Synthesis of 4'-Thiomodified c-di-AMP Analogs.

Cyclic diadenosine monophosphate (c-di-AMP) is a bacterial cyclic dinucleotide (CDN) comprising two adenosine monophosphates covalently linked by two 3',5'-phosphodiester bonds. c-di-AMP works as a second messenger, regulating many biological processes in bacteria such as cell wall homeostasis, DNA integrity, and sporulation via specific protein and/or RNA receptors. Moreover, c-di-AMP can function as an immunomodulatory agent in eukaryote cells via the stimulator of interferon genes (STING) signaling pathway. This protocol describes the chemical synthesis of two c-di-AMP analogs with a sulfur atom at the 4'-position of the furanose ring instead of an oxygen atom: c-di-4'-thioAMP (1) and cAMP-4'-thioAMP (2). Analogs 1 and 2 have resistance to phosphodiesterase-mediated degradation and are therefore useful for understanding the diverse biological phenomena regulated by c-di-AMP. In this protocol, two 4'-thioadenosine monomers are initially prepared via a Pummerer-like reaction assisted by hypervalent iodine. The CDN skeleton is then constructed through two key reactions based on phosphoramidite chemistry: dimerization of two appropriately protected nucleoside monomers to produce a linear dinucleotide, followed by macrocyclization of the resulting linear dinucleotide to form the CDN skeleton. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 4'-thioadenosine monomers 13 and 14 Basic Protocol 2: Preparation of c-di-4'-thioAMP (1) and cAMP-4'-thioAMP (2).

Convenient Synthesis of 3-Deazapurine Nucleosides (3-Deazainosine, 3-Deazaadenosine and 3-Deazaguanosine) Using Inosine as a Starting Material.

A convenient synthetic method for preparing 3-deazapurine nucleosides (3-deazainosine, 3-deazaadenosine, and 3-deazaguanosine) from inosine via a 5-ethynyl-1-ß-D-ribofuranosylimidazole-4-carboxamide (EICAR) derivative, which is a key intermediate, is described. A large-scale synthesis of an EICAR derivative starting from inosine was achieved in six steps via dinitrophenylation at the N1 position followed by ring opening, iodination of the resulting 5-amino group, and a palladium-catalyzed cross-coupling reaction. The resulting EICAR derivative was then converted into 3-deazainosine, 3-deazaadenosine, and 3-deazaguanosine. This route enabled us to synthesize 3-deazapurine nucleosides conveniently in good yields. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 5-ethynyl-1-ß-D-ribofuranosylimidazole-4-carboxamide (EICAR) derivative 6 Basic Protocol 2: Preparation of 3-deazapurine nucleosides 8, 11, and 14.

A Unique Gene-Silencing Approach, Using an Intelligent RNA Expression Device (iRed), Results in Minimal Immune Stimulation When Given by Local Intrapleural Injection in Malignant Pleural Mesothelioma.

BACKGROUND: We have recently introduced an intelligent RNA expression device (iRed), comprising the minimum essential components needed to transcribe short hairpin RNA (shRNA) in cells. Use of iRed efficiently produced shRNA molecules after transfection into cells and alleviated the innate immune stimulation following intravenous injection. METHODS: To study the usefulness of iRed for local injection, the engineered iRed encoding luciferase shRNA (Luc iRed), complexed with cationic liposomes (Luc iRed/liposome-complexes), was intrapleurally injected into an orthotopic mesothelioma mouse model. RESULTS: Luc iRed/liposome-complexes markedly suppressed the expression of a luciferase marker gene in pleurally disseminated mesothelioma cells. The suppressive efficiency was correlated with the expression level of shRNA within the mesothelioma cells. In addition, intrapleural injection of iRed/liposome-complexes did not induce IL-6 production in the pleural space and consequently in the blood compartment, although plasmid DNA (pDNA) or dsDNA (the natural construct for iRed) in the formulation did. CONCLUSION: Local delivery of iRed could augment the in vivo gene silencing effect without eliciting pronounced innate immune stimulation. Our results might hold promise for widespread utilization of iRed as an RNAi-based therapeutic for intracelial malignant cancers.

