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Patent US6794499 - Oligonucleotide analogues - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsNovel oligomers, and synthesis thereof, comprising one or more bi-, tri, or polycyclic nucleoside analogues are disclosed herein. The nucleoside analogues have a “locked” structure, termed Locked Nucleoside Analogues (LNA). LNA's exhibit highly desirable and useful properties. LNA's are capable of...http://www.google.com/patents/US6794499?utm_source=gb-gplus-sharePatent US6794499 - Oligonucleotide analoguesAdvanced Patent SearchPublication numberUS6794499 B2Publication typeGrantApplication numberUS 09/152,059Publication dateSep 21, 2004Filing dateSep 11, 1998Priority dateSep 12, 1997Fee statusPaidAlso published asUS6670461, US7034133, US20020068708, US20030134808, US20030144231Publication number09152059, 152059, US 6794499 B2, US 6794499B2, US-B2-6794499, US6794499 B2, US6794499B2InventorsJesper Wengel, Poul NielsenOriginal AssigneeExiqon A/SExport CitationBiBTeX, EndNote, RefManPatent Citations (4), Non-Patent Citations (83), Referenced by (191), Classifications (7), Legal Events (3) External Links: USPTO, USPTO Assignment, EspacenetOligonucleotide analogues
US 6794499 B2Abstract
Novel oligomers, and synthesis thereof, comprising one or more bi-, tri, or polycyclic nucleoside analogues are disclosed herein. The nucleoside analogues have a “locked” structure, termed Locked Nucleoside Analogues (LNA). LNA's exhibit highly desirable and useful properties. LNA's are capable of forming nucleobase specific duplexes and triplexes will single and double stranded nucleic acids. These complexes exhibit higher thermostability than the corresponding complexes formed with normal nucleic acids. The properties of LNA's allow for a wide range of uses such as diagnostic agents and therapeutic agents in a mammal suffering from or susceptible to, various diseases.
What is claimed is: 1. An oligomer comprising at least one LNA nucleoside of the general formula I wherein
X is selected from —O—; B is selected from hydrogen, hydroxy, optionally substituted C1-4-alkoxy, optionally substituted C1-4-alkyl, optionally substituted C1-4-acyloxy, nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands; P designates the radical position for an internucleoside linkage to a succeeding monomer, or a 5′-terminal group, such internucleoside linkage or 5′-terminal group optionally including the substituent R5; one of the substituents R2, R2*, R3, and R3* is a group P* which designates an internucleoside linkage to a preceding monomer, or a 3′-terminal group, one pair of non-geminal substituents R4*, and R2*, designating a biradical consisting of 2-5 groups/atoms selected from —(CR*R*)r—Y—(CR*R*)s—, —(CR*R*)r—Y—(CR*R*)s—Y—, —Y—(CR*R*)r+s—Y—, —Y—(CR*R*)r—Y—(CR*R*)s—, —(CR*R*)r+s—, each R* is independently selected from hydrogen, halogen, hydroxy, mercapto, amino, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, Y is —O—, —S—, 0 (zero) or —N(RN)—, and each of r and s is 0-4 with the proviso that the sum r+s is 1-4, and provided that when the biradical is —(CR*R*)r—Y—(CR*R*)s—, then Y is —S— or —N(RN′)—; and each of the substituents R1*, R2, R3, R5, and R5*, which are present and not involved in P, P* is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarycarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted; and basic salts and acid addition salts thereof.
2. An oligomer of claim 1 wherein the oligomer comprises 1 to 10000 nucleosides of the formula I and 0-10000 nucleosides selected from naturally occurring nucleosides and nucleoside analogues, with the proviso that the sum of the number of nucleosides and the number of LNA(s) is at least 2.
3. An oligomer of claim 2 wherein at least one LNA nucleoside comprises a nucleobase as the substituent B.
4. An oligomer of claim 1 wherein one of the substituents R3 and R3* designates P*.
5. An oligomer of 1 wherein one or more nucleosides have the following formula Ia. wherein P, P*, B, X, R1*,R2, R2*, R1, R4*, R5 and R5* are as defined in claim 1.
6. An oligomer of claim 5 wherein R3* designates P*.
7. An oligomer of claim 6 wherein the oligomer comprises one biradical constituted by two non-geminal substituents.
8. An oligomer of claim 1 wherein R3* designates P*.
9. An oligomer of claim 8 wherein, R2 is selected from hydrogen, hydroxy, and optionally substituted C1-4-alkoxy, and R3*, R3, R5, and R5* designate hydrogen.
10. An oligomer of claim 9 wherein B is selected from nucleobases.
11. An oligomer of claim 10 wherein the oligomer comprises at least one LNA nucleoside wherein B is selected from adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4, N6-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 2,6-diaminopyrimidino,2,6-diaminopyrazine,1-methyl-pyrazolo[4,3-d]pyrimidino-5,7(4H,6H)-dione, 1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, and inosine.
12. An oligomer according to claim 1 wherein any internucleoside linkage of the one or more LNA nucleosides is selected from linkages consisting of 2 to 4 groups/atoms selected from —CH2—, —O—, —S—, —NRH—, >C═O, >NRH, >C═S, —Si(R″)2— —SO—, —S(O)2—, —P(O)2—, —P(O,S)—, —P(S)2—, —PO(R″)—, —PO(OCH2)—, and —PO(NHRH)—, where RH is selected form hydrogen and C1-4-alkyl, and R″ is selected from C1-6-alkyl and phenyl.
