Facile Method for the Preparation of 7-Methyl-8-oxoguanosines as an Immunomodulator¶

Abstract

Reaction of 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-7-methylguaninium iodide (2a) with hydrogen peroxide in acetic acid gave the corresponding 7-methyl-8-oxoguanosine derivative (3a) in good yield. Deprotection of 3a easily gave 7-methyl-8-oxoguanosine (1), which is well-known as an immunomodulator. Substitution of acetyl group at the N-position of guanine ring accelerated the oxidation reaction of the 7-methylguaninium iodide.

     

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.     

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 2′-Deoxy-9-deazaadenosine and an N-7 Blocked Derivative Useful for the Synthesis of Modified Oligonucleotides Containing 2′-Deoxy-9-deazaadenosine

Abstract

As an epimerization resistant synthon in the synthesis of oligo-nucleotides consisting of C-nucleoside analogues, hitherto unknown 5-benzyloxy-methyl-3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[3,2-d]pyrimpyrimidine (7-benzyloxymethyl-2′-deoxy-9-deazaadenosine) was prepared in seven steps from the known 3-amino-2-cyano-4-(2,3-O-isopropylidene-5-O-trityl-β-D-ribofuranosyl)-pyrrolpyrrole (1). Treatment of 1 with benzyl chloromethyl ether in the presence of potassium t-butoxide and 18-crown-6 afforded the N-protected pyrrole 2, which was converted into the 9-deazapurine derivative 3 in high yield by heating in EtOH. 7-Benzyloxymethyl-9-deazaadenosine 4 was obtained from 3 by acid hydrolysis in 2.5% methanolic hydrogen chloride. After protection of the hydroxyl groups of 4 with Markievicz's reagent, the product 5 was converted into the 2′-O-phenoxythiocarbonyl derivative 6. Reduction of 6 with butyltin hydride in the presence of 2,2′-azobis(2-methylpropionitrile), followed by desilylation with triethylammonium fluoride, afforded the desired 7-benzyloxymethyl-2′-deoxy-9-deazaadenosine (8) in high overall yield. The benzyloxymethyl group of 8 was removed by hydrogenolysis over palladium hydroxide (Degussa type) to give 2′-deoxy-9-deazaadenosine (9) in quantitative yield. The structure of 9 is discussed.     

Preparation of Oligomeric 2′-Deoxy-5-Fluorouridylate of Defined Length and Backbone Composition: A Novel Pro-Drug form of the Potent Anti-Cancer Drug 2′-Deoxy-5-Fluorouridylate

Abstract

A recursive protection scheme for FdUMP is employed by synthesizing oligonucleotides consisting of only FdU. 3′-O-exonuclease action releases FdUMP and a shortened oligonucleotide from which further exonuclease action releases more FdUMP. Such oligonucleotides are more cytotoxic to H4IIe, mouse fibroblast, and metastatic mouse rumor cells than are equivalent concentrations of FdU monomer.     

Acycloadenosine Derivatives as Inhibitors of 5′-Deoxy-5′-Methylthioadenosine Phosphorylase (MesADo PASE)

Abstract

Four classes of acyclo amlogs of 5′-methylthioadenosine were synthesized and tested as inhibitors of mammalian methylthicadenosine phosphorylase. Halogenated dihydroxypropyl acycloadenosires were most potent, i.e. K_i = 0.2 - 0.7 uM.     

Synthesis of O-Benzyl Derivatives of 2′-Deoxy-5-Trifluoromethyluridine for Antitumor Agents

Abstract

A practical synthesis of 3′-O-benzyl-2′-deoxy-5-trifluoromethyluridine was established which involves a selective 3′-O-benzylation of 2′-deoxy-5′-O-trityl-5-iodouridine followed by a cross-coupling with trifluoromethylcopper.     

