C1′ Acylated Derivatives of 2′-Deoxyuridine. Photolabile Precursors of 2′-Deoxyuridin-1′-yl

Abstract

ABSTRACT

C1′ acylated derivatives of 2′-dcoxyuiidinc (1a-c) were synthesised from 1-[3-deoxy-β-D-psieofiiraiiosylliii.acil (6). The acyl group is introduced via the C1′ aldehyde (11). Following nucleophilic addition, the ketones (1a-c) are obtained via periodinane oxidation and desilylation with NH₄F.     

Synthesis of 3′-Amino-3′-deoxyguanosine 5′-Triphosphate

Abstract

Phosphorylation of 2′-0-acetyl-3′-trifluoroacetamido-3′-deoxy-N²-palmitoylguanosine with N-morpholino-O, O-bis(1-benzotriazolyl)phos-phate gives a 5′-phosphotriester. Removal of the benzotriazolyl group and addition of pyrophosphoric acid gave, after deblocking all protecting groups, GTP(3′NH₂).     

Concise and Efficient Synthesis of 3′-O-Tetraphosphates of 2′-Deoxyadenosine and 2′-Deoxycytidine

We describe concise and efficient synthesis of biologically very important 3′-O-tetraphosphates namely 2′-deoxyadenosine-3′-O-tetraphosphate (2′-d-3′-A4P) and 2′-deoxycytidine-3′-O-tetra-phosphate (2′-d-3′-C4P). N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine was converted into N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine-3′-O-tetraphosphate in 87% yield using a one-pot synthetic methodology. One-step concurrent deprotection of N⁶-benzoyl and 5′-O-levulinoyl groups using concentrated aqueous ammonia resulted 2′-d-3′-A4P in 74% yield. The same synthetic strategy was successfully employed to convert N⁴-benzoyl-5′-O-levulinoyl-2′-deoxycytidine into 2′-d-3′-C4P in 68% yield.     

Synthesis of 3′-N-Substituted 3′-Amino-3′-Deoxythymidine Derivatives

Abstract

A series of 3′-N-substituted 3′-amino-3′-deoxythymidine derivatives with alkyl, alkenyl and alkylaryl substituents was synthesized by two methods. The first method involved the reaction of 1-(2,3-dideoxy-3-0-mesyl-5-0-trityl-β-D-threo-pentofuranosyl)thymine with an appropriate amine. In the second method, 3′-amino-5′-0-trityl-3′-deoxy-thymidine served as a synthetic precursor which was reacted with an appropiate aldehyde or ketone followed by sodium borohydride reduction. An improved synthesis of 3′-amino-3′-deoxythymidine from 3′ -azido-5′-0-trityl-3′-deoxythymidine using sodium borohydride was also described.     

Efficient Synthesis of 2′-Amino-2′-deoxypyrimidine 5′-Triphosphates

Abstract

The synthesis of 2′-amino-2′-deoxypyrimidine 5′-triphosphates is described. The 2′-amino-2′-deoxyuridine 5′-triphosphate is obtained from uridine in four steps with 25% overall yield. The 2′-amino-2′-deoxycytidine 5′-triphosphate is obtained from uridine in seven steps with 13% overall yield.     

The Crystal and Molecular Structure of the Complex of 2′3′-Didehydro-2′3′-Dideoxyguanosine with Pyridine

Abstract

The structure of 2′,3′-didehydro-2′,3′-dideoxyguanosine was determined by X-ray crystallographic analysis of the complex with pyridine. The two independent nucleoside molecules have similar, commonly observed glycosyl link (x = -102.3° and -94.2°) and 5′-hydroxyl (y = 54.0° and 47.6°) conformations. The five-membered rings are very planar with r.m.s. deviations from planarity of less than 0.015 A. 2′,3′-Didehydro-2′,3′-dideoxyadenosine has a similar glycosyl link conformation but a different 5′-hydroxyl group orientation and a slightly less planar 5-membered ring.     

Synthesis of Analogues of 3′-Deoxypsicothymidine

Abstract

Preparation of 3′-deoxypsicothymidines bearing a tether group at O1′ is described. Selective protection of the primary hydroxy functions of the starting nucleoside is briefly discussed.     