13. An oligomer of claim 12 wherein any internucleoside linkage of the one or more LNA nucleosides is selected from —CH2—CH2—CH2—, —CH2—CO—CH2—CH2—CHOH—CH2—, —O—CH2—O—, —O—CH2—CH2—, —O—CH2—CH═, —CH2—CH2—O—, —NRH—CH2—, —CH2—CH2—NRH—, —CH2—NRH—CH2—, —O—CH2—CH2NRH—, —NRH—CO—O—, —NRH—CO—NRH—, —NRH—CS—NRH—, —NRH—C(═NRH)—NRH—, —NRH—CO—CH2—NRH—, —O—CO—O—, —O—CO—CH2—O—, —O—CH2—CO—O—, —CH2—CO—NRH—, —O—CO—NRH—, —NRH—CO—CH2—, —O—CH2—CO—NRH—, —O—CH2—CH2—NRH—, —CH═N—O—, —CH2—NRH—O—, —CH2—O—N═, —CH2—O—NRH—, —CO—NRH—CH2—, —CH2—NRH—O—, —CH2—NRH—CO—, —O—NRH—CH2—, —O—NRH—, —O—CH2—S—, —S—CH2—O—, —CH2—CH2—S—, —O—CH2—CH2—S—, —S—CH2—CH═, —S—CH2—CH2—, —CH2—CH2—O—, —S—CH2—CH2—S—, —CH2—S—CH2—, —CH2—SO—CH2—, —CH2—SO2—CH2—, —O—SO—O—, —S(O)2—O—, —O—S(O)2—CH2—, —O—S(O)2—NRH—, —NRH—S(O)2—CH2—, —O—S(O)2—CH2—, —O—P(O)2—O—, —O—P(O,S)—O—, —O—P(S)2—O—, —S—P(O)2—O—, —S—P(O,S)—O—, —S—P(S)2—O—, —O—P(O)2—S—, —O—P(O,S)—S—, —O—P(S)2—S—, —S—P(O)2—S—, —S—P(O,S)—S—, —S—P(S)2—S—, —O—PO(R″)—O—, —O—PO(OCH3)—O—, —O—PO(BH3)—O—, —O—PO(NHRH)—O—, —O—P(O)2—NRH—, —NRH—P(O)2—O—, —O—P(O,NRH)—O—, and —O—Si(R″)2—O—.
14. An oligomer of claim 13 wherein any internucleoside linkage of the one or more LNA nucleosides is selected from —CH2—CO—NRH—, —CH2—NRH—O—, —S—CH2—O—, —O—P(O)2—, —O—P(O,S)—O—, —O—P(S)2—O—, —NRH—P(O)2—O—, —O—P(O,NRH)—O—, —O—PO(R″)—O—, —O—PO(CH2)—O—, and —O—PO(NHRN)—O—, where RH is selected form hydrogen and C1-4-alkyl, and R″ is selected from C1-6-alkyl and phenyl.
15. An oligomer of claim 1 wherein each of the substituents R1*, R2, R3, R3*, R5, and R5*, of the one or more LNA nucleosides, which are present and not involved in P, P* is independently selected from hydrogen, optionally substituted C1-6-alkyl, optionally substituted C2-6-alkenyl), hydroxy, C1-6-alkoxy, C2-6-alkenyloxy, carboxy, C1-6-alkoxycarbonyl, C1-6-alkylcarbonyl, formyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, C1-6-alkyl-carbonylamino, cabamido, azido, C1-6-alkanoyloxy, sulphono, sulphanyl, C1-6-alkylthio, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and halogen, where two geminal substituents together may designate oxo, and where RN*, when present and not involved in a biradical, is selected from hydrogen and C1-4-alkyl.
16. An oligomer of claim 1 wherein each of the substituents R1*, R2, R1, R3*, R5, and R5*, of the LNA(s), which are present and not involved in P, P* designate hydrogen.
17. An oligomer of claim 1 wherein P is a 5′-terminal group selected from hydrogen, hydroxy, optionally substituted C1-6-alkyl, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkylcarbonyloxy, optionally substituted aryloxy, monophosphate, diphosphate, triphosphate, and —W—A′, wherein W is selected from —O—, —S—, and —N(RH)— where RH is selected from hydrogen and C1-6-alkyl, and where A′ is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.
18. An oligomer of claim 1 wherein P* is a 3′-terminal group selected from hydrogen, hydroxy, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkylcarbonyloxy, optionally substituted aryloxy, and —W—A′, wherein W is selected from —O—, —S—, and —N(RH)— where RH is selected from hydrogen and C1-6-alkyl, and where A′ is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.
