Access to 3-Deazaguanosine Building Blocks for RNA Solid-Phase

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Access to 3‑Deazaguanosine Building Blocks for RNA Solid-Phase Synthesis Involving Hartwig−Buchwald C−N Cross-Coupling Elisabeth Mairhofer, Laurin Flemmich, Christoph Kreutz, and Ronald Micura* Institute of Organic Chemistry and Center for Molecular Biosciences, University of Innsbruck, 6020 Innsbruck, Austria

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S Supporting Information *

ABSTRACT: 3-Deazaguanosine (c3G) and phosphoramidite derivatives thereof that allow incorporation of this modification into RNA are needed for atomic mutagenesis experiments to explore mechanistic aspects of ribozyme catalysis. Here, we report a practical synthesis for c3G phosphoramidites from inexpensive starting materials. The key reaction sequence is a silyl-Hilbert−Johnson nucleosidation followed by Hartwig−Buchwald cross-coupling to achieve the N2-phenoxyacetyl protected c3G nucleoside.

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the six-membered-ring. In an alternative route, hydrazine was employed.19 Minakawa and Matsuda reported a synthesis that started from 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide (AICA-riboside) via 5-ethynyl-1-(β-D-ribofuranosyl)-1Himidazole-4-carboxamide or 5-ethynyl-1-(β-D-ribofuranosyl)1H-imidazole-4-carbonitrile.20 To the best of our knowledge, only one synthesis for a phosphoramidite of c3G has been reported so far.13 Therein, relying on the c3G nucleoside route by Robins,15−18 Seela and Beigelman utilized phenoxyacetyl protection at the N2 and a diphenylcarbamoyl residue at the O6 of the nucleobase while the 2′-hydroxyl group was blocked with a triisopropylsilyl group (Scheme 1).13 Access to the naked nucleoside of c3G is one obstacle that must be overcome. The c3G nucleoside is commercially available; however, high prices for minute amounts do make this supply inadequate. Another major limitation is the rather stable diphenylcarbamoyl moiety at the c3G nucleobase that requires harsh basic conditions during RNA deprotection. To address these limitations, we set out to elaborate a viable synthetic path toward 3-deazaguanosine and phosphoramidites thereof. The key reaction sequence of our novel route to c3G phosphoramidites is a silyl-Hilbert−Johnson nucleosidation of 6-benzyloxy-2-bromo-3-deazapurine 1 and 1,2,3,5-tetra-Oacetyl-β- D -ribofuranose in the presence of N,O-bis(trimethylsilyl)acetamide (BSA) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) to give the corresponding 2bromo nucleoside derivative 2 in high yields (Scheme 2). This compound was then reacted with 2-phenoxyactetamide under carefully optimized Buchwald−Hartwig C−N cross-coupling conditions,21−23 using 4,5-bis(diphenylphosphino)-9,9-dime-

he preparation of 3-deazaguanosine building blocks for RNA solid-phase synthesis has remained a challenge in synthetic RNA chemistry. This nucleoside modification is needed for studies that scrutinize the mechanism of phosphodiester cleavage of novel catalytic RNAs, such as hatchet, pistol, twister, and twister sister ribozymes.1,2 Specific guanosines in their active sites have been considered to play crucial mechanistic roles. For instance, structural analyses of the hatchet ribozyme suggested that active site guanosines might be involved in folding and catalysis.3 Similarly, for the twister-sister ribozyme, it has been found that a guanosine in the active site is indispensable for cleavage, presumably by stabilization of the transition state through H-bond networks to the scissile phosphate.4 An earlier prominent example concerns the hammerhead ribozyme where guanosine (G12) becomes properly positioned by forming an N3-mediated trans Hoogsteen/sugar edge base pair5 with adenosine (A9) and hence enables general base catalysis of G12 in phosphodiester cleavage.6,7 Comparative atomic mutagenesis is an important means to investigate RNA catalyzed reactions and to elucidate the underlying chemical mechanisms.8−10 Therefore, 1-deazaguanosine (c1G),11 1-deaza-2′-deoxyguanosine (c1dG),12 3deazaguanosine (c3G),13 and 3-deaza-2′-deoxyguanosine (c3dG),14 and the corresponding phosphoramidites are highly needed nucleoside modifications for the preparation of oligoribonucleotides. Unfortunately, currently available synthetic approaches are inefficient and time-consuming, in particular for c3G. The first syntheses of 3-deazaguanine and 3-deazaguanosine were described by Robins and co-workers in 1975.15−18 They established glycosylation of methyl 5(cyanomethyl)-1H-imidazole-4-carboxylate with 1-O-acetyl2,3,5-tri-O-benzoyl-β-D-ribofuranose in the presence of tin(IV) chloride, and subsequently, used ammonia for the formation of © XXXX American Chemical Society

