Synthesis and Properties of 2′-Deoxyadenosine Mimics Bearing a Thieno[3,2-d]pyrimidine Ring

Abstract

A C-nucleoside with a stable C–C glycosidic bond can be used as a building block for chemically modified oligonucleotides (ONs). In this study, two adenosine-like C-nucleosides (d^SA and d^SO2A) bearing thieno[3,2-d]pyrimidine rings were designed and synthesized. These analogues were synthesized via the Heck reaction, and their properties as monomer nucleosides were investigated. Both the d^SA and d^SO2A monomers were not recognized by adenosine deaminase (ADA). In addition, they exhibited fluorescence emissions in the UV and visible regions of d^SA and d^SO2A, respectively. Subsequently, d^SA was converted into a phosphoramidite compound and incorporated into the ONs. The synthesized d^SA-modified ONs formed a stable duplex with DNA and RNA complements comparable to natural adenosine. Furthermore, the modified ONs exhibited fluorescence emission derived from d^SA.

The glycosidic bond of an N-nucleoside is a C–N bond that is susceptible to enzymatic and acid-catalyzed hydrolysis, whereas that of a C-nucleoside consists of a chemically and biologically stable C–C bond. Several C-nucleosides occur naturally,[1] and pseudouridine,[2] showdomycin,[3] and formycin analogues[4] have been reported. Pseudouridine was isolated as a uridine mimic from transfer RNA and has been utilized recently in mRNA vaccines[5] by incorporating it into the RNA sequence. On the other hand, formycin A is an adenosine-like C-nucleoside (8-aza-9-deazaadenosine) and a potent inhibitor of adenosine-utilizing enzymes.[6] Moreover, formycin A has been reported to adopt a syn conformation in oligonucleotides (ONs), which destabilizes the duplex with pseudouridine-containing ONs.[7] This finding suggests that when incorporating purine-like C-nucleosides into oligonucleotides (ONs), the chemical structure of the Hoogsteen face can have crucial influence on their properties. Numerous artificial C-nucleosides with adenosine- or guanosine-like structures have also been synthesized, and their antiviral activity[8] and fluorescence property[9] have been evaluated using nucleoside monomers. Meanwhile, few reports on the properties of chemically modified ONs containing adenosine- or guanosine-like C-nucleosides exist in the literature.[10] However, if isomorphic C-nucleosides are to be applied in ON technologies such as ON therapeutics, the properties of ONs including these should be investigated. In this study, 2′-deoxyadenosine mimics of C-nucleosides were designed (Figure [1]). The designed C-nucleosides have a thieno[3,2-d]pyrimidine ring (d^SA) or an oxidized (S,S-dioxide) ring (d^SO2A) as their nucleobase. Although several thieno[3,2-d]pyrimidine derivatives with bioactivities such as antibiotic[11] or anticancer activities[12] and apoptosis induction[13] have been reported, their properties as nucleoside mimics are poorly understood despite their purine-like structure. In addition, the designed nucleosides could exhibit fluorescence emission because the fluorescence properties of thieno[3,2-d]pyrimidine derivatives are known.[14] Therefore, we were also interested in the photophysical properties of the nucleosides. In this paper, the synthesis of d^SA and d^SO2A nucleosides, enzymatic reaction using them, their photophysical properties, introduction of the nucleosides into ONs, and the duplex- and triplex-forming ability and photophysical properties of the modified ONs are reported.

