Synthesis of 8-oxo-dGTP and its β,γ-CH₂-, β, γ-CHF-, and β, γ-CF₂- analogues

Abstract

Three novel 8-oxo-dGTP bisphosphonate analogues of 3 in which the bridging β,γ-oxygen is replaced by a methylene, fluoromethylene or difluoromethylene group (4–6, respectively) have been synthesized from 8-oxo-dGMP 2 by reaction of its morpholine 5’-phosphoramidate 14 or preferably, its N-methylimidazole 5’-phosphoramidate 15 with n-tributylammonium salts of the appropriate bisphosphonic acids, 11–13. The latter method also provides a convenient new route to 3. Analogues 4–6 may be useful as mechanistic probes for the role of 3 in abnormal DNA replication and repair.

Graphical Abstract

Introduction

Oxidative DNA damage due to reactive oxygen species (ROS) has been implicated in the pathogenesis of a wide variety of diseases, including cancer,¹ neurodegenerative and neurodevelopmental disorders,² inflammatory disorders³ and aging.⁴ 8-Oxo-2’-deoxyguanosine (8-oxo-dG, 1) is a harbinger of oxidative DNA damage⁵ and has been implicated in carcinogenesis by inducing mutations⁶ as well as by abnormal epigenetic modulation of gene expression.⁷ The mutagenicity of 1 is attributed to a conformational shift of the N9-C1’ glycosidic bond from anti to syn, causing it to mimic a syn thymidine.⁸ As a result of A:8-oxo-G (A:8OG) Hoogsteen base mispairing, replicative DNA polymerases (pols) often insert dATP opposite 1 instead of dCTP.^9,10 Pol β, pol η, REV1, pol ξ and pol κ have all been implicated¹¹ in the incorporation of 8-oxo-dGMP 2 into DNA from 8-oxo-2’-deoxyguanosine-5’-triphosphate (8-oxo-dGTP, 3), present as an ROS in the cellular nucleotide pool.¹² Wilson and co-workers recently described the crystal structure of a pol β DNA complex in which the adenine of a DNA (syn)8OG:A base pair was replaced at the primer terminus by a cytosine.¹³ It is apparent that complementary information about the functional mechanism and transition state (TS) is desirable.

Herein, we report the synthesis of a small toolkit of 8-oxo-dGTP bisphosphonate probes, including an alternate preparation of the reference nucleotide, 3. The toolkit comprises three β,γ-CXY bridged 8-oxo-dGTP analogues: β,γ-methylene- 4, β,γ-monofluoromethylene- 5, and β,γ-difluoromethylene-8-oxo-dGTP 6 (Fig. 1).

Figure 1.

Structures of 8-oxo-dGTP 3, β,γ-CH₂- 4, β,γ-CHF- 5, and β,γ-CF₂-8-oxo-dGTP 6.

A similar toolkit based on the natural nucleotides has been used to study leaving group effects on the nucleotidyl transfer kinetic mechanisms and fidelity of pols^14–18 and other biocatalysts.¹⁹ As the β,γ-bridge atom X and Y substituents become more electronegative, the pK_a4 of the corresponding bisphosphonate leaving group decreases,^{20, 21} stabilizing the conjugate base anion. If the rate-determining step (RDS) involves P-O bond breaking, then a Bronsted plot of the log of the catalytic rate constant (k_pol) versus pK_a4 is predicted to be linear (linear free energy relationship, LFER) with a negative slope reflecting the sensitivity of the TS to anion stabilization.²² The bisphosphonates selected provide a range of pK_a4 of 2.75 units, centered on the pK_a4 of the leaving group in 3, pyrophosphoric acid.²⁰

Results and Discussion

Early oxidative methods^{12, 23} to prepare 3 itself directly from dGTP in low or unstated yield were not reproduced by others,²⁴ as confirmed by own work (data not shown). Direct oxidative methods have a further limitation in that they do not give convenient access to 8-[¹⁷O]- or 8-[¹⁸O]-oxo-guanosine derivatives.²⁵ Einolf described an 8-step synthesis of 3 beginning from dG 7 involving several protection/deprotection steps, culminating in separation of the final compound from mono- and diphosphate 8-oxo-dG byproducts.²⁶ Nampalli and Kumar²⁴ subsequently outlined a gram-scale synthesis of 3 from 8-bromo-dG^{27, 28} 8 in 36% overall yield via conversion to the 8-benzyloxy derivative 9 using sodium/benzyl alcohol in dimethyl sulfoxide, followed by treatment with phosphorus oxychloride (POCl₃)²⁹ in trimethyl phosphate and reaction with bis(tributylammonium) pyrophosphate. The final product was obtained by hydrolysis of the resulting cyclic triphosphate intermediate in aqueous triethylammonium bicarbonate (TEAB) at pH 7.5 (Scheme 1 and 2).²⁴

Scheme 1.