19. An oligomer of claim 1 wherein the oligomer corresponds to the following formula V:
G[Nu-L]n(0)-{[LNA-L]m(q)-[Nu-L]n(q)}q-G* V wherein
q is 1-50; each of n(0), . . . , n(q) is independently 0-10000; each of m(1), . . . , m(q) is independently 1-10000; with the proviso that the sum of n(0), . . . , n(q) and m(1), . . . , m(q) is 2-15000; G designates a 5′-terminal group; each Nu independently designates a nucleoside selected from naturally occurring nucleosides and nucleoside analogues; each LNA independently designates a nucleoside analogue; each L independently designates an internucleoside linkage between two groups selected from Nu and LNA, or L together with G* designates a 3′-terminal group; and each LNA-L independently designates a nucleoside analogue of the general formula I: wherein the substituents B, P, P*, R1*, R2, R2*, R3, R4*, R5, and R5*, and X are as defined in claim 1. 20. An oligomer of claim 1 further comprising a PNA mono- or oligomer segment of the formula wherein B is a defined above for the formula I, AASC designates hydrogen or an amino acid side chain, t is b 1-5, and w is 1-50.
21. An oligomer of claim 1 which has an increased specificity towards complementary ssRNA or ssDNA compared to a corresponding reference oligonucleotide which does not contain any LNA units.
22. An oligomer of claim 1 which has an increased affinity towards complementary ssRNA or ssDNA compared to a corresponding reference oligonucleotide which does not contain any LNA units.
23. An oligomer of claim 1 which is capable of binding to a target sequence in a dsDNA or dsRNA molecule by of strand displacement or by triple helix formation.
24. An oligomer of claim 1 which is more resistant to nucleases than a corresponding reference oligonucleotide which does not contain any LNA units.
25. An oligomer according to claim 1 which has nucleic acid catalytic activity.
26. A diagnostic or analysis kit comprising an oligonucleotide of claim 1.
27. A kit of claim 26 wherein the oligonucleotide is immobilized on a solid support.
28. A diagnostic or analysis kit comprising a reaction body and one or more oligonucleotides of claim 1.
29. The kit of claim 28 wherein the one or more oligonucleotides are immobilized on the reaction body.
30. A diagnostic or analysis kit comprising a reaction body and one or more oligonucleotides of claim 1.
31. The kit of claim 30 wherein the one or more oligonucleotides are immobilized on the reaction body.
32. A diagnostic or analysis kit comprising a reaction body and one or more oligonucleotides of claim 9.
33. The kit of claim 32 wherein the one or more oligonucleotides are immobilized on the reaction body.
This application claims the benefit of the application U.S. provisional applications: U.S. provisional application No. 60/058,541, filed Sep. 12, 1997, now abandoned; U.S. provisional application No. 60/068,293, filed Dec. 19, 1997, now abandoned; U.S. provisional application No. 60/071,682, filed Jan. 16, 1998, now abandoned; U.S. provisional application No. 60/057,591, filed Mar. 3, 1998, now abandoned; U.S. provisional application No. 60/083,507, filed Apr. 29, 1998, now abandoned; U.S. provisional application No. 60/088,309, filed Jun. 5, 1998, now abandoned; U.S. provisional application No. 60/094,355, filed Jul. 28, 1998, now abandoned; all of which are incorporated herein by reference.
The present invention relates to the field of bi- and tricyclic nucleoside analogues and to the synthesis of such nucleoside analogues which are useful in the formation of synthetic oligonucleotides capable of forming nucleobase specific duplexes and triplexes with single stranded and double stranded nucleic acids. These complexes exhibit higher thermostability than the corresponding complexes formed with normal nucleic acids. The invention also relates to the field of bi- and tricyclic nucleoside analogues and the synthesis of such nucleosides which may be used as therapeutic drugs and which may be incorporated in oligonucleotides by template dependent nucleic acid polymerases.
More recently, oligoribonucleotides and oligodeoxyribonucleotides and analogues thereof which combine RNAse catalytic activity with the ability to sequence specifically interact with a complementary RNA target (ribozymes) have attracted much interest as antisense probes. Thus far ribozymes have been reported to be effective in cell cultures against both viral targets and oncogenes.
To completely prevent the synthesis of a given protein by the antisense approach it is necessary to block/destroy all mRNAs that encode that particular protein and in many cases the number of these mRNA are fairly large. Typically, the mRNAs that encode a particular protein are transcribed from a single or a few genes. Hence, by targeting the gene (“antigene” approach) rather than its mRNA products it should be possible to either block production of its cognate protein more efficiently or to achieve a significant reduction in the amount of oligonucleotides necessary to elicit the desired effect. To block transcription, the oligonucleotide must be able to hybridise sequence specifically to double stranded DNA. In 1953 Watson and Crick showed that deoxyribo nucleic acid (DNA) is composed of two strands (Nature, 1953, 171, 737) which are held together in a helical configuration by hydrogen bonds formed between opposing complementary nucleobases in the two strands. The four nucleobases, commonly found in DNA are guanine (G), adenine (A), thymine (T) and cytosine (C) of which the G nucleobase pairs with C, and the A nucleobase pairs with T. In RNA the nucleobase thymine is replaced by the nucleobase uracil (U) which similarly to the T nucleobase pairs with A. The chemical groups in the nucleobases that participate in standard duplex formation constitute the Watson-Crick face. In 1959, Hoogsteen showed that the purine nucleobases (G and A) in addition to their Watson-Crick face have a Hoogsteen face that can be recognised from the outside of a duplex, and used to bind pyrimidine oligonucleotides via hydrogen bonding, thereby forming a triple helix structure. Although making the “antigene” approach conceptually feasible the practical usefulness of triple helix forming oligomers is currently limited by several factors including the requirement for homopurine sequence motifs in the target gene and a need for unphysiologically high ionic strength and low pH to stabilise the complex.
