Highly accurate synthesis of the fully 2′-fluoro-modified oligonucleotide by Therminator DNA polymerases

Oligonucleotides composed of natural nucleotides are inapplicable for biotechnical and therapeutic use due to its instability under biological conditions. Therminator DNA polymerases, mutant DNA polymerases of thermophilic marine archaea, show that they can efficiently synthesize fully 2′-fluoro-modified (2′F-) oligonucleotides. Furthermore, the sequence analysis reveals that the oligonucleotide sequence is highly accurate, especially the fidelity of a 2′F-oligonucleotide synthesized by Therminator II is more accurate than that of natural RNA synthesized by conventional RNA polymerase. These finding would be helpful for the synthesis of chemically modified oligonucleotides, for the use of biotechnical or medical applications.     

Selective Inhibition of DNA Chain Elongation Catalyzed by DNA Polymerases

Abstract

The results of series of works on the properties of a large number of nucleoside 5′-triphosphates analogs in the reaction catalyzed by several DNA polymerases are summarized.

Molecular mechanisms of substrate selection by DNA polymerases are not studied in detail. Therefore we have undertaken a comparative analysis of DNA polymerases from different sources employing nucleoside 5′-triphosphate analogs capable of incorporating into DNA chains terminating these chains elongation. Synthesis of a large line of nucleoside 5′-triphosphate analogs with substitution at the sugar residue has been performed. DNA polymerases have been isolated, and the synthesis of DNA has been studied using phage M13 DNA or phage MS2 RNA with synthetic deoxyoligonucleoti-de primers. The molecular mechanism of the substrate action has been determined by PAG electrophoresis of the reaction     

Synthesis and Evaluation of 2′-Deoxy-2′-Spirodiflurocyclopropyl Nucleoside Analogs

The preparation of 2′-deoxy-2′-siprodifluorocyclopropany-lnucleoside analogs has been achieved from α-d-glucose in several steps. The key step in the synthesis was the introduction of the difluorocyclopropane through a difluorocarbene type reaction at the 2′-position. Then, a series of novel 2′-deoxy-2′-spirodifluorocyclopropanyl nucleoside analogs were synthesized using the Vorbrüggen method. All the synthesized nucleosides were characterized and subsequently evaluated against hepatitis C and influenza A virus strains in vitro.     

New synthetic route to ethynyl-dUTP: A means to avoid formation of acetyl and chloro vinyl base-modified triphosphates that could poison SELEX experiments

5-Ethynyl-2′-deoxyuridine is a common base-modified nucleoside analogue that has served in various applications including selection experiments for potent aptamers and in biosensing. The synthesis of the corresponding triphosphates involves a mild acidic deprotection step. Herein, we show that this deprotection leads to the formation of other nucleoside analogs which are easily converted to triphosphates. The modified nucleoside triphosphates are excellent substrates for numerous DNA polymerases under both primer extension and PCR conditions and could thus poison selection experiments by blocking sites that need to be further modified. The formation of these nucleoside analogs can be circumvented by application of a new synthetic route that is described herein.     

Reactions of adenosine 5′-phosphorimidazolide with adenosine analogs on a polyuridylic acid template: The uniqueness of the 2′-3′-unsubstituted β-ribosyl system

We have studied the reactions between adenosine 5′-phosphorimidazolide and various adenosine analogs on a poly(U) template. The nucleosides were adenosine (I), 2′-deoxyadenosine (II), 3′-deoxyadenosine (III), 2′-O-methyladenosine (IV), 3′-O-methyladenosine (V), 9-β-d-xylofuranosyladenine (VI), and 9-β-d-arabinofuranosyladenine (VII). We find that the various analogs form triple helices with poly(U) which are of comparable stability, but that only the β-riboside takes part in an efficient template-directed condensation.     