Use of modified U1 small nuclear RNA for rescue from exon 7 skipping caused by 5'-splice site mutation of human cathepsin A gene.

Cathepsin A (CTSA) is a multifunctional lysosomal enzyme, and its hereditary defect causes an autosomal recessive disorder called galactosialidosis. In a certain number of galactosialidosis patients, a base substitution from adenine to guanine is observed at the +3 position of the 7th intron (IVS7 +3a>g) of the CTSA gene. With this mutation, a splicing error occurs; and consequently mRNA lacking the 7th exon is produced. This skipping of exon 7 causes a frame shift of the transcripts, resulting in a non-functional CTSA protein and hence galactosialidosis. This mutation seems to make the interaction between the 5'-splice site of intron 7 of pre-mRNA and U1 small nuclear RNA (U1 snRNA) much weaker. In the present study, to produce properly spliced mRNA from the CTSA gene harboring this IVS7 +3a>g mutation, we examined the possible usefulness of modified U1 snRNA that could interact with the mutated 5'-splice site. Toward this goal, we first prepared a model system using a mutant CTSA mini gene plasmid for delivery into HeLa cells. Then, we examined the effectiveness of modified U1 snRNA on the formation of properly spliced mRNA from this mutant CTSA mini gene. As a result, we succeeded in obtaining improved formation of properly spliced CTSA mRNA. Our results suggest the usefulness of modified U1 snRNA for rescue from exon 7 skipping caused by the IVS7 +3a>g mutation of the CTSA gene.

Ribavirin-related compounds exert in vitro inhibitory effects toward rabies virus.

Rabies remains an invariably fatal neurological disease despite the availability of a preventive vaccination and post-exposure prophylaxis that must be immediately administered to the exposed individual before symptom onset. There is no effective medication for treatment during the symptomatic phase. Ribavirin, a guanine nucleoside analog, is a potent inhibitor of rabies virus (RABV) replication in vitro but lacks clinical efficacy. Therefore, we attempted to identify potential ribavirin analogs with comparable or superior anti-RABV activity. Antiviral activity and cytotoxicity of the compounds were initially examined in human neuroblastoma cells. Among the tested compounds, two exhibited a 5- to 27-fold higher anti-RABV activity than ribavirin. Examination of the anti-RABV mechanisms of action of the compounds using time-of-addition and minigenome assays revealed that they inhibited viral genome replication and transcription. Addition of exogenous guanosine to RABV-infected cells diminished the antiviral activity of the compounds, suggesting that they are involved in guanosine triphosphate (GTP) pool depletion by inhibiting inosine monophosphate dehydrogenase (IMPDH). Taken together, our findings underline the potency of nucleoside analogs as a class of antiviral compounds for the development of novel agents against RABV.

Synthesis and evaluation of c-di-4'-thioAMP as an artificial ligand for c-di-AMP riboswitch.

Cyclic-di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that binds to an RNA receptor called riboswitch and regulates its downstream genes involving cell wall metabolism, ion transport, and spore germination. Therefore, the c-di-AMP riboswitch can be a novel target of antibiotics. In this study, we synthesized c-di-4'-thioAMP (1), which possesses a sulfur atom instead of an oxygen atom in the furanose ring, as a candidate of a bioisoster for natural c-di-AMP. The resulting 1 bound to the c-di-AMP riboswitch with a micromolar affinity (34.8µM), and the phosphodiesterase resistance of 1 was >12-times higher than that of c-di-AMP. Thus, 1 can be considered to be a stable ligand against a c-di-AMP riboswitch.

The AMPK/mTOR pathway is involved in D-dopachrome tautomerase gene transcription in adipocytes differentiated from SGBS cells, a human preadipocyte cell line.