Bicyclo[3.3.0] nucleosides (bcDNA) with an additional C-3′,C-5′-ethano-bridge (A and B) have been synthesised with all five nucleobases (G, A, T, C and U) whereas (C) has been synthesised only with T and A nucleobases (M. Tark�y, M. Bolli, B. Schweizer and C. Leumann, Helv. Chim. Acta, 1993, 76, 481; Tark�y and C. Leumann, Angew. Chem., Int. Ed. Engl., 1993, 32, 1432; M. Egli, P. Lubini, M. Dobler and C. Leumann, J. Am. Chem. Soc., 1993, 115, 5855; M. Tark�y, M. Bolli and C. Leumann, Helv. Chim. Acta, 1994, 77, 716; M. Bolli and C. Leumann, Angew. Chem., Int. Ed. Engl., 1995, 34, 694; M. Bolli, P. Lubini and C. Leumann, Helv. Chim. Acta, 1995, 78, 2077; J. C. Litten, C. Epple and C. Leumann, Bioorg. Med. Chem. Lett., 1995, 5, 1231; J. C. Litten and C. Leumann, Helv. Chim. Acta, 1996, 79, 1129; M. Bolli, J. C. Litten, R. Sch�ltz and C. Leumann, Chem. Biol., 1996, 3, 197; M. Bolli, H. U. Trafelet and C. Leumann, Nucleic Acids Res., 1996, 24, 4660). DNA oligonucleotides containing a few, or being entirely composed, of these analogues are in most cases able to form Watson-Crick bonded duplexes with complementary DNA and RNA oligonucleotides. The thermostability of the resulting duplexes, however, is either distinctly lower (C), moderately lower (A) or comparable to (B) the stability of the natural DNA and RNA counterparts. All bcDNA oligomers exhibited a pronounced increase in sensitivity to the ionic strength of the hybridisation media compared to the natural counterparts. The α-bicyclo-DNA (B) is more stable towards the 3′-exonuclease snake venom phosphordiesterase than the β-bicyclo-DNA (A) which is only moderately more stable than unmodified oligonucleotides. Bicarbocyclo[3.1.0]nucleosides with an additional C-1′,C-6′- or C-6′,C-4′-methano-bridge on a cyclopentane ring (D and E, respectively) have been synthesised with all five nucleobases (T, A, G, C and U). Only the T-analogues, however, have been incorporated into oligomers. Incorporation of one or ten monomers D in a mixed poly-pyrimidine DNA oligonucleotide resulted in a substantial decrease in the affinity towards both DNA and RNA oligonucleotides compared to the unmodified reference oligonucleotide. The decrease was more pronounced with ssDNA than with ssRNA. Incorporation of one monomer E in two different poly-pyrimidine DNA oligonucleotides induced modest increases in Tm's of 0.8� C. and 2.1� C. for duplexes towards ssRNA compared with unmodified reference duplexes. When ten T-analogues were incorporated into a 15 mer oligonucleotide containing exclusively phosphorothioate internucleoside linkages, the Tm against the complementary RNA oligonucleotide was increased approximately 1.3� C. per modification compared to the same unmodified phosphorothioate sequence. Contrary to the control sequence the oligonucleotide containing the bicyclic nucleoside E failed to mediate RNAseH cleavage. The hybridisation properties of oligonucleotides containing the G, A, C and U-analogues of E have not been reported. Also, the chemistry of this analogue does not lend itself to further intensive investigations on completely modified oligonucleotides (K.-H. Altmann, R. Kesselring, E. Francotte and G. Rihs, Tetrahedron Lett., 1994, 35, 2331; K.-H. Altmann, R. Imwinkelried, R. Kesselring and G. Rihs, Tetrahedron Lett., 1994, 35, 7625; V. E. Marquez, M. A. Siddiqui, A. Ezzitouni, P. Russ, J. Wang, R. W. Wagner and M. D. Matteucci, J. Med. Chem., 1996, 39, 3739; A. Ezzitouni and V. E. Marquez, J. Chem. Soc., Perkin Trans. 1, 1997, 1073).
A bicyclo[3.3.0] nucleoside containing an additional C-2′,C-3′-dioxalane ring has been synthesised as a dimer with an unmodified nucleoside where the additional ring is part of the internucleoside linkage replacing a natural phosphordiester linkage (F). This analogue was only synthesised as either thymine-thymine or thymine-5-methylcytosine blocks. A 15-mer polypyrimidine sequence containing seven of these dimeric blocks and having alternating phosphordiester- and riboacetal-linkages, exhibited a substantially decreased Tm against complementary ssRNA compared to a control sequence with exclusively natural phosphordiester internucleoside linkages (R. J. Jones, S. Swaminathan, J. F. Millagan, S. Wadwani, B. S. Froehler and M. Matteucci, J. Am. Chem. Soc., 1993, 115, 9816).
The two dimers (G and H) with additional C-2′,C-3′-dioxans rings forming bicyclic[4.3.0]-systems in acetal-type internucleoside linkages have been synthesised as T-T dimers and incorporated once in the middle of 12 mer polypyrimidine oligonucleotides. Oligonucleotides containing either G or H both formed significantly less stable duplexes with complementary ssRNA and ssDNA compared with the unmodified control oligonucleotide (J. Wang and M. D. Matteucci, Bioorg. Med. Chem. Lett., 1997, 7, 229).
