Expanding a fluorescent RNA alphabet: synthesis, photophysics and utility of isothiazole-derived purine nucleoside surrogates

Abstract

A series of emissive ribonucleoside purine mimics, all comprised of an isothiazolo[4,3-d]pyrimidine core, was prepared using a divergent pathway involving a key Thorpe-Ziegler cyclization. In addition to an adenosine and a guanosine mimic, analogues of the noncanonical xanthosine, isoguanosine, and 2-aminoadenosine were also synthesized and found to be emissive. Isothiazolo 2-aminoadenosine, an adenosine surrogate, was found to be particularly emissive and effectively deaminated by adenosine deaminase. Competitive studies with adenosine deaminase with each analogue in combination with native adenosine showed preference for the native substrate while still deaminating the isothiazolo analogues.

Fig. 1. Thiopheno vs. isothiazolo family.

Fig. 2. The synthetic family tree of an isothiazolo alphabet and potential synthetic routes.

Scheme 1. Isothiazolo functional library. Conditions: (a) trifluoroacetic anhydride, Py, 0 °C to rt, 90 min, 72% yield; (b) KOCN, H₂O, acetic acid, rt, 12 h, 70% yield; (c) dimethylformamide dimethyl acetal, DMF, rt, 12 h, 45% yield; (d) chloroformamidine hydrochloride, dimethylsulfone, 125 °C, 1 h, 74% yield; (e) dimethylformamide dimethyl acetal, DMF, rt, 12 h, 68% yield; (f) piv-Cl, Py, rt to 70 °C, 1 h, 61% yield.

Fig. 3. Thorpe–Ziegler cyclization.

Scheme 2. Bromination of derivatives. Reagents and conditions: (a) Br₂, AcOH, 55 °C, 1 h, 73% yield; (b) Br₂, AcOH, 50 °C, 2 h, 41% yield; (c) (i) Br₂, AcOH, 50 °C, 1 h, (ii) TFAA, Py, 0 °C to rt, 9 h, 60% overall; (d) Br₂, AcOH, 50 °C, 2 h, 63% yield.

Scheme 3. Thiol construction and cyclization. Conditions: (a) CH₂I₂, MeLi, toluene, –78 °C, 1 h, 67%; (b) potassium thioacetate, DMF, rt, 6 h, 74%; (c) Et₂O, LiAlH₄, 0 °C to rt, 1 h, >90%; (d) 16a or 16b, morpholine, MeOH, 0 °C to rt, 12 h, 67% (17a), 79% (17b).

Scheme 4. Reduction of lactol. Conditions: (a) 17a, BF₃·OEt₂, triethylsilane, DCM, –78 °C to rt, 4 h, 67% yield; (b) 17b, BF₃·OEt₂, triethylsilane, DCM, –78 °C to rt, 4 h, 79% yield. (c) 17a, (i) NaBH₄, MeOH, 0 °C, 4 h, (ii) DIAD, PPh₃, DCM, 0 °C to rt, 12 h, 30% over 2 steps; (d) (i) 17a, l-selectride, MeOH, 0 °C, 4 h, (ii) DIAD, PPh₃, DCM, 0 °C to rt, 12 h, 40% over 2 steps.

Scheme 5. Protected ^tzG (20), ^tzX (22), ^tz2-AA (23) synthesis. Conditions: (a) (i) ethoxycarbonyl isothiocyanate, MeCN, rt, 6 h; (ii) EDCl, HMDS, rt, 24 h, 63% over 2 steps; (b) 1 M NaOH, MeOH, 80 °C, 1 h; (c) KOCN, H₂O, acetic acid, rt, 12 h, 79%; (d) NaOMe, MeOH, rt, 12 h, 89%; (e) (i) POCl₃, pyridine, 100 °C, 2 h; (ii) NH₃, MeOH, 80 °C, 24 h, 40% over 2 steps.

Scheme 6. Synthesis of protected ^tzA (25) and ^tzisoG (27). Conditions: (a) triethyl orthoformate, acetic anhydride, 100 °C, 69% yield; (b) (i) P₂S₅, Py, 120 °C, (ii) NH₃, MeOH, 80 °C, 80% crude yield; (c) ethoxycarbonyl isothiocyanate, EDC, HMDS, MeCN, rt, 72 h, 40% yield; (d) 1 M NaOH, MeOH, 80 °C, 1 h, 90% crude yield.

Fig. 4. X-ray crystal structures of isothiazolo[3,4-d]pyrimidine analogs: (a) ^tzisoG (b) ^tzX.

Fig. 5. (a) Absorption (dashed lines) and emission (solid lines) spectra of ^tzisoG (magenta), ^tz2-AA (cyan) and ^tzX (brown) in water. (b) Absorption (dashed lines) and emission (solid lines) traces in water, dioxane and mixture thereof for ^tzX. (c) Stokes shift correlation versus solvent polarity (E _T(30)) of water/dioxane mixtures for ^tzisoG (magenta), ^tz2-AA (cyan) and ^tzX (brown). (d) Stokes shift correlation versus solvent polarity (E _T(30)) of water/dioxane mixtures for ^tzX (brown) in comparison to the structural isomer ^tzU (purple).

Fig. 6. (a) Schematic representation of possible isothiazolo-pyrimidine nucleoside tautomers with labelled nitrogen atoms (red) and suggested pK _a values (blue) corresponding to multiple possible protonation/deprotonation sites (parenthesis). (b) Absorption (dashed lines) and emission (solid lines) spectra of ^tzX as a function of water solution pH. (c) Absorption maxima and (d) emission maxima variation versus pH for ^tzisoG (magenta), ^tz2-AA (cyan) and ^tzX (brown).

Fig. 7. (a) ^tzisoG emission spectra at pH 3.49, recorded upon excitation at different wavelengths and normalized for the corresponding absorbance intensity. (b) ^tzisoG emission spectra at pH 3.49, recorded upon excitation at different wavelengths and normalized to unit in intensity. (c) Excitation spectra normalized to unit recorded at selected emission spectra wavelengths, covering the whole emission band of aqueous solutions of ^tzisoG at pH 3.49. (d) Reconstructed excitation spectra of ^tzisoG at pH 3.49, plotting the emission intensities at the relative maxima 415 (red) and 500 (blue) nm upon excitation at different wavelengths. The grey line represents the relative ratio of the emission intensities upon excitation at different wavelengths.

Fig. 8. (a) Enzymatic competitive deamination of ^tz2-AA and native A to provide ^tzG and inosine as monitored by HPLC traces at T = 0 s and T = 600 s. (b) Enzymatic deamination of native 2-AA (grey) and ^tz2-AA (black) monitored by change in absorption spectra intensity at 290 and 368 nm respectively. Inset: ADA-mediated deamination of ^tz2-AA followed by absorption spectroscopy at 368 nm (black), real-time emission at 475 nm (pink) and HPLC relative peak area variation (cyan) at different time-points for ^tz2-AA. (c) Enzymatic competitive deamination of ^tz2-AA (cyan) and native A (green) to provide ^tzG (orange) and inosine (purple) monitored by HPLC relative peak area variation at different time-points. (d) Enzymatic competitive deamination of ^tz2-AA (cyan) and ^tzA (blue) to provide ^tzG (orange) and ^tzI (red) monitored by HPLC relative peak area variation at different time-points. (e) Enzymatic competitive deamination of ^tzA (blue) and native A (green) to provide ^tzI (red) and inosine (purple) monitored by HPLC relative peak area variation at different time-points.