Antiviral Activities of β-Enantiomers of 3′-Substituted-3′-deoxythymidine Analogs

Abstract

Several β-L-3′-substituted-3′-deoxythymidine were stereospecifically synthesized. None of these analogs inhibited HIV-1 nor HBV replication in vitro suggesting that these β-L-pyrimidine derivatives may not be efficiently phosphorylated inside the cells.     

Synthesis of 2′-Deuterio and 3′-Deuterio Cytidine 5′-Diphosphate

Abstract

2′-²H- and 3′-²H-CDP were synthesized from 5′-MMT-3′-O-TBDMS and 2′,5′- O-diTBDMS cytidine derivatives, respectively, by oxidation followed by acidic removal of 5′-protection, reduction with [NaBD(OAc)₃] and finally displacement of a tosyl group by pyrophosphate.     

Synthesis of 3′-Amino and 5′-Amino Hydantoin 2′-Deoxynucleosides

Abstract

3′-Amino and 5′-amino derivatives of hydantoin 2′-deoxynucleosides have been prepared from the corresponding 3′-phthalimido and 5′-azido nucleosides, respectively, which in turn were prepared by condensation of appropriate sugars with 5-benzylidenehydantoin. The amino nucleosides were tested for their potential activity against HIV and HSV.     

Synthesis and Evaluation of Antiviral Activity of 3′-C-Cyano-3′-Deoxynucleosides

Abstract

A series of 3′-C-cyano-3′-deoxy and 3′-C-cyano-2′,3′-dideoxy-nucleoside analogues of thymidine, uridine, cytidine and adenosine have been prepared. Their antiviral activity was assessed in various assay systems and while none of the compounas proved specifically active against human immunodeficiency virus, some compounds had marked activity against other viruses.     

5′-Halogenated Formycins as Inhibitors of 5′-Deoxy-5′-methylthioadenosine Phosphorylase: Protection of Cells Against the Growth-Inhibitory Activity of 5′-Halogenated Adenosines

Abstract

A series of 5′-halogenated formycins, including the chloro-, bromo- and iodo- derivatives, were synthesized. These compounds are competitive inhibitors of 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAPase) with K_i values in the range of 10^?7 M, making them the most potent inhibitors of MTAPase reported to date. These compounds protect cells from the growth-inhibitory action of 5′-halogenated adenosines, which must be activated by MTAPase. The syntheses of 5′-halogenated formycin B derivatives, which inhibit purine nucleoside phosphorylase, are also described.     

An Improved Synthesis of 3′-Keto-5′-Tritylthymidine

Abstract

The title compound is prepared in consistently high yield and purity by molecular sieve catalyzed pyridinium dichromate oxidation of 5′-0-tritylthymidine. Shortcomings of other preparations are described, and properties of the title compound are reported.     

Microbial Synthesis of Purine 2′-Amino-2′-deoxyribosides

The microbial synthesis of some purine 2′-amino-2′-deoxyribonucleosides from purine bases and 2′-amino-2′-deoxyuridine is described. Various bacteria, especially Erwinia herbicola, Salmonella schottmuelleri, Enterobacter aerogenes and Escherichia coli, were able to transfer the aminoribosyl moiety of 2′-amino-2′-deoxyuridine to purine bases (transaminoribosylation) in the presence of inorganic phosphate. The optimum conditions for the reaction were pH 7.0 and 63°C. No reaction was observed in the absence of inorganic phosphate and the optimum concentration of it was around 30 mm. Adenine, guanine, 2-chlorohypoxanthine and hypoxanthine were transformed to the corresponding 2′-amino-2′-deoxyribonucleosides by the catalytic activity of the wet cell paste of Enterobacter aerogenes AJ 11125. The enzymatically synthesized purine 2′-amino-2′-deoxyribonucleosides were isolated and identified by physicochemical means. 2′-Amino-2′-deoxyadenosine strongly inhibited the growth of Hela cells in tissue culture, and the ED⁵⁰ was 2.5μ/ml.     