The designed C-nucleoside was constructed using the Heck reaction[15] as shown in Scheme [1]. 7-Bromothieno[3,2-d]pyrimidine-4-amine (1) was protected with a monomethoxytrityl (MMTr) group to obtain compound 2 in 97% yield. The Heck reaction between compound 2 and 3 [16] was performed in the presence of Pd₂(dba)₃, t-Bu₃P, and DIPEA to produce silyl enol ether intermediate, which was converted into compound 4 by the deprotection of the TBS group in 54% yield over two steps. The reduction of the ketone group using sodium triacetoxyborohydride (NaBH(OAc)₃) stereoselectively proceeded to afford the 3′,5′-diol compound 5 in a quantitative yield. The deprotection of the MMTr group using trifluoroacetic acid (TFA) provided the nucleoside monomer d^SA in 81% yield.[17] The exocyclic amino group of d^SA was protected using dimethylformamide-dimethylacetal (DMF-DMA) to obtain the amidine compound 6 in 74% yield. The 4,4′-dimethoxytrityl (DMTr) protection of the 5′-OH group using 4,4′-dimethoxytrityl chloride (DMTrCl) produced compound 7 in 91% yield. Subsequently, the phosphitylation of the 3′-hydroxy group of compound 7 produced the phosphoramidite compound 8 in 74% yield, which is a building block for solid-phase ON synthesis.

The d^SO2A monomer and the corresponding phosphoramidite compound 12 were synthesized from compound 5 (Scheme [2]). The oxidation of the thiophene ring was performed using Oxone^® in the presence of acetone to obtain the oxidized compound 9 in 94% yield. Similar to d^SA, the deprotection of the MMTr group was achieved using TFA to afford the nucleoside monomer d^SO2A in 62% yield.[18] The DMTr protection of the 5′-OH group produced compound 10 in 99% yield. The protection of the exocyclic amino group using DMF-DMA afforded compound 11 in 93% yield. Subsequently, phosphitylation of the 3′-hydroxy group produced the phosphoramidite compound 12 in 71% yield.

We next investigated whether the synthesized adenosine mimics can be recognized by adenosine-related enzyme. The enzymatic conversion of adenosine (A) into inosine (I) using adenosine deaminase[19] (ADA) was performed using d^SA and d^SO2A (Figure S1). The treatment of d^SA with a catalytic amount of ADA in 50 mM phosphate buffer (pH 7) did not produce the corresponding inosine d^sI.[20] Also, in the case of d^SO2A, no conversion product was observed.[20] On the contrary, 2′-deoxyadenosine (dA) was successfully converted into 2′-deoxyinosine (dI) under the same conditions (Figure S1). Subsequently, the ADA inhibitory activities of d^SA and d^SO2A were evaluated (Figure S2). Consequently, the ADA-catalyzed conversion of dA into dI was maintained in the presence of equivalent concentrations of d^SA and d^SO2A. In contrast, ^thA,[21] the 7-carba-8-thio isomer of d^SA, is an ADA substrate. The enzymatic activity of ADA decreases when using isomorphic adenosine with a bulky group at the pseudoadenine N7 position.[22] Therefore, the bulkiness of the S atom and SO₂ group in d^SA and d^SO2A would prevent recognition by ADA. In other words, d^SA and d^SO2A can act as adenosine mimics with high enzymatic stability.

The photophysical properties of d^SA and d^SO2A were investigated using UV/Vis absorption and fluorescence spectroscopy (Figure [2] and Table [1]). The maximum absorption wavelength in the absorption spectra of d^SA and d^SO2A exhibited bathochromic shifts compared to the maximum absorption wavelength of the natural dA (λ^abs _max = 260 nm in H₂O, Figure S3). Moreover, the maximum absorption wavelength of ^SO2A (λ^abs _max = 331 nm in MeOH) was longer than that of d^SA (λ^abs _max = 298 nm in MeOH), which may be due to the electron push–pull effect between the 4-amino and 5-sulfone groups. The push–pull effect was also observed in the fluorescence spectra; the fluorescence emission maximum of d^SO2A was observed in the visible region (λ^abs _max = 452 nm in MeOH) whereas the emission maximum of d^SA was observed in the UV region (λ^abs _max = 333 nm in MeOH). Relative fluorescence quantum yields of d^SA and d^SO2A were calculated using tryptophan[23] as the standard. The quantum yield of d^SA was higher than that of d^SO2A, which showed a significantly low yield (< 0.01).