In adipose tissue, D-dopachrome tautomerase (DDT), a cytokine with structural similarity to macrophage migration inhibitory factor, is mainly expressed in adipocytes rather than preadipocytes and acts as an anti-obesity adipokine in an autocrine manner. However, its transcriptional regulation is largely unknown. In order to explore molecules affecting DDT transcription, a chemical library screening using HEK293 cells stably expressing a DDT promoter-reporter construct was performed. Several derivatives of 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, were identified as transcriptional activators of the DDT gene. Furthermore, DDT mRNA levels were reduced in SGBS adipocytes treated with compound C, an AMPK inhibitor, suggesting involvement of AMPK in DDT transcription. Overexpression of the FOXO1 constitutive active form reduced transcriptional activity of the DDT gene in SGBS cells, but increased it in HEK293 cells. Cell-type specific effects were also observed in the DDT gene expression of cells treated with AS1842856, a FOXO1 inhibitor. Finally, involvement of the mammalian target of rapamycin (mTOR) signaling in DDT transcription in SGBS adipocytes was investigated. Rapamycin, an inhibitor of mTOR, increased DDT mRNA levels and attenuated the inhibitory effects of compound C on DDT mRNA levels in SGBS adipocytes. In conclusion, DDT transcription may be regulated in a cell-dependent manner, and were enhanced by AMPK activation in SGBS adipocytes through inhibiting the mTOR signaling.

Groove modification of siRNA duplexes to elucidate siRNA-protein interactions using 7-bromo-7-deazaadenosine and 3-bromo-3-deazaadenosine as chemical probes.

Elucidation of dynamic interactions between RNA and proteins is essential for understanding the biological processes regulated by RNA, such as RNA interference (RNAi). In this study, the logical chemical probes, comprising 7-bromo-7-deazaadenosine (Br7C7A) and 3-bromo-3-deazaadenosine (Br3C3A), to investigate small interfering RNA (siRNA)-RNAi related protein interactions, were developed. The bromo substituents of Br7C7A and Br3C3A are expected to be located in the major and the minor grooves, respectively, and to act as a steric hindrance in each groove when these chemical probes are incorporated into siRNAs. A comprehensive investigation using siRNAs containing these chemical probes revealed that (i) Br3C3A(s) at the 5'-end of the passenger strand enhanced their RNAi activity, and (ii) the direction of RISC assembly is determined by the interaction between Argonaute2, which is the main component of RISC, and siRNA in the minor groove near the 5'-end of the passenger strand. Utilization of these chemical probes enables the investigation of the dynamic interactions between RNA and proteins.

Gene Silencing Using 4'-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response.

The development of a versatile technique to induce RNA interference (RNAi) without immune stimulation in vivo is of interest as existing approaches to trigger RNAi, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) expressing short hairpin RNA (shRNA), present drawbacks arising from innate immune stimulation. To overcome them, an intelligent shRNA expression device (iRed) designed to induce RNAi was developed. The minimum sequence of iRed encodes only the U6 promoter and shRNA. A series of iRed comprises a polymerase chain reaction (PCR)-amplified 4'-thioDNA in which any one type of adenine (A), guanine (G), cytosine (C), or thymine (T) nucleotide unit was substituted by each cognate 4'-thio derivatives, i.e., dSA iRed, dSG iRed, dSC iRed, and ST iRed respectively. Each modified iRed acted as a template to transcribe shRNA with RNAi activity. The highest shRNA yield was generated using dSC iRed that exerted gene silencing activity in an orthotopic mouse model of mesothelioma. Reducing the minimal structure required to transcribe shRNA and the presence of the 4'-thiomodification synergistically function to abrogate innate immune response induced by dsDNA. The iRed will introduce a new approach to induce RNAi without inducing a detectable innate immune response.

Practical synthesis of 4'-thioribonucleosides starting from D-ribose.