Synthesis of 8-oxo-dG 1.^{24, 27, 28}

Scheme 2.

‘One-pot’ phosphorylation^{30, 31} synthesis of 8-oxo-dGTP 3 from 8-oxo-dG 1;²⁴ (TEAB: triethylammonium bicarbonate buffer)

8-Benzyloxy-dG 9 can be prepared from 7 in 78% yield,^{27, 28} and we found that formation of an 8-dimsyl-dG byproduct can be avoided by using DMF in place of DMSO as the solvent (Scheme 1). However, in our hands, phosphorylation of 1 or 9 on a small scale using the literature one-pot-three-step procedure^{24, 30, 31} (Scheme 2) gave lower yields than anticipated.

We therefore examined an alternative synthesis of 3 starting from 8-oxo-dGMP 2 (prepared in 33% yield by monophosphorylation of 1 with POCl3 in PO(OMe)₃²⁹ followed by aqueous workup with 0.5 M TEAB) after activation by morpholine³² or N-methylimidazole^{33, 34} to facilitate coupling with pyrophosphate 10, as a method likely to be adaptable to the synthesis of 4–6 from the appropriate bisphosphonate tributylammonium salt 11–13,²⁰ prepared by treatment of commercially available methylenebis(phosphonic acid) or its α-fluorinated derivatives³⁵ with tributylamine in aq. ethanol. 8-Oxo-dGMP-morpholidate 14 gave 3 and the target bisphosphonate nucleotides 4–6, but the reactions were sluggish, with very poor yields (Scheme 3).

Scheme 3.

Synthesis of 3–6 via the 5’-morpholidate 14.

Better results were obtained with N-methylimidazole activation.^{33, 34} Thus, 2 suspended in a mixture of triethylamine and excess trifluoroacetic anhydride in acetonitrile was treated with N-methylimidazole to give the corresponding 8-oxo-dGMP-N-methylimidazolide 15, which was then added to tributylammonium pyrophosphate 10 or the tributylammonium bisphosphonate 11, 12 or 13 in DMF (Scheme 4). Deactivated 8-oxo-dGMP 2 can be recovered during purification of the final products (characterized by ¹H, ³¹P and ¹⁹F NMR, LC-MS and HRMS) via SAX/C-18 preparative HPLC.

Scheme 4.

Synthesis of 3–6 via the 5’-N-methylimidazolide 15.

In the coupling reactions to form 3–6, the reaction time was much shorter with the N-methylimidazolides (2–3 h) compared to the morpholidates (3–5 d).^30–32 It is critical to thoroughly dry the tributylammonium salt of the bisphosphonic acid by repeated coevaporation with anhydrous N,N-dimethylformamide before use to achieve optimal yields.

The proton-decoupled ³¹P NMR spectra of 3–6 are compared in Figure S44. As expected, a dramatic upfield shift of the P_γ and P_β resonances is seen with more electronegative substituents (CF₂ > CHF > CH₂) on the bridging β-methylene group. There is no effect on the P_α resonance which remains constant at about −10 ppm.

Consistent with published data for the β,γ-monofluoro and β,γ-difluoro analogues of dGTP,¹⁵ 5 and 6 exhibit ¹⁹F NMR resonances at δ −217.3 and δ −121. ppm, respectively. The ³¹P_β, ³¹P_γ and ¹⁹F peaks of 5 exhibit a slight broadening consistent with a small Δδ for the R v S diastereomers.¹⁵

Conclusion

In summary, we examined several alternative approaches for the synthesis of 8-oxo-dGTP 3 and three novel bisphosphonate analogues: β,γ-methylene- 4, (R/S)-β,γ-monofhioromethylene- 5, and β,γ-difluoromethylene-8-oxo-dGTP 6. Conjugation of the corresponding bisphosphonic acid tributylammonium salts 11–13 with 8-oxo-dGMP-N-methylimidazolide 15 in anhydrous DMF is a practical route to these compounds, albeit in low (not optimized) yields. The availability of the resulting 8-oxo-dGTP analogue toolkit provides a novel means to probe leaving group effects on the binding and kinetic mechanisms of 3 interacting with nucleic acid polymerases.

Highlights:

1. 8-Oxo-dGTP plays a key role in oxidative DNA mutation pathways.

2. 8-Oxo-GTP and 3 novel β,γ-CXY analogues were synthesized via 5’-P-imidazolides.

3. This leaving group-modified toolkit provides useful probes for DNA polymerases.