7‐Deaza‐2′‐Deoxyadenosine‐5′‐Triphosphate as an Alternative Nucleotide for the Pyrosequencing Technology

A new adenosine nucleotide analog suitable for the Pyrosequencing method is presented. The new analog, 7‐deaza‐2′‐deoxyadenosine‐5′‐triphosphate (c⁷dATP), has virtually the same low substrate specificity for luciferase as the currently used analog, 2′‐deoxyadenosine‐5′‐O‐(1‐thiotriphosphate) (dATPαS). The inhibitory effect dATPαS displays on the nucleotide degrading activity of apyrase was reduced significantly by substituting the c⁷dATP for the dATPαS. Both analogs show high stability after long time storage at + 8°C. Furthermore, with the new nucleotide a read length of up to 100 bases was obtained for several templates from fungi, bacteria and viruses.     

Synthesis of 5′-O-Aminothymidine and 5′-Deoxy-5′-Hydrazinothymidine,Novel Nucleoside Derivatives

Abstract

Two novel nucleoside derivatives,5′-0-aminothymidine and 5′-deoxy-5′-hydrazinothymidine were synthesised as potential thymidine kinase inhibitors. The former was made from N-hydroxyu re thane and 5′-0-tosylthymidine and the latter from benzyl carbazate and 5′-0-tosylthymidine.     

Methyl esterification of m7G5′p reversibly blocks its activity as an analog of eukaryotic mRNA 5′-caps

The methyl ester of m⁷G^5′ p was synthesized by a carbodiimide-catalyzed reaction of G^5′ p with methanol followed by dimethylsulfate alkylation. Comparative spectral analyses indicated that m⁷Gp · methyl ester retained the rigid conformation characteristic of the messenger RNA cap analog, m⁷G^5′ p but not its strong inhibitory activity against initiation of capped mRNA translation. Attachment of reovirus mRNA to wheat germ ribosomes, crosslinking of capbinding protein to the 5′-end of oxidized mRNA, and stimulation by this protein of capped mRNA translation in HeLa cell extract were all several-fold more sensitive to inhibition by m⁷G^5′ p than to m⁷Gp · methyl ester. Conversion of the esterified analog to m⁷G^5′ p by digestion with venom phosphodiesterase restored completely the ability to inhibit initiation complex formation. The results indicate that structural features of the 5′-terminal m⁷G cap of mRNA over and above preferred conformation are recognized during eukaryotic protein synthesis.     

The effect of the 3′ → 5′ exonuclease of T7 DNA polymerase on frameshifts and deletions

Both spontaneous frameshift mutation and deletion mutation were measured in a T7 phage deficient in the 3′ → 5′ exonuclease of T7 DNA polymerase. It was found that the absence of this exonuclease caused a marked increase in the revision of both plus one and minus one mutations. The exonuclease deficiency caused essentially no effect on the frequency of deletion between 10-bp direct repeats even when the segment between the direct repeats contained a 25-bp palindrome.     

Nucleosides. 145. Synthesis of 2,5′-Anhydro-2-Thiouridine and its Conversion to 3′-O-Acetyl-2,2′-Anhydro-5′-Chloro-5′-Deoxy-2-Thiouridine. Studies Directed Toward the Synthesis of 2′-Deoxy-2′-Substituted arabino Nucleosides (7)

Abstract

In an attempt to introduce a substituent at C-2′ in the “up” arabino configuration directly by nucleophilic displacement reaction of a preformed pyrimidine ribonucleoside, we synthesized 2,5′-anhydro-5′-deoxy-2-thiouridine (6) in three steps from uridine. Compound 6 was converted into the 3′-O-acetyl derivative 7. Upon treatment of 7 with triflyl chloride in methylene chloride in the presence of triethylamine and p-dimethylaminopyridine, 2,2′-anhydro-1-(3-O-acetyl-5-chloro-2,5-dideoxy-β-D-arabinofuranosyl)-2-thiouracil (9) was obtained as the only isolable product. Obviously, the intermediate 3′-O-acetyl-2,5′-anhydro-2′-O-triflyl-2-thiouridine (8) was attacked by the chloride nucleophile at C-5′ first giving the 2′-O-triflyl-2-thiouridine intermediate from which 9 was formed by intramolecular nucleopilic reaction.     

