A Nucleotide Dimer Synthesis Without Protecting Groups Using Montmorillonite as Catalyst

A synthesis has been developed providing nucleotide dimers comprising natural or unnatural nucleoside residues. A ribonucleoside 5′-phosphorimidazolide is added to a nucleoside adsorbed on montmorillonite at neutral pH with the absence of protecting groups. Approximately 30% of the imidazolide is converted into each 2′-5′ dimer and 3′-5′ dimer with the rest hydrolyzed to the 5′-monophosphate. Experiments with many combinations have suggested the limits to which this method may be applied, including heterochiral and chimeric syntheses. This greener chemistry has enabled the synthesis of dimers from activated nucleotides themselves, activated nucleotides with nucleosides, and activated nucleotides with nucleotide 5′-monophosphates.

[Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides & Nucleic Acids to view the free supplemental files.]     

DIMERIZATION IN HIGHLY CONCENTRATED SOLUTIONS OF PHOSPHOIMIDAZOLIDE ACTIVATED MONONUCLEOTIDES

Phosphoimidazolide activated ribomononucleotides (*pN) are useful substrates for the non-enzymatic synthesis of polynucleotides. However, dilute neutral aqueous solutions of *pN typically yield small amounts of dimers and traces of polymers; most of *pN hydrolyzes to yield nucleoside 5-monophosphate. Here we report the self-condensation of nucleoside 5-phosphate 2-methylimidazolide (2-MeImpN with N = cytidine, uridine or guanosine) in the presence of Mg2+ in concentrated solutions, such as might have been found in an evaporating lagoon on prebiotic Earth. The product distribution indicates that oligomerization is favored at the expense of hydrolysis. At 1.0 M, 2-MeImpU and 2-MeImpC produce about 65% of oligomers including 4% of the 3,5-linked dimer. Examination of the product distribution of the three isomeric dimers in a self-condensation allows identification of reaction pathways that lead to dimer formation. Condensations in a concentrated mixture of all three nucleotides (U,C,G mixtures) is made possible by the enhanced solubility of 2-MeImpG in such mixtures. Although percent yield of internucleotide linked dimers is enhanced as a function of initial monomer concentration, pyrophosphate dimer yields remain practically unchanged at about 20% for 2-MeImpU, 16% for 2-MeImpC and 25% of the total pyrophosphate in the U,C,G mixtures. The efficiency by which oligomers are produced in these concentrated solutions makes the evaporating lagoon scenario a potentially interesting medium for the prebiotic synthesis of dimers and short RNAs.     

Bacterial Synthesis of Nucleotides

General properties of bacterial nucleoside phosphotransferase were demonstrated. Nucleoside phosphotransferase activity was observed somewhere in cells, and the activity and the specificity for donor and product in this reaction are described to be due to the basic character of strains. Such aromatic phosphates as p-nitrophenylphosphate, phenylphosphate, benzylphosphate and the nucleotides were apparent to be useful for nucleotide synthesis, and the ability as donor did not always depend upon the energy consideration. The product specificity of this reaction was confirmed to correlate with nucleotide isomer added as donor; that is, the bacteria characterized to phosphorylate at 5′-position of nucleoside catalyzed the interconversion of phosphoryl or phosphate radical between 5′-nuclotides and those characterized to do at 3′(& 2′)-position of nucleoside catalyzed the interconversion of that between 3′(& 2′)-nucleotides. The phosphoryl or phosphate transfer reaction using nucleotide as donor is reversible but that using p-nitrophenylphosphate as donor is irreversible. The factors to get a good yield on the synthesis of 5′-inosinic acid were discussed, then the maximum yield was accounted to 80%.     