Dimers containing a bicyclo[3.1.0]nucleoside with a C-2′,C-3′-methano bridge as part of amide- and sulfonamide-type (I and J) internucleoside linkages have been synthesised and incorporated into oligonucleotides. Oligonucleotides containing one ore more of these analogues showed a significant reduction in Tm compared to unmodified natural oligonucleotide references (C. G. Yannopoulus, W. Q. Zhou, P. Nower, D. Peoch, Y. S. Sanghvi and G. Just, Synlett, 1997, 378).
An attempt to make the bicyclic uridine nucleoside analogue Q planned to contain an additional O-2′,C-4′-five-membered ring, starting from 4′-C-hydroxymethyl nucleoside P, failed (K. D. Nielsen, Specialerapport (Odense University, Denmark), 1995).
Until now the pursuit of conformationally restricted nucleosides useful in the formation of synthetic oligonucleotides with significantly improved hybridisation characteristics has met with little success. In the majority of cases, oligonucleotides containing these analogues form less stable duplexes with complementary nucleic acids compared to the unmodified oligonucleotides. In other cases, where moderate improvement in duplex stability is observed, this relates only to either a DNA or an RNA target, or it relates to fully but not partly modified oligonucleotides or vice versa. An appraisal of most of the reported analogues are further complicated by the lack of data on analogues with G, A and C nucleobases and lack of data indicating the specificity and mode of hybridisation. In many cases, synthesis of the reported monomer analogues is very complex while in other cases the synthesis of fully modified oligonucleotides is incompatible with the widely used phosphoramidite chemistry standard.
Thus, the present invention relates to oligomers comprising at least one nucleoside analogue (hereinafter termed “LNA”) of the general formula I wherein
−X is selected from —O—, —S—, —N(RN*)—, —C(R8R6*)—, —O—C(R7R7*)—, —C(R5R6*)—O—, —S—C(R7R7*)—, —C(R6R6*)—S—, —N(RN*)—C(R7R7*)—, —C(R6R6*)—N(RN*)—, and —C(R6R6*)—C(R7R7*)—;
one or two pairs of non-geminal substituents selected from the present substituents of R1*, R4*, R5, R5*, R6, R6*, R7, R7*, RN*, and the ones of R2, R2*, R3, and R3* not designating P* each designates a biradical consisting of 1-8 groups/atoms selected from —C(RaRb)—, —C(Ra)═C(Ra)—, —C(Ra)═N—, —O—, —Si(Ra)2—, —S—, —SO2—, —N(Ra)—, and >C═Z, wherein Z is selected from —O—, —S—, and —N(Ra)—, and Ra and Rb each is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents Ra and Rb together may designate optionally substituted methylene (═CH2), and wherein two non-geminal or geminal substitutents selected from Ra, Rb, and any of the substituents R1*, R2, R2*, R3, R3*, R4*, R5, R5*, R6 and R6*, R7, and R7* which are present and not involved in P, P* or the biradical(s) together may form an associated biradical selected from biradicals of the same kind as defined before; said pair(s) of non-geminal substituents thereby forming a mono- or bicyclic entity together with (i) the atoms to which said non-geminal substituents are bound and (ii) any intervening atoms; and
each of the substituents R1*, R2, R2*, R3, R4*, R5, R5*, R6 and R6*, R7, and R7* which are present and not involved in P, P* or the biradical(s), is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene, or together may form a spiro biradical consisting of a 1-5 carbon atom(s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected from —O—, —S—, and —(NRN)— where RN is selected from hydrogen and C1-4-alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and RN*, when present and not involved in a biradical, is selected from hydrogen and C1-4-alkyl;
(iii) R1* and R6* do not together designate a biradical —CH2— when LNA is a bicyclic nucleoside analogue; and
(iv) R4* and R5* do not together designate a biradical —CH2— when LNA is a bicyclic nucleoside analogue.