We tried to synthesize the ONs containing d^SA and d^SO2A using compounds 8 and 12 to evaluate their properties. d^SA-Modified ONs were successfully obtained using an automated DNA synthesizer via standard phosphoramidite chemistry (Table S1); however, no d^SO2A-modifed ONs could be obtained, and complex mixtures were observed after cleavage from a solid support using an ammonia solution. Thus, d^SA and d^SO2A monomers were treated under basic conditions (28% aq. NH₃ at room temperature for 2 h or 50 mM of K₂CO₃ in MeOH at room temperature for 4 h), which are generally used to cleave ONs from solid supports. Consequently, although d^SA was stable under these conditions, d^SO2A decomposed to produce multiple products (Figure S4). Although the structures of the decomposed products were undetermined, a nucleophilic attack on the α,β-unsaturated sulfone in d^SO2A might have occurred.

The duplex-forming ability of d^SA-modified ONs (ON1–4) with single-stranded DNA (ssDNA) and ssRNA was evaluated by UV melting experiments (Table [2] and Figure S5) and compared with that of the corresponding natural ON (ON5). The T _m values of the duplexes of ON1–4 with ssRNA ranged from 45.9 °C to 46.5 °C, which are slightly higher than the T _m value of the natural ON (44.7 °C). The ΔT _m value per modification (ΔT _m/mod.) of d^SA reached up to 1.3 °C. On the other hand, in terms of the thermal stability of the duplexes for complementary ssDNA, the T _m values for d^SA-modified ONs were comparable or slightly lower (T _m = 48.5–50.4 °C) than the T _m value of the natural DNA duplex (50.8 °C). In fact, the ΔT _m/mod. value of d^SA for DNA ranged from –1.2 °C to –0.4 °C. These results imply the d^SA modifications of ONs can maintain duplex-forming ability with ssRNA and ssDNA. We also assessed the nucleobase discrimination ability of the d^SA modifications by measuring the T_m values of mismatched DNA–RNA and DNA–DNA duplexes (Table [3]). The T_m values of the mismatched duplexes of d^SA-modified ONs with ssRNA (T _m = 35.5–37.8 °C) and ssDNA (T _m = 39.9–45.0 °C) were much lower than those of the complementary duplexes and the same tendency was observed for the natural matched and mismatched duplexes. Therefore, d^SA modification also maintained sufficient nucleobase discrimination ability, similar to natural ON. These results demonstrate that d^SA behaves like an adenosine when forming DNA–RNA and DNA–DNA duplexes.

Because d^SA monomer exhibits fluorescence emission, the fluorescence spectra of d^SA-modified ONs (ON1–4) were measured (Figure [3]). Significant changes in the fluorescence intensities of the ONs were observed (ON1 > ON3 > ON2 > ON4), indicating that the fluorescence emission of d^SA inside the ONs is susceptible to the sequence of bases around d^SA. Specifically, the fluorescence intensity of ON2 with a neighboring G base was lower than that of ON1. In addition, the fluorescence intensity of ON4 with d^SA modifications at each side of the G base was the lowest among ON1–4. Therefore, the fluorescence emission of d^SA is quenched by the neighboring G base, which is known as a fluorescence quencher.[24] In addition, the results imply that d^SA also works as a quencher in the cases of ON3 and ON4 which include two d^SA modifications. The fluorescence spectra of matched and mismatched duplexes with ON1 were also obtained (Figure S6) and showed fluorescence quenching upon duplex formation. The fluorescence experiment results suggest that the fluorescence of d^SA is sensitive to neighboring bases and can discriminate between single- and double-stranded structures. Thus, d^SA can become a fluorescent nucleoside that responds to the environment.