Use of A Uridine Nucleotide as an Affinity Handle and 5′ Phosphorylating Agent for Synthetic DNA

Abstract

The 5′-(O-cyanoethyl N, N-diisopropyl phosphoramidite) of 2′,3′-O-bis(4,4′-dimethoxytrityl)uridine can be used to attach a uridine residue through a 5′-5′ phosphodiester linkage to a synthetic oligodeoxyribonucleotide. This 5′-terminal structure allows the oligomer to be selectively retarded on a chromatographic support containing dihydroxyboryl substituents, and to be converted upon periodate oxidation and p-elimination to the form possessing a 5′ phosphate group.     

A Direct and Efficient Synthesis of 5′-Deoxy-2′, 3′-

Abstract

A direct and efficient synthesis of 5′-deoxy-2′,3′-O-isopropylideneinosine, 7, from readily available inosine is described. An example of a potentially general synthesis of N -substituted-5′-deoxyadenosines from 7 is also described.     

Integrated mechanism for the generation of the 5′ junctions of LINE inserts

To elucidate the molecular mechanism of the integration of long interspersed elements (LINEs), we characterized the 5′ ends of more than 200 LINE de novo retrotransposition events into chicken DT40 or human HeLa cells. Human L1 inserts produced 15-bp target-site duplications (TSDs) and zebrafish ZfL2-1 inserts produced 5-bp TSDs in DT40 cells, suggesting that TSD length depends on the LINE species. Further analysis of 5′ junctions revealed that the 5′-end-joining pathways of LINEs can be divided into two fundamental types—annealing or direct. We also found that the generation of 5′ inversions depends on host and LINE species. These results led us to propose a new model for 5′-end joining, the type of which is determined by the extent of exposure of 3′ overhangs generated after the second-strand cleavage and by the involvement of host factors.     

Formation of an Unexpected 2-Deoxy-α-D-Threo-Pentofuranosyl Azide by Reaction of O2,3′-Anhydro-5′-O-trityl-2′-deoxycytidine with Lithium Azide

Abstract

Reaction of 0²,3′-anhydro-5′-0-trityl-2′-deoxycytidine (1) with LiN₃s in DMF resulted in the formation of 1-(3-azido-2,3-dideoxy-5-0-trityl-β-D-erythro-pentofuranosyl) cytosine (2) and 3-0-(4-amino-1,3-pyrimidin-2-yl)-5-0-trityl-2-deoxy-α-D-threo-pentofuranosyl azide (3) (2:3 = 1:1) in 88% yield. Compound 3 was deprotected with 80% aqueous AcOH yielding 4     

Most Anti-BrdU Antibodies React with 2′-Deoxy-5-Ethynyluridine — The Method for the Effective Suppression of This Cross-Reactivity

5-Bromo-2′-deoxyuridine (BrdU) and 2′-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU. Except for one sample all the others evinced reactivity with EdU. A high level of EdU persists in nuclear DNA even after the reaction of EdU with fluorescent azido-dyes if the common concentration of dye is used. Although a ten-time increase of azido-dye concentration resulted in a decrease of the signal provided by anti-BrdU antibodies, it also resulted in a substantial increase of the non-specific signal. We have shown that this unwanted reactivity is effectively suppressed by non-fluorescent azido molecules. In this respect, we have tested two protocols of the simultaneous localisation of incorporated BrdU and EdU. They differ in the mechanism of the revelation of incorporated BrdU for the reaction with antibodies. The first one was based on the use of hydrochloric acid, the second one on the incubation of samples with copper(I) ions. The use of hydrochloric acid resulted in a significant increase of the non-specific signal. In the case of the second method, no such effect was observed.     

Ex-Novo and Revisum Procedures for the Preparation of C-1′ Branched Nucleosides

Abstract

C-1′ acylated derivatives of 2′-deoxyuridine were obtained either by revising the existing procedures or by introducing a new methodology.