each of Q and Q* is independently selected from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O—, Act-O—, mercapto, Prot-S—, Act-S—, C1-6-alkylthio, amino, Prot-N(RH)—, Act-N(RH)—, mono- or di(C1-6-alkyl)amino, optionally substituted C1-8-alkoxy, optionally substituted C1-5-alkyl, optionally substituted C2-6-alkenyl, optionally substituted C2-6-alkenyloxy, optionally substituted C2-5-alkynyl, optionally substituted C2-6-alkynyloxy, monophosphate, diphosphate, triphosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH2—, Act-O—CH2—, aminomethyl, Prot-N(RH)—CH2—, Act-N(RH)—CH2—, carboxymethyl, sulphonomethyl, where Prot is a protection group for —OH, —SH, and —NH(RH), respectively, Act is an activation group for —OH, —SH, and —NH(RH), respectively, and RH is selected from hydrogen and C1-6-alkyl;
(i) R2* and R4* together designate a biradical selected from —O—, —(CR*R*)r+s+1—, —(CR*R*)r—O—(CR*R*)s—, —(CR*R*)r—S—(CR*R*)s—, —(CR*R*)r—N(R*)—(CR*R*)s—, —O—(CR*R*)r+s—O—, —S—(CR*R*)r+s—O—, —O—(CR*R*)r+s—S—, —N(R*)—(CR*R*)r+s—O—, —O—(CR*R*)r+s—N(R*)—, —S—(CR*R*)r+s—S—, —N(R*)—(CR*R*)r+s—N(R*)—, —N(R*)—(CR*R*)r+s—S—, and —S—(CR*R*)r+s—N(R*)—;
(ii) R2 and R3 together designate a biradical selected from —O—, —(CR*R*)r+s—, —(CR*R*)r—O—(CR*R*)s, —(CR*R*)r—S—(CR*R*)s—, and —(CR*R*)r—N(R*)—(CR*R*)s—;
each of the substituents R1, R2, R2*, R3, R4*, R5, and R5*, which are not involved in Q, Q* or the biradical, is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-8-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene, or together may form a spiro biradical consisting of a 1-5 carbon atom(s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected from —O—, —S—, and —(NRN)— where RN is selected from hydrogen and C1-4-alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and RN*, when present and not involved in a biradical, is selected from hydrogen and C1-4-alkyl;
with the first proviso that,
(i) R3 and R5 do not together designate a biradical selected from —CH2—CH2—, —O—CH2—, and —O—Si(iPr)2—O—Si(iPr)2—O—;
FIGS. 1A and 1B illustrate known conformationally restricted nucleotides.
FIG. 5 illustrates that LNA modified oligonucleotides, immobilised on a solid surface, function efficiently in the sequence specific capture of a PCR amplicon.
FIG. 8 illustrates that LNA modified oligonucleotides can functions as primers in target amplification processes.
FIG. 9 illustrates that LNA modified oligonucleotides carrying a 5′anthraquinone can be covalently immobilised on a solid support by irradiation and that the immobilised oligomer is efficient in the capture of a complementary DNA oligo.
FIGS. 18 and 19 illustrate the use of [α33P] ddNTP's and ThermoSequenase™ DNA Polymerase to sequence DNA templates containing LNA T monomers.
When used herein, the term “LNA” (Locked Nucleoside Analogues) refers to the bi- and tricyclic nucleoside analogues of the invention, either incorporated in the oligomer of the invention (general formula I) or as discrete chemical species (general formula II). The term “monomeric LNA” specifically refers to the latter case.
As mentioned above, the present invention i.a. relates to novel oligomers (oligonucleotides) comprising one or more bi-, tri-, or polycyclic nucleoside analogues (hereinafter termed “LNA”). It has been found that the incorporation of such LNAs in place of, or as a supplement to, e.g., known nucleosides confer interesting and highly useful properties to an oligonucleotide. Bi- and tricyclic, especially bicyclic, LNAs seem especially interesting within the scope of the present invention.
Each of the possible LNAs incorporated in an oligomer (oligonucleotide) has the general formula I wherein X is selected from —O— (the furanose motif), —S—, —N(RN*)—, —C(R6R6*)—, —O—C(R7R7*)—, —C(R6R6*)—O—, —S—C(R7R7*)—, —C(R6R6*)—S—, —N(RN*)—C(R7R7*)—, —C(R6R6*)—N(RN*)—, and —C(R6R6*)—C(R7R7*)—, where R6, R6*, R7, R7*, and RN* are as defined further below. Thus, the LNAs incorporated in the oligomer may comprise an either 5- or 6-membered ring as an essential part of the bi-, tri-, or polycyclic structure. It is believed that 5-membered rings (X=—O—, —S—, —N(RN*)—, —C(R6R6*)—) are especially interesting in that they are able to occupy essentially the same conformations (however locked by the introduction of one or more biradicals (see below)) as the native furanose ring of a naturally occurring nucleoside. Among the possible 5-membered rings, the situations where X designates —O—, —S—, and —N(RN*)— seem especially interesting, and the situation where X is —O— appears to be particularly interesting.
The substituent B may designate a group which, when the oligomer is completing with DNA or RNA, is able to interact (e.g. by hydrogen bonding or covalent bonding or electronic interaction) with DNA or RNA, especially nucleobases of DNA or RNA. Alternatively, the substituent B may designate a group which acts as a label or a reporter, or the substituent B may designate a group (e.g. hydrogen) which is expected to have little or no interactions with DNA or RNA. Thus, the substituent B is preferably selected from hydrogen, hydroxy, optionally substituted C1-4-alkoxy, optionally substituted C1-4-alkyl, optionally substituted C1-4-acyloxy, nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.
In the present context, the terms “nucleobase” covers naturally occurring nucleobases as well as non-naturally occurring nucleobases. It should be clear to the person skilled in the art that various nucleobases which previously have been considered “non-naturally occurring” have subsequently been found in nature. Thus, “nucleobase” includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Illustrative examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynyleytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the “non-naturally occurring” nucleobases described in Benner et al., U.S. Pat. No. 5,432,272. The term “nucleobase” is intended to cover every and all of these examples as well as analogues and tautomers thereof. Especially interesting nucleobases are adenine, guanine, thymine, cytosine, and uracil, which are considered as the naturally occurring nucleobases in relation to therapeutic and diagnostic application in humans.