A practical synthesis of 4'-thioribonucleosides, i.e., 4'-thiouridine, -cytidine, -adenosine, and -guanosine, which are versatile units for nucleic acids-based therapeutics, is described. Large-scale synthesis of 4-thiosugar starting from D-ribose was achieved (33%) in eight steps and with only three chromatographic purifications. After the appropriate chemical conversion of the 4-thiosugar, the resulting sulfoxide was subjected to the Pummerer reaction in the presence of silylated nucleobases. In reactions with silylated pyrimidine bases, the desired 4'-thioribonucleoside derivatives were obtained in good yield and ß-selectively. On the other hand, N-7 isomers were obtained mainly in the Pummerer reaction with purine bases under the same conditions. However, the desired N-9 isomers were obtained in moderate yields when the reaction mixtures were subsequently heated under reflux. As a result, effective synthesis of 4'-thioribonucleosides was accomplished.

Chemistry, properties, and in vitro and in vivo applications of 2'-O-methoxyethyl-4'-thioRNA, a novel hybrid type of chemically modified RNA.

We report the synthesis, properties, and in vitro and in vivo applications of 2'-O-methoxyethyl-4'-thioRNA (MOE-SRNA), a novel type of hybrid chemically modified RNA. In its hybridization with complementary RNA, MOE-SRNA showed a moderate improvement of Tm value (+3.4 °C relative to an RNA:RNA duplex). However, the results of a comprehensive comparison of the nuclease stability of MOE-SRNA relative to 2'-O-methoxyethylRNA (MOERNA), 2'-O-methyl-4'-thioRNA (Me-SRNA), 2'-O-methylRNA (MeRNA), 4'-thioRNA (SRNA), and natural RNA revealed that MOE-SRNA had the highest stability (t1/2 >48 h in human plasma). Because of the favorable properties of MOE-SRNA, we evaluated its in vitro and in vivo potencies as an anti-microRNA oligonucleotide against miR-21. Although the in vitro potency of MOE-SRNA was moderate, its in vivo potency was significant for the suppression of tumor growth (similar to that of MOERNA).

The systemic administration of an anti-miRNA oligonucleotide encapsulated pH-sensitive liposome results in reduced level of hepatic microRNA-122 in mice.

Efficient delivery continues to be a challenge in microRNA (miRNA) therapeutics. We utilized a pH-sensitive multifunctional envelope-type nano device (MEND) containing a pH-sensitive lipid YSK05 (YSK05-MEND) to regulate liver specific miRNA-122 (miR-122). Anti-microRNA oligonucleotides including 2'-OMe and phosphorothioate modifications against miR-122 (AMO122) were encapsulated in the YSK05-MEND. Despite the lower uptake, the YSK05-MEND showed a higher activity in liver cancer cells than Lipofectamine2000 (LFN2k) due to efficient endosomal escape. Cytotoxicity was minimal at 100 nM of AMO122 in YSK05-MEND treated cells, but LFN2k showed toxicity at 50 nM. When mice were administrated with free AMO122, it was eliminated via the kidney due to its molecular weight, and lesser amounts were detected in the liver. Conversely, the YSK05-MEND delivered higher amounts of the AMO122 to the liver. Systemic administration of YSK05-MEND induced the knockdownofmiR-122andan increase in target genes inthe liver, and a subsequent reduction in plasma cholesterol at a dose of 1mgAMO/kgwhile free AMO122 showed no activity at the same dose. The effect ofAMO122 delivered by YSK05-MEND persisted for over 2 weeks. These results suggest that YSK05-MEND is a promising system for delivering AMOs to the liver.

Investigations toward the selection of fully-modified 4'-thioRNA aptamers: optimization of in vitro transcription steps in the presence of 4'-thioNTPs.