Received: March 8, 2019

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DOI: 10.1021/acs.orglett.9b00855 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Scheme 1. Earlier Strategy for c3G Synthesis

Scheme 3. Synthesis of Glycosyl Acceptor 1.a

Scheme 2. Key Steps of c3G Phosphoramidite Synthesisa

Finally, the 6-bromo substituent was replaced in highly regioselective manner using freshly prepared sodium benzyloxide in benzyl alcohol. The desired glycosyl acceptor 6benzyloxy-2-bromo-3-deazapurine 1 was thus obtained in 27% overall yield, and the 8-step reaction sequence was conducted at multigram scales without the need for chromatographic purifications. At this point, we also note that the dibromo precursor 7 can be readily reacted with either 1,2,3,5-tetra-O-acetyl-β-Dribofuranose or 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose in silyl-Hilbert−Johnson nucleosidations. This affords the corresponding nucleoside derivatives 8 and 9 (Supporting Information), which represent valuable intermediates to synthesize 2,6-diamino-3-deazapurine nucleosides. Moreover, we showed that nucleoside 3 is readily converted into the free nucleoside, 3-deazaguanosine 10, by deacylation and Pd/Ccatalyzed hydrogenation (Scheme 4) and we also demonScheme 4. Access to c3G Nucleoside 10 and Nucleobase 11

a Abbreviations: BSA = N,O-bis(trimethylsilyl)acetamide; TMSOTf = trimethylsilyl trifluoromethanesulfonate; Xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene; and Pd2dba3 = tris(dibenzylideneacetone)dipalladium(0).

thylxanthene (Xantphos), Cs2CO3, and tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3). Nucleoside 3 carries the N2-phenoxyacetyl protecting group that is required in the final c3G building block for RNA synthesis. The yield of the C−N cross-coupling step was nearly quantitative, according to thin layer chromatography; however, because of partial cleavage of O-acetyl protecting groups at the ribose moiety during extended reaction times, O-deacetylated byproducts co-occurred. These byproducts were also utilized in the subsequent reaction step of ribose deacetylation; hence, compound 4 was obtained in 83% yield over the two steps starting from 2. Our route to the novel nucleobase precursor 7 started from 2,6-dibromo-3-nitro-pyridin-4-ylamine 5, itself readily accessible in five steps from 2,6-dibromopyridine (see Scheme 3, as well as the Supporting Information).24 Derivative 5 was then efficiently reduced to 2,6-dibromopyridine-3,4-diamine 6 by Fe0 in acetic acid. Treatment with trimethyl orthoformate and acetic anhydride at elevated temperature resulted in formation of the imidazo moiety to furnish 2,6-dibromo-3-deazapurine 7.

strated that nucleobase 1 can be efficiently transformed in three steps into the free nucleobase, 3-deazaguanine 11 (Scheme 4); both 10 and 11 are valuable compounds known for their antiviral activity25 and high potential in cancer research.26 At this point, we note that the analysis of nucleosides 8, 9, and 10 by ROESY and HMBC NMR spectroscopy was consistent with the structures of the desired β-N9 isomers (see the Supporting Information). To further transform the c3G derivative 3 (obtained via silylHilbert−Johnson nucleosidation and Buchwald−Hartwig amidation; see Scheme 2) into a powerful phosphoramidite B

DOI: 10.1021/acs.orglett.9b00855 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Scheme 5. Synthesis of c3G Phosphoramidite 17a

a

Abbreviations: Pac = phenoxyacetyl; TIPDSCl2 = 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane; TfOH = trifluoromethanesulfonic acid; NIS = Niodosuccinimide; THF = tetrahydrofuran; Cem = 2-cyanoethoxymethyl; DMTrCl = 4,4′-dimethoxytriphenylmethyl chloride; BTT = 5(benzylthio)-1H-tetrazole; and CEP(iPr2N)2 = 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite.