In the present context, the term “reporter group” means a group which is detectable either by itself or as a part of an detection series. Examples of functional parts of reporter groups are biotin, digoxigenin, fluorescent groups (groups which are able to absorb electromagnetic radiation, e.g. light or X-rays, of a certain wavelength, and which subsequently reemits the energy absorbed as radiation of longer wavelength; illustrative examples are dansyl (5-dimethylamino)-1-naphthalenesulfonyl), DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, acridines, coumarins, Cy3 and Cy5 (trademarks for Biological Detection Systems, Inc.), erytrosine, coumaric acid, umbelliferone, texas red, rhodamine, tetramethyl rhodamine, Rox, 7-nitrobenzo-2-oxa-1-diazole (NBD), pyrene, fluorescein, Europium, Ruthenium, Samarium, end other rare earth metals), radioisotopic labels, chemiluminescence labels (labels that are detectable via the emission of light during a chemical reaction), spin labels (a free radical (e.g. substituted organic nitroxides) or other paramagnetic probes (e.g. Cu2+, Mg2+) bound to a biological molecule being detectable by the use of electron spin resonance spectroscopy), enzymes (such as peroxidases, alkaline phosphatases, β-galactosidases, and glycose oxidases), antigens, antibodies, haptens (groups which are able to combine with an antibody, but which cannot initiate an immune response by itself, such as peptides and steroid hormones), carrier systems for cell membrane penetration such as: fatty acid residues, steroid moieties (cholesteryl), vitamin A, vitamin D, vitamin E, folic acid peptides for specific receptors, groups for mediating endocytose, epidermal growth factor (EGF), bradykinin, and platelet derived growth factor (PDGF). Especially interesting examples are biotin, fluorescein, Texas Red, rhodamine, dinitrophenyl, digoxigenin, Ruthenium, Europium, Cy5, Cy3, etc.
In the present context “ligand” means something which binds. Ligands can comprise functional groups such as: aromatic groups (such as benzene, pyridine, naphtalene, anthracene, and phenanthrene), heteroaromatic groups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane, and pyrimidine), carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, C1-C20 alkyl groups optionally interrupted or terminated with one or more heteroatoms such as oxygen atoms, nitrogen atoms, and/or sulphur atoms, optionally containing aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly-β-alanine, polyglycine, polylysine, peptides, oligo/polysaccharides, oligo/polyphosphates, toxins, antibiotics, cell poisons, and steroids, and also “affinity ligands”, i.e. functional groups or biomolecules that have a specific affinity for sites on particular proteins, antibodies, poly- end oligosaccharides, and other biomolecules.
It will be clear for the person skilled in the art that the above-mentioned specific examples under DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands correspond to the “active/functional” part of the groups in question. For the person skilled in the art it is furthermore clear that DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands are typically represented in the form M—K— where M is the “active/functional” part of the group in question and where K is a spacer through which the “active/functional” part is attached to the 5- or 6-membered ring. Thus, it should be understood that the group B, in the case where B is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, has the form M—K—, where M is the “active/functional” part of the DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group, and ligand, respectively, and where K is an optional spacer comprising 1-50 atoms, preferably 1-30 atoms, in particular 1-15 atoms, between the 5- or 6-membered ring and the “active/functional” part.
In the oligomers of the present invention (formula I), P designates the radical position for an internucleoside linkage to a succeeding monomer, or a 5′-terminal group. The first possibility applies when the LNA in question is not the 5′-terminal “monomer”, whereas the latter possibility applies when the LNA in question is the 5′-terminal “monomer”. It should be understood (which also will be clear from the definition of internucleoside linkage and 5′-terminal group further below) that such an internucleoside linkage or 5′-terminal group may include the substituent R5 (or equally applicable: the substituent R5*) thereby forming a double bond to the group P. (5′-Terminal refers to the position corresponding to the 5′ carbon atom of a ribose moiety in a nucleoside.)
In the groups constituting the biradical(s), Z is selected from —O—, —S—, and —N(Ra)—, and Ra and Rb each is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino-C1-5-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands (where the latter groups may include a spacer as defined for the substituent B), where aryl and heteroaryl may be optionally substituted. Moreover, two geminal substituents Ra and Rb together may designate optionally substituted methylene (═CH2 optionally substituted one or two times with substituents as defined as optional substituents for aryl), and two non-geminal or geminal substituents selected from Ra, Rb, and any of the substituents R1*, R2, R2*, R3, R3*, R4*, R5, R5*, R6 and R6*, R7, and R7* which are present and not involved in P, P* or the biradical(s) may together form an associated biradical selected from biradicals of the same kind as defined before. It will be clear that each of the pair(s) of non-geminal substituents thereby forms a mono- or bicyclic entity together with (i) the atoms to which the non-geminal substituents are bound and (ii) any intervening atoms.
It is believed that biradicals which are bound to the ring atoms of the 5- or 6-membered rings are preferred in that inclusion of the substituents R5 and R5* may cause an undesired sterical interaction with internucleoside linkage. Thus, it is preferred that the one or two pairs of nongeminal substituents, which are constituting one or two biradical(s), respectively, are selected from the present substituents of R1*, R4*, R6, R6*, R7, R7*, RN*, and the ones of R2, R2*, R3, and R3* not designating P*.