We describe herein a method for isolating fully-modified 4'-thioRNA aptamers against human alpha-thrombin using the SELEX protocol. In order to isolate the desired aptamers, in vitro transcription was examined in the presence of four kinds of 4'-thioribonucleoside triphosphates (4'-thioNTPs) in an effort to afford the fully-modified 4'-thioRNA. The transcription efficiency of the 4'-thioNTPs was first compared with that of the nucleoside 5'-(alpha-thio)triphosphates (alphaSNTPs) and found to be less effective than that of the alphaSNTPs, especially when GTP and/or ATP were substituted for 4'-thioGTP and/or 4'-thioATP. Further attempts to improve its efficiency, including the use of a mutant RNA polymerase instead of the wild type, various additives, and 4'-thioNTP concentrations were unsuccessful. Accordingly, the transcription was performed in the presence of 4'-thioNTPs together with the natural GTP and ATP at the appropriate concentrations. Although this attempt furnished a highly-modified 4'-thioRNA, but not a fully-modified 4'-thioRNA, we eventually succeeded in isolating the fully-modified 4'-thioRNA aptamers by SELEX using optimized transcription conditions, followed by post-modification of the resulting aptamers.

Synthesis and properties of 2'-modified-4'-thioRNA.

As a part of our ongoing research projects, we envisioned the synthesis of 2'-modified-4'-thioRNA, such as 2'-fluoro-4'-thioRNA (2'-F-4'-thioRNA) and 2'-O-methyl-4'-thioRNA (2'-OMe-4'-thioRNA) to enhance the potential of 4'-thioRNA. Appropriately protected 2'-deoxy-2'-fluoro-4'-thiouridine (6), -cytidine (10), and - adenosine (18), substrates for the synthesis of novel modified RNAs were successfully synthesized. The fullymodified RNA consisting of 2'-deoxy-2'-fluoro-4'-thionucleosides was synthesised, and we examined its abilities of hybridization and stability against nucleases. It was found that 2'-F-4'-thioRNA shows high hybridization ability to complementary RNA. Furthermore, 2'-F-4'-thioRNA has strong resistance toward degradation in 50% human plasma.

Recognition of nucleotide analogs containing the 7,8-dihydro-8-oxo structure by the human MTH1 protein.

The MTH1 protein catalyzes hydrolysis of oxidatively damaged purine nucleotides including 8-hydroxy-dGTP to the monophosphates. The MTH1 protein seems to act as an important defense system against mutagenesis, carcinogenesis, and cell death induced by oxidized purine nucleotides. We previously reported that the functional groups at the 2- and 6-positions of the purine ring affect the recognition by the human MTH1 protein. 8-Hydroxy-dGTP and 8-hydroxy-dATP are substrates of MTH1, and both have the "7,8-dihydro-8-oxo structure." In this study, three nucleotide analogs containing this motif were examined. A synthetic purine analog containing the 7,8-dihydro-8-oxo structure and the 2-amino function (dJTP) was hydrolyzed to the monophosphate with high efficiency by MTH1. On the other hand, two analogs that lack the two-ring system of their bases [formamidopyrimidine-dGTP (FAPY-dGTP) and 2-OH-dYTP] were poor substrates. FAPY-dGTP is a mixture of conformers and was hydrolyzed more than ten-fold less efficiently than 8-hydroxy-dGTP. These results clarify the effects of the 2-amino group and the two-ring system of the purine base on the recognition by the human MTH1 protein.

Synthesis of 3'-beta-carbamoylmethylcytidine (CAMC) and its derivatives as potential antitumor agents.

3'-beta-Carbamoylmethylcytidine (CAMC) and its derivatives were synthesized using an intramolecular Reformatsky-type reaction promoted by SmI2 as the key step. In vitro tumor cell growth inhibitory activity was evaluated and CAMC was found to exhibit potent cytotoxicity against various human tumor cell lines. From a structure-activity relationship study it was postulated that the cytotoxic mechanism of action of CAMC did not require phosphorylation at the 5'-hydroxyl group. This study provides a novel strategy for the development of a new type of antitumor nucleoside.

Synthesis and physical and physiological properties of 4'-thioRNA: application to post-modification of RNA aptamer toward NF-kappaB.