The 5′-Nor Aristeromycin Analogues of 5′-Deoxy-5′-Methylthioadenosine and 5′-Deoxy-5′-Thiophenyladenosine

To extend the potential of 5′-noraristeromycin (and its enantiomer) as potential antiviral candidates, the enantiomers of the carbocyclic 5′-nor derivatives of 5′-methylthio-5′-deoxyadenosine and 5′-phenylthio-5′-deoxyadenosine have been synthesized and evaluated. None of the compounds showed meaningful antiviral activity.     

Synthesis of 5′-Deoxy-5′-nucleosideacetic Acid Derivatives

Abstract

The synthesis of several new 5′-deoxy-5′-nucleosideacetic acid derivatives by the reactions of alkoxycarbonylmethylene triphenylphosphoranes with nucleoside 5′-aldehydes is described.     

V-cGAPs: attenuators of 3′3′-cGAMP signaling

Cyclic GMP-AMPs (cGAMPs) are new members of the cyclic dinucleotide family of second messenger signaling molecules identified in both bacteria and mammalian cells. A recent study by Gao et al. published in Cell Research has identified and characterized three 3′3′-cGAMP-specific phosphodiesterases (termed as V-cGAP1/2/3) in V. cholerae, thereby providing mechanistic insights into the function of these enzymes that degrade cGAMPs.Despite their indispensable roles in the composition of DNA and RNA, as well as serving as energy sources, nucleotides are also well known as crucial signaling molecules in all domains of life. Cyclic dinucleotides (CDNs) represent an important and growing family of second messengers, which have been previously recognized as key modulators governing a variety of cellular activities in bacteria, and more recently, in mammalian cells. c-di-GMP and c-di-AMP, the first two members of the CDN family, have been implicated in central bacterial processes, and likely act as universal bacterial secondary messengers^1,2. The latest addition to the bacterial CDN family is 3′3′-cGAMP, a hybrid molecule that is synthesized from ATP and GTP by DncV (a cyclase from V. cholerae) and shown to promote intestinal colonization of V. cholerae by downregulating chemotaxis³. Predicted homologs of DncV are present in many other bacterial species³, indicating that 3′3′-cGAMP may also regulate a wide range of cellular functions, similar to c-di-GMP and c-di-AMP. The research on CDNs as second messengers reached new heights following the recent identification of 2′3′-cGAMP, a noncanonical CDN in mammalian cells containing mixed 2′,5′ (at GpA step) and 3′,5′ (at ApG step) linkages, which is synthesized by cGAMP synthase (cGAS) in response to the presence of DNA in the cytosol^4,5,6. A remarkable set of new discoveries have revealed that all the CDNs described above are able to bind and activate STING, the central adaptor in the cytosolic DNA sensing pathway, thereby promoting the innate immune response in mammalian cells by inducing the expression of Type I interferon (IFN)^7,8,9.Given their critical roles in a variety of important cellular processes, the cellular levels of CDNs have to be tightly controlled by the coordinated action of counteracting cyclases and degradation enzymes. To date, several phosphodiesterases (PDEs) have been found to hydrolyze c-di-GMP (EAL or HD-GYP domain-containing enzymes)¹ and c-di-AMP (DHH-DHHA or HD domain-containing enzymes)^2,10 (Figure 1). In addition, recent research reported that ENPP1 (ecto-nucleotide pyrophosphatase/phosphodiesterase) is the dominant 2′3′-cGAMP hydrolyzing enzyme in mammalian cells¹¹ (Figure 1). A new study by Gao et al.¹² has now identified the first three 3′3′-cGAMP-specific PDEs in V. cholerae and provided detailed insights into their enzymatic mechanisms.Open in a separate windowFigure 1Schematic representation of degradation enzymes identified for different cyclic dinucleotides and the related hydrolysis products. The various protein domains are highlighted by different shapes and colors. Note that the newly identified V-cGAPs belong to the HD-GYP domain-containing PDEs.There are a total of 36 potential PDE genes (containing EAL, HD-GYP or DHH domains) in the V. cholerae genome. To search for 3′3′-cGAMP-specific PDE(s), Gao et al.