To investigate the interactions at the Hoogsteen face of d^SA, the triplex-forming ability of d^SA-modified duplexes (ON6–8) with triplex-forming ONs (TFOs) was determined using UV melting experiments (Figure S7 and Table S2). In a parallel motif triplex, a pyrimidine-rich TFO binds to the purine strand of the duplex on the Hoogsteen face to form T-AT and protonated C (C⁺H)-GC triplets. Natural duplex (ON9) formed a parallel triplex with TFO at pH values of 6.0 and 7.0 (T _m = 44.2 and 28.8 °C for pH = 6.0 and 7.0, respectively). Not surprisingly, the duplex containing one d^SA modification (ON6) showed decreased thermal stability of the parallel triplex at both pH values of 6.0 and 7.0 (T _m = 36.8 and 21.5 °C for pH = 6.0 and 7.0, respectively). In addition, the triplex-forming ability of duplexes containing three d^SA modifications (ON7 and ON8) was either considerably decreased or not observed (Table S2). The destabilization of the triplex by d^SA was due to the S atom on the Hoogsteen face, which could not form a hydrogen bond. Therefore, d^SA-modified duplexes are less likely to form higher-order structures via hydrogen-bonding interactions at the Hoogsteen face.

In summary, we developed two 2′-deoxyadenosine mimics (d^SA and d^SO2A) with thieno[3,2-d]pyrimidine rings and investigated their properties. In the enzymatic A-to-I conversion reaction using d^SA and d^SO2A, both nucleoside monomers were intact in the enzyme. Both d^SA and d^SO2A monomers showed fluorescence emission in solution, and the fluorescence quantum yield of d^SA was higher than that of d^SO2A. Although the d^SO2A monomer is labile to the basic conditions generally used in solid-phase ON synthesis, d^SA could be incorporated successfully into the ONs. The duplex-forming ability of the d^SA-modified ONs was similar to that of natural adenosine. Furthermore, the modified ONs showed fluorescence emission derived from d^SA, which was affected by neighboring bases and duplex formation. In addition, the triplex-forming ability of the duplex containing the d^SA modification decreased. This study demonstrated that d^SA can be used as an adenosine congener with high enzymatic stability and fluorescence properties in ON technologies.

Conflict of Interest

The authors declare no conflict of interest.