We report herein full details of the preparation of 4'-thiouridine, -cytidine, -adenosine and -guanosine phosphoramidites based on our synthetic protocol via the Pummerer reaction. Fully modified 4'-thioRNAs containing four kinds of 4'-thioribonucleoside units were prepared according to the standard RNA synthesis. The T(m) values and thermodynamic parameters of a series of duplexes were determined by UV melting and differential scanning calorimetry (DSC) measurements. The resulting overall order of thermal stabilities for the duplexes was 4'-thioRNA:4'-thioRNA >> 4'-thioRNA:RNA > RNA:RNA > RNA:DNA > 4'-thioRNA:DNA. In addition, it was shown that the dominant factor in the stability of the duplexes consisting of 4'-thioRNA was enthalpic in character. The CD spectra of not only 4'-thioRNA:RNA and 4'-thioRNA:4'-thioRNA but also 4'-thioRNA:DNA were all similar to those of duplexes in the A-conformation. The stability of 4'-thioRNA in human serum was 600 times greater than that of natural RNA. Neither the RNA:RNA nor the 4'-thioRNA:4'-thioRNA duplexes were digested under the same conditions. The first example of a post-modification of an RNA aptamer by 4'-thioribonucleoside units was demonstrated. Full modification of the aptamer thioRNA3 resulted in complete loss of binding activity. In contrast, modifications at positions other than the binding site were tolerated without loss of binding activity. The post-modified RNA aptamer thioRNA5 was thermally stabilized and resistant toward nuclease digestion. The results presented in this paper will, it is hoped, contribute to the development of 4'-thioRNA as a new generation of artificial RNA.

Identification of A-minor tertiary interactions within a bacterial group I intron active site by 3-deazaadenosine interference mapping.

The A-minor motifs appear to be the most ubiquitous helix packing elements within RNA tertiary structures. These motifs have been identified throughout the ribosome and almost every other tertiary-folded RNA for which structural information is available. These motifs utilize the packing of the donor adenosine's N1, N3, and/or 2'-OH against the 2'-OHs and minor groove edge of the acceptor base pair. The ability to identify biochemically which adenosines form A-minor motifs and which base pairs they contact is an important experimental objective. Toward this goal, we report the synthesis and transcriptional incorporation of 5'-O-(1-thio)-3-deazaadenosine triphosphate and its use in Nucleotide Analogue Interference Mapping (NAIM) and Nucleotide Analogue Interference Suppression (NAIS). This analogue makes it possible for the first time to explore the functional importance of the N3 imino group of adenosine in RNA polymers. Interference analysis of the group I self-splicing introns from Tetrahymena and Azoarcus indicates that A-minor motifs are integral to the helix packing interactions that define the 5'-splice site of the intron. Specifically, Azoarcus A58 in the J4/5 region contacts the G.U wobble pair at the cleavage site in the P1 helix, and Azoarcus A167 in the J8/7 region contacts the C13-G37 base pair in the P2 helix. Both of these structural features are conserved between the eukaryotic and bacterial introns. These results suggest that nucleotide analogue interference patterns can identify and distinguish A-minor interactions in RNA tertiary structure, particularly the most prevalent type I and type II varieties. Furthermore, clustering of 3-deazaadenosine interferences is suggestive of A patches, in which a series of consecutive A-minor motifs mediate helix packing. Biochemical identification of these interactions may provide valuable constraints for RNA structure prediction.

New neplanocin analogues. 12. Alternative synthesis and antimalarial effect of (6'R)-6'-C-methylneplanocin A, a potent AdoHcy hydrolase inhibitor.

An improved method for the synthesis of (6'R)-6'-C-methylneplanocin A (RMNPA, 2), a potent S-adenosyl-L-homocysteine (AdoHcy) hydrolase inhibitor, was developed via a chelation-controlled stereoselective addition of MeTiCl(3) to the neplanocin A 6'-aldehyde derivative 6. Compound 2 effectively inhibited the growth of malaria parasites both in vitro and in vivo. The antimalarial EC(50) value of 2 against Plasmodium berghei in mice was 1.0 mg/kg/day, which was superior to that of chloroquine (EC(50) = 1.8 mg/kg/day).