Modified Nucleosides, Nucleotides and Nucleic Acids via Click Azide-Alkyne Cycloaddition for Pharmacological Applications

Abstract

The click azide = alkyne 1,3-dipolar cycloaddition (click chemistry) has become the approach of choice for bioconjugations in medicinal chemistry, providing facile reaction conditions amenable to both small and biological molecules. Many nucleoside analogs are known for their marked impact in cancer therapy and for the treatment of virus diseases and new targeted oligonucleotides have been developed for different purposes. The click chemistry allowing the tolerated union between units with a wide diversity of functional groups represents a robust means of designing new hybrid compounds with an extraordinary diversity of applications. This review provides an overview of the most recent works related to the use of click chemistry methodology in the field of nucleosides, nucleotides and nucleic acids for pharmacological applications.

Keywords: 1,2,3-trizole; azide-alkyne cycloaddition; bioisosteres; click chemistry; nucleic acid; nucleosides; oligonucleotides; pharmaceutical approach.

Figure 1

Synthetic routes to 1,2,3-triazoles: (A): R₂ = H, alkyl or aryl group; (B,C): R₂ = H; (D): R₁ = -(CH₂)₃CH(R₃)-R₂-, R₂ = -(CH₂)₂-R₁-, R₃ = alkyl or aryl group.

Figure 2

Molecular structure of nucleosides functionalized with alkyne- or azide-reactive handles: (a) commercially available; (b) reported in Reference [29].

Scheme 1

Synthetic scheme for the preparation of adenosine-based bile acid conjugates. Reagents and conditions: (a) CuSO₄·5H₂O, sodium ascorbate, 1:1:1.5 H₂O/t-BuOH /THF (v/v), 25 °C, 18 h (15a 72% yield, 15b 78% yield).

Scheme 2

Synthetic scheme for preparation of hybrid 17. Reaction conditions: (a) CuSO₄·5H₂O, sodium ascorbate, 1:1:1.5 H₂O/t-BuOH/THF (v/v), 25 °C, 18 h (56% yield).

Scheme 3

Synthetic scheme for preparation of hybrid 20 and 21. Reaction conditions: (a) sodium ascorbate, CuSO₄·5H₂O, EtOH/H₂O, r. t., 16 h; (20 51% yield; 21 42% yield).

Scheme 4

Synthetic scheme for the preparation of compound 24. Reaction conditions: (a) sodium ascorbate (1.2 eq), CuSO₄ (0.2 eq.), DMF/H₂O, 24 h; (14% yield).

Scheme 5

Synthetic scheme for preparation of compound 27a,b and 28a,b. Click reaction conditions: DIPEA, sodium ascorbate (0.3 eq.), CuSO₄·5H₂O (0.1 eq.), t-BuOH/H₂O 3:1, r.t., 16 h, (50–60% yield).

Scheme 6

Synthetic scheme for preparation of compound 31 (a) CuI (1.5 eq.), DIPEA (3 eq.), toluene, 50 °C, 16 h, (68% yield).

Scheme 7

Reagents and conditions: (a) for β-34a,b: propargyl alcohol, (EtO)₃P·CuI, 1:1 toluene/THF, 24 h, reflux; for α-35a: propargyl alcohol, CuSO₄ (0.2 eq.), sodium ascorbate (0.4 eq.), THF, H₂O, 20–25 min, reflux; for α-35b: propargyl alcohol, CuSO₄ (0.2 eq.), sodium ascorbate (0.4 eq.), t-BuOH, H₂O, 20 h, 50 °C; (b) 40, DCI, MeCN, 45 min, r. t. (c) t-BuOOH, decane, MeCN, 45 min, r.t.; then Et₂NH, MeCN, 45 min, r.t.; (d) NH₄OH, MeCN, 18 h, r.t. DCI: 4,5-dicyanoimidazole.