Solid-phase synthesis of 5′-triphosphate 2′-5′-oligoadenylates analogs with 3′-O-biolabile groups and their evaluation as RNase L activators and antiviral drugs

5′-Triphosphate 2′-5′-oligoadenylate (2–5A) is the central player in the 2–5A system that is an innate immunity pathway in response to the presence of infectious agents. Intracellular endoribonuclease RNase L activated by 2–5A cleaves viral and cellular RNA resulting in apoptosis. The major limitations of 2–5A for therapeutic applications is the short biological half-life and poor cellular uptake. Modification of 2–5A with biolabile and lipophilic groups that facilitate its uptake, increase its in vivo stability and release the parent 2–5A drug in an intact form offer an alternative approach to therapeutic use of 2–5A. Here we have synthesized the trimeric and tetrameric 2–5A species bearing hydrophobic and enzymolabile pivaloyloxymethyl groups at 3′-positions and a triphosphate at the 5′-end. Both analogs were able to activate RNase L and the production of the trimer 2–5A (the most active) was scaled up to the milligram scale for antiviral evaluation in cells infected by influenza virus or respiratory syncytial virus. The trimer analog demonstrated some significant antiviral activity.     

Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

DNA polymerase α₁, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α₁ and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α₁ activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.     

Inhibition of tubulin polymerization with ribose-modified analogs of GDP and GTP reduced inhibition with microtubule-associated proteins and magnesium

Inhibitory effects of ribose-modified GDP and GTP analogs on tubulin polymerization were examined to explore nucleotide structural requirements at the exchangeable GTP binding site. With microtubule-associated proteins and Mg²⁺, GTP-supported polymerization was only modestly inhibited by GDP, and still weaker inhibitory activity was found with two analogs, dGDP and 9-β-D-arabinofuranosylguanine-5′-diphosphate (araGDP). Omission of Mg²⁺ significantly enhanced the inhibitory effects of GDP, dGDP and araGDP and resulted in weak inhibition of the reaction by several other GDP analogs. The relative inhibitory activity of the GDP analogs had no discernable relationship to the relative activity of cognate GTP analogs in supporting microtubule-associated protein-dependent polymerization. One GTP analog, 2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), supports polymerization both with and without microtubule-associated proteins. The inhibitory activity of GDP and GDP analogs in ddGTP-supported polymerization was much greater in the absence of microtubule-associated proteins than in their presence; and both reactions were more readily inhibited than was microtubule-associated protein-dependent, GTP-supported polymerization. Microtubule-associated protein-independent, ddGTP-supported polymerization was also potently inhibited by GTP and a number of GTP analogs. GTP was in fact twice as inhibitory as GDP. The relative inhibitory activity of the GTP analogs was comparable to the relative inhibitory activity of the cognate GDP analogs and very different from their relative activity in supporting polymerization.     

ANALOGS OF GUANINE NUCLEOSIDE TRIPHOSPHATES FOR SEQUENCING APPLICATIONS

We have synthesized more than 30 different deoxyribonucleosides and triphosphates with modifications either in the base or the phosphate moiety as analogs of 2′-dGTP for DNA sequencing applications. All the modified nucleoside triphosphates were tested as substrates for DNA polymerases, including Sequenase? T7 DNA polymerase or Thermo Sequenase? DNA polymerase. Two of the analogs, 7-ethyl-7-deaza-dGTP and 7-hydroxymethyl-7-deaza- dGTP meet our requirements as better sequencing reagents.     

Novel Method for the Synthesis of 2′ -Phosphorylated Oligonucleotides

We have developed a new method for the preparation of oligodeoxyribonucleotides and oligo(2′-O-methylribonucleotides) that contain a 2′-phosphorylated ribonucleoside residue, and optimized it to avoid 2′ -3′ -isomerization and chain cleavage. Structures of the 2′ -phosphorylated oligonucleotides were confirmed by MALDI-TOF MS and enzymatic digestion, and the stability of their duplexes with DNA and RNA was investigated. 2′-Phosphorylated oligonucleotides may be useful intermediates for the introduction of various chemical groups for a wide range of applications.     

Synthesis and Anti-HIV Activity of β-D-3′-Azido-2′,3′-unsaturated Nucleosides and β-D-3′-Azido-3′-deoxyribofuranosylnucleosides

Since the discovery of 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) as potent and selective inhibitors of the replication of human immunodeficiency virus (HIV), there has been a growing interest for the synthesis of 2′,3′-didehydro-2′,3′-dideoxynucleosides with electron withdrawing groups on the sugar moiety. Here we described an efficient method for the synthesis of such nucleoside analogs bearing structural features of both AZT and d4T. The key intermediate, 3-azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-D-ribofuranose, 5 was synthesized from commercially available D-xylose in five steps, from which a series of pyrimidine and purine nucleosides were synthesized in high yields. The resultant protected nucleosides were converted to target nucleosides using appropriate chemical modifications. The final nucleosides were evaluated as potential anti-HIV agents.     

