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.     

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).     

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.     

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 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.     

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.     

An efficient synthesis of 3′-amino-3′-deoxythymidine derivatives

An efficient method of reduction of 3-azido-3-deoxythymidine and its 5-protected derivatives to 3-aminothymidine derivatives on a palladium catalyst using ammonium formate as a source of hydrogen was suggested.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 147–150.Original Russian Text Copyright © 2005 by Seregin, Chudinov, Yurkevich, Shvets.     

Preparation,Hydrolysis and Intramolecular Transesterification of 3′-Deoxy-3′-thioinosine 3′-S-Dimethylphosphorothiolate

Abstract

The hydrolytic reactions of the dimethyl ester of 3′-deoxy-3′-thioinosine 3′-S-phosphorothiolate have been followed over a wide aciditty range by HPLC. At pH > 3, only hydroxide ion catalyzed isomerization to the 2′-dimethylphosphate takes place, whereas under more acidic conditions hydrolysis to the 2′-monomethylphosphate and 3′-S-monomethylphosphorothiolate competes. The latter is the only product accumulating in very acidic solutions (1 M hydrochloric acid). Mechanisms of the reactions are discussed.     

New Phosphonoformic Acid Derivatives of 3′-Azido-3′-Deoxythymidine

New 5-alkyl ethoxy- and aminocarbonylphosphonates of 3-azido-3-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity.     

Synthesis of Some 2′,3′-Dideoxy-3′-C-Fluoromethyl and 3′-C-Azidomethyl Nucleosides

Abstract

The synthesis of 3′-C-fluoromethyl and 3′-C-azidomethyl nucleosides is reported. The 3′-C-fluoromethyl furanoside 4 was synthesized via fluoride ion induced displacement of the corresponding trifluoromethanesulfonate. The 3′-C-hydroxymethyl furanoside 3 was converted to the corresponding 3′-C-azidomethyl furanoside 6 using triphenylphosphine-carbon tetrabromide-lithium azide. The 3′-C-fluoromethyl furanoside derivative 5 and the 3′-C-azidomethyl furanoside derivative 7 were subsequently condensed with silylated purine and pyrimidine bases. Deblocking and separation of the anomers by chromatography afforded the α- and β-nucleoside analogues. The nucleosides were tested for inhibition of HIV multiplication in vitro and were found to be inactive in the assay.     

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 L-3′-Hydroxymethylribonucleosides

Abstract

The target compounds were synthesized via the key intermediate carbohydrate 8, which was synthesized by first selectively protecting the 1′ - and 2′- hydroxyl groups followed by selective tosylation of the 5′ -hydroxyl group to obtain compound 3. The tosyl moiety was then replaced by a benzyl ether to obtain 4. Compound 4 underwent Dess-Martin oxidation to afford the ketone 5. Compound 5 was subjected to Wittig olefination to afford the alkene 6 followed by regioselective hydroboration to obtain 7. Compound 7 was fully acetylated using acetic acid, acetic anhydride and sulfuric acid to obtain the key intermediate 8.     

Synthesis of 2′,3′-Didehydro-2′,3′-Dideoxy-3′-C-Methyl Substituted Nucleosides

Abstract

1-(2,3-Dideoxy-3-C-hydroxmethyl-β-D-threo-pentofuranosyl) -,1- (2,3-didehydro-2,3-dideoxy-3-C-hydroxymethyl-β-D-glycero- pentofuranosyl) -and 1-(3-C-azidomethyl-2,3-dideoxy-3-C-hydroxymethyl-β-D-glycero- pentofuranosyl)uracil, thymine and cytosine were synthesized and evaluated for anti-HIV activity. The synthetic strategy was based on an allylic alcohol transposition of the corresponding 3′-C-methylene-nucleoside analogues.     

A New Synthesis of 2′,3′-Didehydro-3′-deoxy-3-Alkylthymidine

Abstract

The preparation of 3-alkyl D4T derivatives has been carried out starting from the corresponding 5′-O-t-butyldimethylsilyl-3′-O-methanesulfonylthymidine 2 by way of deprotection-elimination and succesive alkylation reactions.     

Synthesis and Antiviral Evaluation of 3′-Substituted Thymidine Analogues Derived from 3′-Amino-3′-deoxythymidine

Abstract

To assess the structure-activity relationship for antiviral activity, a series of 3′-deoxy-3′-N-functionalized thymidine analogues were synthesized. Several of these thymidine analogues show moderate in vitro activity against HIV-1 and HIV-2.     

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.     

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 and evaluation of sulfonylethyl-containing phosphotriesters of 3′-azido-3′-deoxythymidine as anticancer prodrugs

A series of bis(sulfonylethyl) and mono(sulfonylethyl) phenyl phosphotriesters of zidovudine (3′-azido-3′-deoxythymidine, AZT) were synthesized as potential anticancer prodrugs that liberate AZT monophosphate via nonenzymatic β-elimination mechanism. Stability studies demonstrated that all the synthesized prodrugs spontaneously liberate AZT monophosphate with half-lives in the range of 0.07–278.8 h under model physiological conditions in 0.1 M phosphate buffer at pH 7.4 and 37 °C. Analogous to aldophosphamide, the elimination rates were accelerated in the presence of reconstituted human plasma under the same conditions. Among the compounds, 3, 4, 8, and 10 were comparable or superior to AZT against five established human cancerous cell lines in vitro. Moreover, the selected compounds were equally sensitive to both the wild-type osteosarcoma 143B and the thymidine kinase-deficient 143B/TK⁻ cell lines. The findings are consistent with that these compounds deliver AZT monophosphate intracellularly.