Solid-phase synthesis of two short RNAs, 5′-UA(c3G)CG and 5′-GGCUA(c3G)CC, was performed with the novel building block 17 in combination with N-phenoxyacetyl protected 2′-O-tBDMS nucleoside phosphoramidites (see Figure 1, as well as Figure S1 in the Supporting Information).

building block for RNA solid-phase synthesis, we envisaged a concept that retains the N2-phenoxyacetyl protection, utilizes a free nucleobase lactam moiety, and applies the 2-cyanoethoxymethyl group (Cem)27−29 for ribose 2′-O protection. The latter was considered advantageous over 2′-O-tert.-butyldimethylsilyl (tBDMS) or 2′-O-[(triisopropylsilyl)oxy]methyl (TOM) protection, with respect to installation at the nucleoside level, because of our experience with the related 3-deazaadenosine derivatives,30 where we encountered hardly any product formation under conditions that are generally used for the introduction of 2′-O-tBDMS or 2′-O-TOM groups. Based on this observation, we eluded to the bulky 2′-O-TIPS protection in the case of c3A phosphoramidites.30 Here, however, we decided to explore the less bulky Cem group for two reasons: (1) higher coupling yields that were expected during RNA synthesis, and (2) the anticipated compatibility with 2′-O-tBDMS or 2′-O-TOM RNA chemistry. The functionalization of nucleoside 4 into the desired c3G phosphoramidite 17 involved six high-yielding transformations, which are summarized in Scheme 5. Our route began with the simultaneous protection of the 3′ and 5′-hydroxyl groups of 4 by applying 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane. Then, the Cem group was introduced into the 3′,5′-di-Oprotected ribonucleoside 12 in good yield via reaction with 2cyanoethyl methylthio-methyl ether28 as the alkylating agent and N-iodosuccinimide as the activator in the presence of trifluoromethanesulfonic acid to provide nucleoside 13. Selective removal of the 3′,5′-O-tetraisopropyldisiloxane was accomplished with triethylamine trihydrofluoride in tetrahydrofuran and gave derivative 14, leaving the 2′-O-Cem group unaffected. Nucleoside 14 was then transformed to the dimethoxytritylated compound 15, using 4,4′-dimethoxytriphenylmethyl chloride in pyridine, and subsequently, the O6benzyl group was cleaved by Pd/C-catalyzed hydrogenation to provide 16. Finally, phosphitylation was executed with 2cyanoethyl-N,N,N′,N′-bisdiisopropylphosphorodiamidite in the presence of 5-(benzylthio)-1H-tetrazole. Starting from nucleoside 4, our route provides phosphoramidite 17 with 26% overall yield in six steps involving six chromatographic purifications; in total, 0.42 g of compound 17 was obtained during the course of this study.

Figure 1. Characterization of a c3G-modified RNA by (A, B) anionexchange HPLC and (C) LC−ESI mass spectrometry. HPLC conditions: Dionex DNAPac column (4 mm × 250 mm), 80 °C, 1 mL min−1, 0−60% B in A within 45 min; buffer A: Tris-HCl (25 mM), urea (6 M), pH 8.0; buffer B: Tris-HCl (25 mM), urea (6 M), NaClO4 (0.5 M), pH 8.0. See the Supporting Information for LC− ESI MS conditions.

Using standard setups of the oligonucleotide synthesizer, coupling yields for the c3G building block 17 were >98%. Release of the synthetic RNA strands from the solid support and their deprotections was performed either following the recommended protocols for Cem deprotection (first, ammonia in ethanol, followed by TBAF in dry DMSO, and traces of CH3NO2),27,28 or alternatively, by applying standard deprotection conditions for tBDMS or TOM RNA chemistry.31 The crude products gave a major product peak in anion-exchange (AE) and reversed-phase (Rp) HPLC analysis and were purified using the same chromatographic systems.31 The molecular weights of the purified RNAs were confirmed by liquid chromatography−electrospray ionization (LC−ESI) mass spectrometry (MS) (see Figure 1, as well as Figure S1). C

DOI: 10.1021/acs.orglett.9b00855 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