Synthesis and antibacterial activity of 5′-tetrachlorophthalimido and 5′-azido 5′-deoxyribonucleosides

Reported is an efficient synthesis of adenyl and uridyl 5′-tetrachlorophthalimido-5′-deoxyribonucleosides, and guanylyl 5′-azido-5′-deoxyribonucleosides, which are useful in solid-phase synthesis of phosphoramidate and ribonucleic guanidine oligonucleotides. Replacement of 5′-hydroxyl with tetrachlorophthalimido group was performed via Mitsunobu reaction for adenosine and uridine. An alternative method was applied for guanosine which replaced the 5′-hydroxyl with an azido group. The resulting compounds were converted to 5′-amino-5′-deoxyribonucleosides for oligonucleotide synthesis. Synthetic intermediates were tested as antimicrobials against six bacterial strains. All analogs containing the 2′,3′-O-isopropylidine protecting group demonstrated antibacterial activity against Neisseria meningitidis, and among those analogs with 5′-tetrachlorophthalimido and 5′-azido demonstrated increased antibacterial effect.     

An efficient synthesis of 3′-O-triazole modified guanosine-5′-O-monophosphate using click chemistry

First chemical synthesis of 3′-O-1,2,3-triazolyl-guanosine-5′-O-monophosphate by copper catalyzed click chemistry is described. The present cycloaddition reaction involves, in situ generation of azide from the corresponding bromide followed by copper catalyst cycloaddition with 3′-O-propargyl guanosine monophosphate in water, in the presence of catalytic amount of β-cyclodextrin. The CuAAC reaction is highly regioselective forming 1,4-cycloadduct with good yield and high purity. The final compound, 3′-O -triazole substituted guanosine monophosphate has the potential to use in various biomolecules such as labeled nucleic acids, mRNA dinucleotide cap analogs for molecular biology and their applications in the therapeutic field.     

Synthesis and properties of 4′-C-aminoalkyl-2′-fluoro-modified RNA oligomers

Synthesis and properties of double-stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) containing 4′-C-aminoethyl-2′-deoxy-2′-fluorouridine are described. Thermal denaturation studies showed that incorporation of 4′-C-aminoethyl-2′-fluoro analog improved the thermal stabilities of dsRNAs and siRNAs compared to the corresponding 4′-C-aminoethyl-2′-O-methyl analog. siRNA incorporating eight 4′-aminoethyl-2′-fluoro analogs in the passenger strand showed sufficient RNAi activity at 1?nM concentration, which was similar to that of the unmodified siRNA. Furthermore, the siRNA containing the 4′-C-aminoethyl-2′-fluoro analog exhibited high stability in a buffer containing 20% bovine serum. Forty-eight percent of the siRNA remained intact after 48?h of incubation. Thus, modification of siRNAs by the 4′-C-aminoethyl-2′-fluoro analog would be useful for the development of therapeutic siRNA molecules.     

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.     

An AZT Analog with Strongly Pairing Ethynylpyridone Nucleobase and Its Antiviral Activity against HSV1

Challenges resulting from novel viruses or new strains of known viruses call for new antiviral agents. Nucleoside analogs that act as inhibitors of viral polymerases are an attractive class of antivirals. For nucleosides containing thymine, base pairing is weak, making it desirable to identify nucleobase analogs that pair more strongly with adenine, in order to compete successfully with the natural substrate. We have recently described a new class of strongly binding thymidine analogs that contain an ethynylmethylpyridone as base and a C-nucleosidic linkage to the deoxyribose. Here we report the synthesis of the 3′-azido-2′,3′-deoxyribose derivative of this compound, dubbed AZW, both as free nucleoside and as ProTide phosphoramidate. As a proof of principle, we studied the activity against Herpes simplex virus type 1 (HSV1). Whereas the ProTide phosphoramidate suffered from low solubility, the free nucleoside showed a stronger inhibitory effect than that of AZT in a plaque reduction assay. This suggests that strongly pairing C-nucleoside analogs of pyrimidines have the potential to become active pharmaceutical ingredients with antiviral activity.     