Scheme 8

Click reaction conditions: CuI, DIPEA, H₂O/t-BuOH/CH₃CN (43a 42% yield, 43b 49% yield).

Scheme 9

(a) Methyl propiolate, CuI/DIPEA, HOAc, CH₂Cl₂, 0 °C to r.t., 1 h (95% yield); (b) NH₃·MeOH, r.t., 48 h; (c) PPh₃/I₂/imidazole, CH₂Cl₂, 0–35 °C, 4 h; (d) DBU, THF, 65 °C, 24 h; (e) ICl/NaN₃, THF, 0 °C to r.t., 8 h; (f) BzCl, DMAP, Et₃N, CH₂Cl₂, 0 °C, 20 min; (g) m-CPBA, m-CBA, n-Bu₄NHSO₄/K₂HPO₄, CH₂Cl₂, 0 °C to r.t., 16 h; (h) Ni(CH₃COO)₂·4H₂O, HOAc, reflux, 7 h; (i) NH₃/MeOH, 24 h.

Scheme 10

Reaction conditions: (a) Ph-Me, 110 °C, 24 h, (95% yield); (b) NH₃ aq., 1,4 dioxane, reflux, 48 h, (43% yield); (c) Dowex 500WX8, MeOH/H₂O, 50 °C, 12 h, (72% yield).

Scheme 11

Reaction conditions: (a) Ph-Me, 110 °C, 24 h.

Scheme 12

Synthetic scheme for the preparation of hybrid 56 and 58. Yields after RP HPLC purification 56: 98%; 58: 96%.

Scheme 13

Synthetic scheme for preparation of 61. Reaction conditions: (a) DMSO, r.t., 5 min, (70% yield).

Scheme 14

Synthetic scheme for preparation of 64. Reaction conditions: (a) sodium ascorbate, CuSO₄·5H₂O; (b) (COCl)₂, DMSO, TEA; (c) CH₃COC(=N)₂PO(OCH₃)₂ (Ohira-Bestmann reagent), K₂CO₃, MeOH; (d) Pd(PPh₃)₄, TEA, DMF.

Scheme 15

Best reaction conditions for the synthesis of 65: (a): sodium ascorbate, CuSO₄, DMF/H₂O (9:1), 90 °C, 5 h; (62% yield).

Scheme 16

Synthetic scheme for preparation of hybrids 68–70. Reaction conditions: (a) sodium ascorbate, CuSO₄·5H₂O, t-BuOH/THF/H2O (3:1:1); (68: 63% yield; 69: 49% yield; 70: 68% yield).

Figure 3

Structure of natural nucleic acids (A) and triazole-linked analogues (B). The natural O-P-O phosphate linkage is substituted by an artificial N-C-C triazole linkage.

Scheme 17

Synthesis of triazole-linked dinucleotides. Reaction conditions: (a) CuBr·SMe₂, t-BuOH, DMF, rt; (b) EtN(i-Pr)₂, ClP(OCH₂CH₂CN)N(i-Pr)₂, CH₂Cl₂, r.t. The triazole linkage is represented as t.

Scheme 18

Synthesis of the triazole-linked morpholino dinucleotide. Reaction conditions: (a) CuI, THF:t-BuOH:H₂O (3:2:1), μW, 80 °C, 1 h; (b) EtN(i-Pr)₂, ClP(OCH₂CH₂CN)N(i-Pr)₂, DMAP, THF, r.t.

Scheme 19

Synthesis of triazole-linked LNA dinucleotides. Reaction conditions: (a) CuSO₄·5H₂O, sodium ascorbate, THF:t-BuOH:H₂O (1:1:1), r.t (yield: 91–92%); (b) DMTrCl, pyridine, r.t. (yield: 77–83%); (c) EtN(i-Pr)₂, ClP(OCH₂CH₂CN)N(i-Pr)₂, CH₂Cl₂, r.t.

