Modified internucleoside linkages for nuclease-resistant oligonucleotides

Abstract

In the past few years, several drugs derived from nucleic acids have been approved for commercialization and many more are in clinical trials. The sensitivity of these molecules to nuclease digestion in vivo implies the need to exploit resistant non-natural nucleotides. Among all the possible modifications, the one concerning the internucleoside linkage is of particular interest. Indeed minor changes to the natural phosphodiester may result in major modifications of the physico-chemical properties of nucleic acids. As this linkage is a key element of nucleic acids' chemical structures, its alteration can strongly modulate the plasma stability, binding properties, solubility, cell penetration and ultimately biological activity of nucleic acids. Over the past few decades, many research groups have provided knowledge about non-natural internucleoside linkage properties and participated in building biologically active nucleic acid derivatives. The recent renewing interest in nucleic acids as drugs, demonstrated by the emergence of new antisense, siRNA, aptamer and cyclic dinucleotide molecules, justifies the review of all these studies in order to provide new perspectives in this field. Thus, in this review we aim at providing the reader insights into modified internucleoside linkages that have been described over the years whose impact on annealing properties and resistance to nucleases have been evaluated in order to assess their potential for biological applications. The syntheses of modified nucleotides as well as the protocols developed for their incorporation within oligonucleotides are described. Given the intended biological applications, the modifications described in the literature that have not been tested for their resistance to nucleases are not reported.

Scheme 1. General representation of the hydrolysis of an ODN by a 5′-PDE.

Fig. 1. Bridging and non-bridging modifications of the internucleoside phosphodiester linkage. Nomenclature used for the replacements of non-bridging oxygen atoms in modified internucleoside linkages.

Scheme 2. Eckstein synthesis of 5′-phosphorothioate thymidine (2) and dithymidine phosphorothioate (3).

Scheme 3. Sequence and chemical structure of Mipomersen.

Scheme 4. Chemical structures of S_p and R_p phosphorothioate chiral linkages.

Scheme 5. Automated synthesis cycle for stereoregular PS-ODN. Chemical structures of S_p (4a–d) and R_p (5a–d) OAP monomers.

Scheme 6. Synthesis cycle of stereoregular all-S_P-PS-ODN using ψ reagent. Structures of (+/−)-ψ reagents 7 and 8.

Scheme 7. Letsinger and Kool templated formation of thiophosphate internucleoside linkages.

Scheme 8. Enzymatic resistance of the 5′-SP linkage to NsiI endonuclease.

Scheme 9. Chemical structures of 3′-SP and 5′-SP linkages.

Fig. 2. Chemical structures of the first dinucleotide analogues bearing a selenium atom.

Scheme 10. Synthesis of phosphoroselenoate building blocks 16a–d.

Fig. 3. Chemical structures of phosphoramidates 17, 18 and 19 and NP modified diadenosines 20 and 21 studied by Letsinger et al.

Scheme 11. Synthesis cycle of N3′ → P5′ PN-ODN.

Fig. 4. Oligonucleotides studied by Shaw et al. and chemical structure of the P-NH₂-NP linkage.

Fig. 5. Chemical structures of modified ODN: LNA, LMP, N5′ → P3′ PN, 2′,4′-BNA and 2′,4′-BNA-NP.

Scheme 12. General route for the synthesis of MP-dinucleotides 25a–f.

Fig. 6. Chemical structures of S_p and R_p methylphosphonate chiral linkages.

Fig. 7. Chemical structures of backbone-modified oligonucleotides containing chiral R_pMP linkages: R_pMP/MP, R_pMP/PO and 2′-O-methyl-R_pMP/PO backbones.

Scheme 13. Chemical structures of S_p and R_p PyrP chiral linkages. Synthesis of PyrP phosphoramidite building blocks 28a–c.

Scheme 14. Chemical structures of S_p and R_p 2-AEP chiral linkages. Synthesis of the protected 2-(3,4,5,6-tetrabromophthalimido) or 2-(3,4,5,6-tetrachlorophthalimido) phosphoramidite building block 30.

Fig. 8. Chemical structures of 3′- and 5′-DHMT linkages obtained from 3′-DHMT 31 and 5′-DHMT 32 phosphoramidite building blocks.

Fig. 9. Chemical structures of regioisomeric 3′-phosphonate (Bpc-B) and 5′-phosphonate (B-pcB) linkages obtained from Bpc-B 33a–d and B-pcB 34a–d building blocks.

Fig. 10. Chemical structures of the 5′-ethylphosphonate linkage obtained from C-phosphonate building block 35.

Scheme 15. Synthesis of VP-dithymidine phosphoramidite 39.

Fig. 11. Chemical structures of EP-linked phosphoramidite thymidine dimers 40 and 41.

Scheme 16. Synthesis of the phosphoramidite thymidine dimer 40.

Fig. 12. (A) Chemical structures of the chimeric ODN used for in cellulo penetration experiments obtained from the protected phosphoramidite building blocks 47a–d; and (B) Chemical structures of the 2′-O-methyl-AcPS-ORN used for in cellulo penetration experiments as inhibitors of HTT expression.