This being said, it should be understood (especially with due consideration of the known bi- and tricyclic nucleoside analogues—see “Background of the Inventions”) that the present invention does not relate to oligomers comprising the following bi- or tricyclic nucleosides analogues:
(i) R2 and R3 together designate a biradical selected from —O—CH2—CH2— and —O—CH2—CH2—CH2— when LNA is a bicyclic nucleoside analogue;
(ii) R3 and R5 together designate a biradical selected from —CH2—CH2—, —O—CH2—, when LNA is a bicyclic nucleoside analogue;
(iii) R3, R5, and R5* together designate a triradical —CH2—CH(—)—CH2— when LNA is a tricyclic nucleoside analogue;
(iv) R1* and R6* together designate a biradical —CH2— when LNA is a bicyclic nucleoside analogue; or
(v) R4* and R6* together designate a biradical —CH2— when LNA is a bicyclic nucleoside analogue;
except where such bi- or tricyclic nucleoside analogues are combined with one or more of the novel LNAs defined herein.
Considering the numerous interesting possibilities for the structure of the biradical(s) in LNA(s) incorporated in oligomers according to the invention, it is believed that the biradical(s) constituted by pair(s) of non-geminal substituents preferably is/are selected from —(CR*R*)r—Y—(CR*R*)s—, —(CR*R*)r—Y—(CR*R*)s—Y—, —Y—(CR*R*)r+s—Y—, —Y—(CR*R*)r—Y—(CR*R*)s—, —(CR*R*)r+s—, —Y—, —Y—Y—, wherein each Y is independently selected from —O—, —S—, —Si(R*)2—, —N(R*)—, >C═O, —C(═O)—N(R*)—, and —N(R*)—C(═O)—, each R* is independently selected from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono- or di(C1-6-alkyl)amino, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and/or two adjacent (non-geminal) R* may together designate a double bond; and each of r and s is 0-4 with the proviso that the sum r+s is 1-5. Particularly interesting situations are those wherein each biradical is independently selected from —Y—, —(CR*R*)r+s—, —(CR*R*)r—Y—(CR*R*)s—, and —Y—(CR*R*)r+s—Y—, wherein and each of r and s is 0-3 with the proviso that the sum r+s is 1-4.
Considering the positioning of the biradical in the LNA(s), it is believed (based on the preliminary findings (see the examples)) that the following situations are especially interesting, namely where: R2* and R4* together designate a biradical selected from —Y—, —(CR*R*)r+s+1—, —(CR*R*)r—Y—(CR*R*)s—, and —Y—(CR*R*)r+s—Y—; R2 and R3 together designate a biradical selected from —Y—, —(CR*R*)r+s—, —(CR*R*)r—Y—(CR*R*)s—, and —Y—(CR*R*)r+s—Y—; R2* and R3 together designate a biradical selected from —Y—, —(CR*R*)r+s—, —(CR*R*)r—Y—(CR*R*)s—, and —Y—(CR*R*)r+s—Y—; R3 and R4* together designate a biradical selected from —Y—, —(CR*R*)r+s—, —(CR*R*)r—Y—(CR*R*)s—, and —Y—(CR*R*)r+s—Y—; R3 and R5 together designate a biradical selected from —Y′—, —(CR*R*)r+s+1—, —(CR*R*)r—Y—(CR*R*)s—, and —Y—(CR*R*)r+s—Y—; R1* and R4* together designate a biradical selected from —Y′—, —(CR*R*)r+s+1—, —(CR*R*)r—Y—(CR*R*)s—, and —Y—(CR*R*)r+s—NR*—; or where R1* and R2* together designate a biradical selected from —Y—, —(CR*R*)r+s—, —(CR*R*)r—Y—(CR*R*)s—, and —Y—(CR*R*)r+s—Y—; wherein each of r and s is 0-3 with the proviso that the sum r+s is 1-4, Y is as defined above, and where Y′ is selected from —NR*—C(═O)— and —C(═O)—NR*—.
Particularly interesting oligomers are those wherein one of the following criteria applies for at least one LNA in an oligomer: R2* and R4* together designate a biradical selected from —O—, —S—, —N(R*)—, —(CR*R*)r+s+1—, —(CR*R*)r—O—(CR*R*)s—, —(CR*R*)r—S—(CR*R*)s—, —(CR*R*)r—N(R*)—(CR*R*)s—, —O—(CR*R*)r+s—O—, —S—(CR*R*)r+s—O—, —O—(CR*R*)r+s—S—, —N(R*)—(CR*R*)r+s—O—, —O—(CR*R*)r+s—N(R*)—, —S—(CR*R*)r+s—S—, N(R*)—(CR*R*)r+s—N(R*)—, —N(R*)—(CR*R*)r+s—S—, and —S—(CR*R*)r+s—N(R*)—; R2 and R3 together designate a biradical selected from —O—, —(CR*R*)r+s—, —(CR*R*)r—O—(CR*R*)s—, —(CR*R*)r—S—(CR*R*)s—, and —(CR*R*)r—N(R*)—(CR*R*)s—; R2* and R3 together designate a biradical selected from —O—, —(CR*R*)r+s—, —(CR*R*)r—O—(CR*R*)s—, —(CR*R*)r—S—(CR*R*)s—, and —(CR*R*)r—N(R*)—(CR*R*)s—; R<