Scheme 20

(A): Synthesis of triazole 3′-LNA: (B): Synthesis of triazole 5′-LNA. Reaction conditions: (a) CuSO₄, sodium ascorbate, tris-(3-hydroxypropyltriazolylmethyl)amine (TBTA).

Scheme 21

Split-and-click approach to modified sgRNAs. Click reaction conditions: (a) MgCl₂, fresh ascorbic acid, CuSO₄- tris-(3-hydroxypropyltriazolylmethyl)amine, triethylammonium acetate buffer (pH 7): DMSO (1:1), r.t., 1–2 h.

Scheme 22

Solid-phase synthesis of 5′ alkyne modified oligonucleotide and CuAAC reaction: (a) alkynyl linker, PvCl, MeCN/Py (3:1), 5 min, r.t.; (b) I₂/Py/H₂O, 15 min, r.t. or S₈, Et₃N, BSA, r.t. 30 min; (c) 3.5% DCA in DCM, 2 min, r.t.; (d) HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), NMM (N-Methylmorpholine), DMF, 30 min, r.t.; (e) azide (2 eq.), CuBr·Me₂S (2.5 eq.),. DIPEA, (1,25 eq.) tBuOH/H₂O/DMSO (1:1:1) r.t. 2 min.; (f) NH₃ in MeOH, r.t. 16 h.

Scheme 23

Solid-phase CuAAC conjugation of oligonucleotides. Reagents and conditions: (a) solid-phase synthesis at 1 µmol scale, tetraazide (20 eq.), CuI·P(EtO)₃ (2 eq.), 2 h, r.t. (b) solid-phase synthesis at 1 µmol scale, GalNAc-alkyne (10 eq.), CuI·P(EtO)₃ (2 eq.), 2 h, r.t. (76.5% overall yield). (c) 1,2-ethylenediamine 2 h. (d) solid-phase synthesis at 1 µmol scale, tris-GalNAc-alkyne, (10 eq.), CuI·P(EtO)₃, (2 eq.)2 h, r.t.

Scheme 24

Conjugation of squalene and solanesol to small interfering RNA through SPAAC. Reaction conditions: (a) azide (50 eq.), DMSO/H₂O/acetone, r.t. 12 h for 95 and 18 h for 96 (95% conversion).

Scheme 25

A representative example of orthogonal amidation and cycloaddition approach to heavy modified siRNA: (a) amidation. (b) Deprotection and resin cleavage. (c) Click reaction: CuBr·Me₂S, ACN/H₂O (2:8) 50 °C, 8 h.

Scheme 26

Conjugation of vitamin B₁₂ with 2′OMeRNA. Click reaction conditions: (a) N₃-RNA. (0.5 eq.), Cu-TBTA (1.5 eq.), ascorbic acid (6 eq.), DMSO/triethylammonium acetate buffer (pH 7), (1:1) r.t. 20 h.

Scheme 27

Synthesis of boron-cluster-modified anti-EGFR antisense oligonucleotides. Click reaction conditions: azide (50 eq.), CuSO₄/TBTA (1:1) (25 eq.), NaAsc (40 eq.), DMSO/tBuOH/H₂O (3:1:1), 4 h, r.t. (61% yield).

Scheme 28

Synthesis of modified Pip6a-PMO. Reaction conditions: (a) Cu-TBTA (12 eq.), 113a–b (1.2 eq.), NaAsc (10 eq.), r.t., 6 h.

Figure 4

DART-Nrf2 multifunctional oligonucleotide.

Scheme 29

Nucleobase-modified aptamers. (A) reaction conditions: (a) 1:4 CuSO₄/THPTA (0.1 eq. in respect to 114), phosphate buffer/DMSO (pH 7), 37 °C, 1 h; (B): click-SELEX cycle representation.

Scheme 30

Gint4.T-MP conjugation. Click reaction conditions: (a) CuSO₄·5H₂O, ascorbic acid, PMDETA, Tris HCl, pH 7.5 buffer, r.t. 2 h, argon atmosphere.