(9) Lang, K.; Erlacher, M.; Wilson, D. N.; Micura, R.; Polacek, N. Chem. Biol. 2008, 15, 485−492. (10) Polacek, N. Chimia 2013, 67, 322−326. (11) Schelling, J. E.; Salemink, C. A. Recueil des Travaux Chimiques des Pays-Bas 1975, 94, 153−156 (c1G) . (12) Kojima, N.; Inoue, K.; Nakajima-Shibata, R.; Kawahara, S.; Ohtsuka, E. Nucleic Acids Res. 2003, 31, 7175−7188 (c1dG) . (13) Seela, F.; Debelak, H.; Andrews, L.; Beigelman, L. Helv. Chim. Acta 2003, 86, 2726−2740 (c3G) . (14) Suzuki, A.; Yanagi, M.; Takeda, T.; Hudson, R. H. E.; Saito, Y. Org. Biomol. Chem. 2017, 15, 7853−7859 (c3dG) . (15) Robins, R. K.; Horner, J. K.; Greco, C. V.; Noell, C. W.; Beames, C. G. J. Org. Chem. 1963, 28, 3041−3046. (16) Cook, P. D.; Rousseau, R. J.; Mian, A. M.; Meyer, R. B.; Dea, P.; Ivanovics, G.; Streeter, D. G.; Witkowski, J. T.; Stout, M. G.; Simon, L. N.; Sidwell, R. W.; Robins, R. K. J. Am. Chem. Soc. 1975, 97, 2916−2917. (17) Cook, P. D.; Rousseau, R. J.; Mian, A. M.; Dea, P.; Meyer, R. B.; Robins, R. K. J. Am. Chem. Soc. 1976, 98, 1492−1498. (18) Srivastava, P. C.; Robins, R. K. J. Heterocycl. Chem. 1979, 16, 1063−1064. (19) Revankar, G. R.; Gupta, P. K.; Adams, A. D.; Dalley, N. K.; McKernan, P. A.; Cook, P. D.; Canonico, P. G.; Robins, R. K. J. Med. Chem. 1984, 27, 1389−1396. (20) Minakawa, N.; Matsuda, A. Tetrahedron 1993, 49, 557−570. (21) Ruiz-Castillo, P.; Buchwald, S. L. Chem. Rev. 2016, 116, 12564−12649. (22) Caner, J.; Vilarrasa, J. J. Org. Chem. 2010, 75, 4880−4883. (23) Ghodke, P. P.; Bommisetti, P.; Nair, D. T.; Pradeepkumar, P. I. J. Org. Chem. 2019, 84, 1734. (24) Hay, D. A.; Adam, F. M.; Bish, G.; Calo, F.; Dixon, R.; Fray, M. J.; Hitchin, J.; Jones, P.; Paradowski, M.; Parsons, C. G.; Proctor, K. J. W.; Pryde, D. C.; Smith, N. N.; Tran, T.-D. Tetrahedron Lett. 2011, 52, 5728−5732. (25) Allen, L. B.; Huffman, J. H.; Cook, P. D.; Meyer, R. B., Jr.; Robins, R. K.; Sidwell, R. W. Antimicrob. Agents Chemother. 1977, 12, 114−119. (26) Saunders, P. P.; Tan, M.-T.; Spindler, C. D.; Robins, R. K. Cancer Res. 1989, 49, 6593−6599. (27) Shiba, Y.; Masuda, H.; Watanabe, N.; Ego, T.; Takagaki, K.; Ishiyama, K.; Ohgi, T.; Yano, J. Nucleic Acids Res. 2007, 35, 3287− 3296. (28) Ohgi, T.; Kitagawa, H.; Yano, J. Curr. Protoc. Nucleic Acid Chem. 2008, 2.15.1−2.15.19. (29) Kremser, J.; Strebitzer, E.; Plangger, R.; Juen, M. A.; Nußbaumer, F.; Glasner, H.; Breuker, K.; Kreutz, C. Chem. Commun. 2017, 53, 12938−12941. (30) Mairhofer, E.; Fuchs, E.; Micura, R. Beilstein J. Org. Chem. 2016, 12, 2556−2562. (31) Micura, R. Angew. Chem., Int. Ed. 2002, 41, 2265−2269.

In summary, we have reported a practical route toward a 3deazaguanosine building block for RNA solid-phase synthesis. The glycosyl acceptor 1 was obtained in three steps in 75% yield from a known dibromopyrimidine derivative. The key reaction sequence employs a silyl-Hilbert−Johnson nucleosidation followed by Hartwig−Buchwald amidation, which introduces the exocyclic N2 and the right protecting group of the phosphoramidite building block 17 in one step. Furthermore, we preferred the 2′-O-Cem group over 2′-OTIPS13 protection because of efficient introduction at the nucleoside level, higher coupling yields during RNA solidphase synthesis, and compatibility with tBDMS and TOM RNA chemistry.31 The developed route is advantageous over the previously described synthesis,13 in terms of overall efficiency and ease of handling.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00855. Experimental procedures and characterization of compounds including NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Website: https://www. uibk.ac.at/organic/micura ORCID

Christoph Kreutz: 0000-0002-7018-9326 Ronald Micura: 0000-0003-2661-6105 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Daniel Fellner and Martin Schwienbacher (University of Innsbruck) for their assistance in the synthesis of 2,6-dibromo-3-deazapurine, Johannes Kremser and Felix Nussbaumer (University of Innsbruck) for discussion and a generous gift of 2-cyanoethyl methylthiomethyl ether. Research Funds from the Austrian Science Fund FWF (Nos. I1040, P27947, P31691 to R.M. and Project Nos. P28725 and P30370 to C.K.), and the Austrian Research Promotion Agency FFG [West Austrian BioNMR 858017], are acknowledged.

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REFERENCES

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DOI: 10.1021/acs.orglett.9b00855 Org. Lett. XXXX, XXX, XXX−XXX