• References and Notes

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Corresponding Author

Publication History

Received: 24 July 2023

Accepted after revision: 12 September 2023

Article published online:
18 October 2023

© 2023. Thieme. All rights reserved

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References and Notes

• 1 Zhang M, Kong L, Gong R, Iorio M, Donadio S, Deng Z, Sosio M, Chen W. Microb. Cell Fact. 2022; 21: 1
  CrossrefPubMedSearch in Google Scholar
• 2a Cohn WE. J. Biol. Chem. 1960; 235: 1488
  CrossrefPubMedSearch in Google Scholar
• 2b Cohn WE. Biochim. Biophys. Acta 1959; 32: 569
  CrossrefPubMedSearch in Google Scholar
• 3a Nishimura H, Mayama M, Komatsu Y, Kato H, Shimaoka N, Tanaka Y. J Antibiot. 1964; 17: 148
  Search in Google Scholar
• 3b Nakagawa Y, Kano H, Tsukuda Y, Koyama H. Tetrahedron Lett. 1967; 8: 4105
  CrossrefPubMedSearch in Google Scholar
• 4a Koyama G, Maeda K, Umezawa H, Iitaka Y. Tetrahedron Lett. 1966; 7: 597
  CrossrefPubMedSearch in Google Scholar
• 4b Robins RK, Townsend LB, Cassidy F, Gerster JF, Lewis AF, Miller RL. J. Heterocycl. Chem. 1966; 3: 110
  CrossrefSearch in Google Scholar
• 4c Wang SA, Ko Y, Zeng J, Geng Y, Ren D, Ogasawara Y, Irani S, Zhang Y, Liu HW. J. Am. Chem. Soc. 2019; 141: 6127
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• 5a Morais P, Adachi H, Yu YT. Front. Cell Dev. Biol. 2021; 9: 1
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• 5b Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, Weissman D. Mol. Ther. 2008; 16: 1833
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• 6a Bzowska A, Kulikowska E, Shugar D. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1992; 1120: 239
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• 6b Long MC, Parker WB. Biochem. Pharmacol. 2006; 71: 1671
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• 6c Wierzchowski J, Antosiewicz JM, Shugar D. Mol. Biosyst. 2014; 10: 2756
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• 7a Ward DC, Reich E. Proc. Natl. Acad. Sci. U.S.A. 1968; 61: 1494
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• 9b Rovira AR, Fin A, Tor Y. J. Am. Chem. Soc. 2015; 137: 14602
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• 17 Synthesis of d^SA To a solution of compound 5 (38 mg, 70 μmol) in CH₂Cl₂ (1 mL), TFA (0.2 mL) was added at room temperature under an argon atmosphere. The reaction mixture was stirred for 17 h followed by addition of MeOH. The resulting mixture was concentrated in vacuo. Subsequently, CHCl₃ was added to the residue, and the resulting precipitate was collected by filtration. The collected solid was dried in vacuo to obtain compound d^SA (15 mg, 81%) as a white solid. IR (ATR): 3329, 3200, 2924, 1643, 1579, 1513 cm^–1. ¹H NMR (500 MHz, CD₃OD): δ = 8.33 (1 H, s), 7.98 (1 H, s), 5.49 (1 H, dd, J = 11.0, 5.5 Hz), 4.46 (1 H, d, J = 5.5 Hz), 4.06–4.05 (1 H, m), 3.81 (1 H, dd, J = 12.0, 3.0 Hz), 3.71 (1 H, dd, J = 12.0, 3.0 Hz), 2.42 (1 H, ddd, J = 13.0, 11.0, 5.5 Hz), 2.22 (1 H, dd, J = 13.0, 5.5 Hz). ¹³C NMR (125 MHz, CD₃OD): δ = 160.4, 157.7, 154.8, 138.1, 131.4, 117.4, 89.7, 77.9, 75.2, 64.5, 58.3, 49.8, 43.0, 18.4. HRMS (ESI-TOF): m/z [M – H]^– calcd for C₁₁H₁₂N₃O₃S: 266.0599; found: 266.0599.
• 18 Synthesis of d^SO2A To a solution of compound 9 (289 mg, 0.5 mmol) in CH₂Cl₂ (5 mL), TFA (0.25 mL) was added at room temperature under an argon atmosphere. The reaction mixture was stirred for 2 days and then quenched with NaHCO₃ and MeOH. The resulting mixture was filtered, and then the filtrate was concentrated in vacuo. Subsequently, a solution of CHCl₃/MeOH (1:1) was added to the residue, and the resulting precipitate was collected by filtration. The collected solid was dried in vacuo to obtain compound d^SO2A (92 mg, 62%) as a gray solid. IR (ATR): 3508, 3376, 3331, 3135, 3084, 1670, 1591, 1537, 1499 cm^–1. ¹H NMR (500 MHz, DMSO-d ₆): δ = 8.58 (1 H, s), 7.95 (2 H, br s), 7.46 (1 H, d, J = 2.0 Hz), 5.15 (1 H, d, J = 5.0 Hz), 5.12 (1 H, ddd, J = 10.0, 6.0, 2.0, Hz), 4.77 (1 H, t, J = 6.0 Hz), 4.19–4.18 (1 H, m), 3.80 (1 H, dt, J = 5.0, 2.0 Hz), 3.45–3.36 (2 H, m), 2.31 (1 H, ddd, J = 13.0, 6.0, 2.0 Hz), 1.79 (1 H, ddd, J = 13.0, 10.0, 5.0 Hz). ¹³C NMR (125 MHz, DMSO-d ₆): δ = 162.2, 158.1, 154.4, 148.1, 129.2, 109.0, 87.6, 72.9, 71.8, 62.0, 40.5. HRMS (ESI-TOF): m/z [M + Na]⁺ calcd for C₁₁H₁₃N₃O₅NaS: 322.0468; found: 322.0474.
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