¹² established an efficient and sensitive eukaryotic screening system by taking advantage of the ability of 3′3′-cGAMP to activate STING and induce type I IFN expression in mammalian cells. By overexpressing the 3′3′-cGAMP synthetase DncV together with the 36 potential PDEs in 293 cells, the authors could monitor IFN-β promoter activation to identify the PDE(s) that could degrade 3′3′-cGAMP. To exclude false-positives, Gao et al. further purified the PDEs that potentially target 3′3′-cGAMP based on the initial screening, and incubated these enzymes with chemically synthesized 3′3′-cGAMP. The treated 3′3′-cGAMP molecules were further assayed by either adding to PFO-permeabilized THP-1 cells to examine IRF3 phosphorylation levels or through loading on HPLC to monitor the generation of new products. As a result of the screening and validation, the authors successfully identified three HD-GYP domain-containing proteins that could degrade 3′3′-cGAMP, named VCA0681, VCA0210 and VCA0931 (designated as V-cGAP1, 2 and 3, respectively).To determine the substrate specificity of V-cGAPs, different cGAMP linkage isomers (3′3′-, 3′2′-, 2′3′-, and 2′2′-cGAMPs) were incubated with the purified V-cGAPs. The results of both IRF3 phosphorylation in THP-1 cells and HPLC assays clearly indicated that V-cGAPs only degrade 3′3′-cGAMP, but not other cGAMP linkage isomers. The 3′3′-cGAMP PDE activity of V-cGAPs was further confirmed by dosage- and time-dependent enzymatic assays. By using mutant proteins, the authors also confirmed that both the HD and GYP motifs within V-cGAPs are critical for PDE activity.Combining detailed HPLC analysis, mass spectrometry and enzymatic treatment, Gao et al. definitively established that 3′3′-cGAMP is first hydrolyzed by all three V-cGAPs to generate linear 5′-pApG, which is further hydrolyzed into 5′-ApG only by V-cGAP1. These results show that V-cGAP2 and V-cGAP3 have only PDE activity, while V-cGAP1 has both PDE and 5′-nucleotidase activities. The authors also found that V-cGAP1 has a much higher activity for linearization of 3′3′-cGAMP to 5′-pApG than V-cGAP2 and 3, with the later two V-cGAPs exhibiting similar kinetics of degradation.The cellular level of 3′3′-cGAMP has to be tightly regulated by a combination of counteracting synthesis and degradation enzymes. Since the expression level of DncV was found to be inducible by outside signals to enhance intestinal colonization and infectivity, it is very likely that the expression level of V-cGAPs will also be regulated by 3′3′-cGAMP production. Indeed, the authors proved that V-cGAP expression is greatly and readily enhanced after arabinose-induced DncV expression in a ΔdncV mutant V. cholerae strain, at both mRNA (by qRT-PCR) and protein (by immunoblot analysis) levels. To confirm the in vivo function of V-cGAPs, the authors performed both “chemotactic” and “infant mouse colonization competition” assays by using V-cGAP1/2/3 single-, double-, or triple-deletion V. cholerae strains. All the in vivo data clearly established that V-cGAPs counteract DncV function and exert a crucial role in regulating bacterial infectivity.The large amount of insightful data presented by Gao et al. has elucidated detailed information regarding the identification and characterization of 3′3′-cGAMP-specific phosphodiesterases, thereby providing valuable insights into our understanding of the regulatory mechanisms of cGAMP signaling in bacteria. Clearly, further structural work will be necessary to understand the intermolecular interactions between 3′3′-cGAMP and V-cGAPs, and provide insights into the mechanism by which V-cGAPs preferentially attack the phosphodiester bond at the GpA step.     

Synthesis and reactions of cyclic acetal derivatives of 6,6′-dichloro-6,6′-dideoxysucrose

Treatment of 6,6′-dichloro-6,6′-dideoxysucrose with a combination of 2,2-dimethoxypropane, N,N-dimethylformamide, and toluene-p-sulphonic acid (reagent A), followed by acetylation, gave the 1′,2:3,4-diacetal 1 (39%) and the 1′,2-acetal 2 (37%). A similar reaction of methyl 6-chloro-6-deoxy-α-D-glucopyranoside with reagent A yielded the corresponding 2,3- and 3,4-acetal derivatives in yields of 29% and 9%, respectively. The structures of 1 and 2 have been confirmed by ¹H-n.m.r. spectroscopy and by chemical transformations.