Steric and Electrostatic Effects at the C2 Atom Substituent Influence Replication and Miscoding of the DNA Deamination Product Deoxyxanthosine and Analogs by DNA Polymerases

Deoxyinosine (dI) and deoxyxanthosine (dX) are both formed in DNA at appreciable levels in vivo by deamination of deoxyadenosine (dA) and deoxyguanosine (dG), respectively, and can miscode. Structure-activity relationships for dA pairing have been examined extensively using analogs but relatively few studies have probed the roles of the individual hydrogen-bonding atoms of dG in DNA replication. The replicative bacteriophage T7 DNA polymerase/exonuclease and the translesion DNA polymerase Sulfolobus solfataricus pol IV were used as models to discern the mechanisms of miscoding by DNA polymerases. Removal of the 2-amino group from the template dG (i.e., dI) had little impact on the catalytic efficiency of either polymerase, as judged by either steady-state or pre-steady-state kinetic analysis, although the misincorporation frequency was increased by an order of magnitude. dX was highly miscoding with both polymerases, and incorporation of several bases was observed. The addition of an electronegative fluorine atom at the 2-position of dI lowered the oligonucleotide T_m and strongly inhibited incorporation of dCTP. The addition of bromine or oxygen (dX) at C2 lowered the T_m further, strongly inhibited both polymerases, and increased the frequency of misincorporation. Linear activity models show the effects of oxygen (dX) and the halogens at C2 on both DNA polymerases as mainly due to a combination of both steric and electrostatic factors, producing a clash with the paired cytosine O2 atom, as opposed to either bulk or perturbation of purine ring electron density alone.     

Di- and triphosphate derivatives of acyclo- and arabinosylguanine effects on the polymerization of purified tubulin

Glutamate- and nucleotide-dependent polymerization of purified calf brain tubulin was used as a model system to study interactions of ribose-modified GDP and GTP analogs with tubulin. Earlier studies (Hamel, E., and Lin, C.M. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3368–3372) were extended to three additional sets of analogs: the di- and triphosphate derivatives of 9-β-D-arabinofuranosylguanine (araGDP and araGTP) and acycloguanosine (9-(2-hydroxyethoxymethyl)guanine) (acycloGDP and acycloGTP), as well as the periodate-oxidized and borohydride-reduced derivatives of GDP and GTP (ox-redGDP and ox-redGTP). Disruption of the ribose ring in ox-redGTP resulted in major loss of activity relative to GTP in supporting tubulin polymerization, although the analog's deficiency may result from an inability to displace GDP from the exchangeable site rather than a direct effect on the polymerization reaction itself. The poor activity of ox-redGTP could be largely reversed if nucleoside diphosphate kinase was added to the reaction mixture. Removal of the 2′ and 3′ carbons entirely, in the form of acycloGTP, resulted in only minimal loss of activity relative to GTP. AraGTP, on the other hand, was more active than GTP in supporting tubulin polymerization. All three GDP analogs were much less effective than GDP in inhibiting tubulin polymerization, although araGDP was significantly more inhibitory than acycloGDP or ox-redGDP. Relative inhibitory activity of these and additional GDP analogs was the same whether GTP or a GTP analog was used to support tubulin polymerization.     

Study on Disulfur-Backboned Nucleic Acids: Part 3. Efficient Synthesis of 3′,5′-Dithio-2′-Deoxyuridine and Deoxycytidine

A general method is described for synthesizing 3′,5′-dithio-2′-deoxypyrimidine nucleosides 6 and 13 from normal 2′-deoxynucleosides. 2,3′-Anhydronucleosides 2 and 9 are applied as intermediates in the process to reverse the conformation of 3′-position on sugar rings. The intramolecular rings of 2,3′-anhydrothymidine and uridine are opened by thioacetic acid directly to produce 3′-S-acetyl-3′-thio-2′-deoxynucleosides 3 or 5. To cytidine, OH^? ion exchange resin was used to open the ring and 2′-deoxycytidine 10 was abtained in which 3′-OH group is in threo-conformation. The 3′-OH is activated by MsCl, and then substituted by potassium thioacetate to form the S,S′-diacetyl-3′,5′-dithio-2′-deoxycytidine 12. The acetyl groups in 3′,5′ position are removed rapidly by EtSNa in EtSH solution to afford the target molecules 6 and 13. The differences of synthetic routes between uridine and cytidine are also discusssed.