0 2 0 lower alkyl/aryl, SO 2 NR'R", NH 2 , N- lower alkyl/aryl, and NHC(O) lower alkyl/aryl, and at least one of the substituents R1-R5 is a nitro, a nitrosyl, or a diazo group; two or more of the substituents R1, R2, R3, R4, and R5 can form one or several rings which may be further substituted with groups defined as for RI, R2, R3, R4, or R5; R6 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(0) lower alkyl/aryl, 0- lower alkyl/aryl, OC(O) lower alkyVaryl, S- lower alkyl/aryl, S0 3 H, S0 2 0- lower alkyl/aryl, SO 2 NR'R", N- lower alkyl/aryl, and NHC(O) lower alkyl/aryl; U, V, W are forming a chain which replaces one of the substituents R1 - R5 on one end and one of the substituents R7 - R1 1 on the other end; U, V, W can independently be absent, or be an alkylene (-R-), cycloalkylene (-R-), or arylene (-Ar-) group, -0-, -S-, -NR'-, -C(O)-, -C(0)0-, -C(O)NR'-, -OC(O)O-, OC(O)NR'-, -NR'C(0)NR"-, -OC(S)NR'-, -NR'C(S)NR"-, -S(O)-, -S(0 2 )-, -S(0 2 )NR'-, - WO 2007/082713 PCT/EP2007/000337 - 32 OP(O 2 )O-, and may contain a label or fluorophore or a group which serves to improve the pharmacological profile of the oligonucleotide. R7, R8, R9, R10, and RI 1 are independently chosen form the group consisting of, hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, halogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, OH, 0- lower alkyl/aryl, OC(O) lower alkyl/aryl, SH, S- lower alkyl/aryl, SO 3 H, S0 2 0 lower alkyl/aryl, SO 2 NR'R", NH2, N- lower alkyl/aryl, NHC(O) lower alkyl/aryl and at least one of the substituents R7-R1 1 is a nitro, a nitrosyl, or a diazo group; R12 is chosen from the group consisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkylaryl, lower alkylhalogen, CN, COOH, C(O)O lower alkyl/aryl, CONR'R", CHO, C(O) lower alkyl/aryl, 0- lower alkyl/aryl, OC(O) lower alkyl/aryl, S lower alkyl/aryl, SO 3 H, S20- lower alkyl/aryl, SO 2 NR'R", N- lower alkyl/aryl, NHC(O) lower alkyl/aryl; Y is 0, N, or S; Z is a phosphoramidite, a phosphonate, or a phosphotriester group able to form a phosphodiester or phosphorothioate linkage to the growing oligonucleotide chain or an amine, an activated carboxylic ester, an isocyanate or an isothiocyanate which is able to form an amide, a urea or a thiourea linkage to the growing oligonucleotide chain.

16. A compound according to claim 15, wherein; PG is dimethoxytriphenylmethyl; X is O; R1 is a nitro group; R3 is -CHrO-P(N[iPr] 2 )-O-CHrCH2-CN); WO 2007/082713 PCT/EP2007/000337 -33 R2, R4, R5, and R6 are hydrogen;