Scheme 17. Chemical structures of phosphonoformate linkages obtained from phosphoramidite building blocks 48a–d.

Scheme 18. Synthesis of the alkyne phosphoramidite building blocks 51a–d used for triazolylphosphonate ODN synthesis.

Scheme 19. Synthesis of PT-dinucleotides 53a–d.

Fig. 13. Chemical structures of the R_p54 and S_p55 PT stereoisomers.

Fig. 14. Chemical structures of PT modified dinucleotides 56 and 57 studied by Letsinger et al.

Scheme 20. Conversion of PSS-ODN or SATE-ODN into natural PO-ODN in (A) a reducing environment and (B) in the presence of carboxyesterase respectively.

Scheme 21. Synthesis cycle of diPO-ODN.

Scheme 22. Synthesis of BP-dithymidine, 61.

Scheme 23. Synthesis of BP-diuridine diastereoisomers 64 and 65.

Scheme 24. Solid supported synthesis of BP-ODN.

Scheme 25. Synthesis cycle of chimeric BP/PO-ODN.

Fig. 15. Chemical structures of the 5′-(α-P-borano)triphosphates 68a–d synthesised.

Scheme 26. Synthesis of BP-diadenosines 74 and 75.

Scheme 27. Synthesis cycle of chimeric stereoregular BP/PO-ODN. Structures of the (R_p) 76a–d and (S_p) 77a–d oxazaphospholidine monomers.

Scheme 28. Synthesis cycle of MPS-ODN.

Scheme 29. Synthesis of SPS phosphoramidite building block 82.

Fig. 16. Chemical structures of PS, NP and NPS linkages.

Scheme 30. Synthesis cycle of NPS-ODN.

Scheme 31. Synthesis of NMP-dithymidine phosphoramidite building blocks 86 and 87.

Scheme 32. Synthesis of BMP-dithymidine 90.

Scheme 33. Synthesis cycle of chimeric BMP/PO- or BMP-PS-ODN. Structures of the methylphosphinoamidite building blocks 91 and 92 used for BMP-ODN elongation.

Fig. 17. Chemical structures of 1,5-triazole linkages.

Scheme 34. Synthesis of TR₁-dithymidine phosphoramidite building block 98.

Scheme 35. Synthesis of TR₂-dithymidine phosphoramidite building block 103.

Fig. 18. Chemical structures of T^L_TrT (104), T^L_TrT^xylo-L (105) and T^L_TrT^L (106) TR-LNA-dithymidines.

Scheme 36. Synthesis of 5′-azido-ODN 109.

Scheme 37. Synthesis of TR-LNA-ODN 115.

Fig. 19. Chemical structures of PO- and TR-DNA and LNA backbones investigated.

Scheme 38. Synthesis of S-dithymidine 119 (T_ST).

Scheme 39. Synthesis of SUL-dithymidine phosphoramidite building block 124 used for ODN elongation.

Scheme 40. Temperature and pH driven boronic ester chemical ligation.

Scheme 41. Synthesis of piperazine-dithymidine phosphoramidite building blocks 130 and 133 (PI_c and PI_co).

Scheme 42. Synthesis of N-substituted guanidine-dithymidine phosphoramidite building blocks 136a–j.

Scheme 43. Synthesis of acetylguanidine-dithymidine phosphoramidite building block 141.

Scheme 44. Synthesis cycle of chimeric MMI/PO-ODN.

Scheme 45. Synthesis of amide-dithymidine phosphoramidite building blocks 148a–c.

Fig. 20. Chemical structures of the AM-dithymidine phosphoramidite building blocks 149–153 synthesised by Just and De Mesmaeker.

Scheme 46. Synthesis of amide-dithymidine phosphoramidite building blocks 156a–d.

Scheme 47. Synthesis cycle of chimeric AM/PO-ODN.

Scheme 48. Synthesis cycle of chimeric AM/PO-ODN.

Scheme 49. Synthesis of urea-dithymidine phosphoramidite building block 167.

Scheme 50. Synthesis of urea-dithymidine phosphoramidite building blocks 171a–d.

Scheme 51. Synthesis of morpholino cytidine oligomers 176 with carbamate internucleoside linkages.

Fig. 21. Chemical structure of the phosphorodiamidate morpholino linkage.

Scheme 52. Iterative procedure for MU-homothymidylate 180 synthesis.

Scheme 53. Synthesis of S-methylthiourea-dithymidine phosphoramidite building block 184.

Scheme 54. Synthesis of 5′-N-CA trinucleoside 187.

Scheme 55. Synthesis of 3′-N-CA-dithymidine phosphoramidite building block 190.

Fig. 22. Chemical structures of the 6 CA-dithymidine phosphoramidite building blocks studied by the group of Brown bearing either 5′-N-CA or 3′-N-CA linkages.

Fig. 23. Timeline representing the first publication of the internucleoside linkages described in this review.