Synthesis of Amide-Linked [(3′)CH2CO-NH(5′)] Nucleoside Analogues of Small Oligonucleotides

Abstract

We report syntheses of new amide-linked (di-penta)nucleoside analogues of antisense oligonucleotide components. Solution-phase coupling of 3′-(carboxymethyl)-3′-deoxy- and 5′-amino-5′-deoxynucleoside derivatives provides amide dimers. Activated [3′-(carboxymethyl)-3′-deoxy] units with a 5′-azido-5′-deoxy function provide “masked” 5′-amino-5′-deoxy residues for chain extension, and a 5′-O-DMT-protected unit provides the 5′-terminus for attachment to a phosphodiester linkage.     

5′-Phosphorylation of Oligonucleotides with Phosphorous Acid in Automated DNA Synthesis

Abstract

Coupling of phosphorous acid in automated DNA synthesis using H-phosphonate methodology leads to 5′-5′ linked dimers and 5′-H-phosphonates. The yield is dependent on the phosphorous acid concentration, chain length of the oligomer, and pore size of the support. 5′-Phosphate oligomers are obtained from the H-phosphonate oligomers by silylation and oxidation.     

Synthesis and P2Y receptor activity of nucleoside 5′-phosphonate derivatives

Ribose-based nucleoside 5′-diphosphates and triphosphates and related nucleotides were compared in their potency at the P2Y receptors with the corresponding nucleoside 5′-phosphonate derivatives. Phosphonate derivatives of UTP and ATP activated the P2Y₂ receptor but were inactive or weakly active at P2Y₄ receptor. Uridine 5′-(diphospho)phosphonate was approximately as potent at the P2Y₂ receptor as at the UDP-activated P2Y₆ receptor. These results suggest that removal of the 5′-oxygen atom from nucleotide agonist derivatives reduces but does not prevent interaction with the P2Y₂ receptor. Uridine 5′-(phospho)phosphonate as well as the 5′-methylenephosphonate equivalent of UMP were inactive at the P2Y₄ receptor and exhibited maximal effects at the P2Y₂ receptor that were ?50% of that of UTP suggesting novel action of these analogues.     

Phosphopentomutase of Bacillus stearothermophilus TH6-2: The Enzyme and Its Gene ppm

Phosphopentomutase catalyzes the transfer of an intramolecular phosphate on ribose or deoxyribose, and is involved in the salvage pathway of nucleoside synthesis. We identified a sequence 5′-upstream of the genes for the nucleoside phosphorylases of Bacillus stearothermophilus as the phosphopentomutase (ppm) gene. The novel gene corresponded to an open reading frame of 1,179 nucleotides that is translated into a putative 393-amino acid protein with a molecular weight of 43,735. The gene product, partially purified from ppm-overexpressing Escherichia coli cells, was judged to be a monomer of a 44-kDa polypeptide. The phosphopentomutase was found to catalyze the phosphotransfer on not only ribose or deoxyribose but also arabinose or dideoxyribose.     

Methanol esterification reactions catalyzed by snake venom and bovine intestinal 5'-nucleotide phosphodiesterases. Formation of nucleoside 5'-monophosphate methyl esters from guanosine 5'-triphosphate and other nucleoside 5'-polyphosphates.

It is not known whether the enzymes 5'-nucleotide phosphodiesterase/nucleotide pyrophosphatase (EC 3.1.4.1/EC 3.6.1.9) catalyze the transfer of nucleotides to acceptors other than water. We have investigated the action of snake venom and bovine intestinal mucosa phosphodiesterases on nucleoside 5'-polyphosphates in the presence of methanol. In those conditions, GTP was converted by snake venom phosphodiesterase to a mixture of GMP and another compound with a different retention time in reverse-phase high-performance liquid chromatography. That compound, by ultraviolet, 1H- and 13C-nuclear magnetic resonance spectroscopic analysis, and by enzyme analysis, was characterized as the methyl ester of GMP (GMP-OMe). The molar fraction [GMP-OMe]/[GMP + GMP-OMe] formed was higher than the molar fraction of methanol as a solvent in reaction mixtures. Similar reactions took place at comparable rates with snake venom and bovine intestinal mucosa phosphodiesterases using several nucleoside 5'-polyphosphates as substrates. The ability of 5'-nucleotide phosphodiesterases to catalyze transfer reactions to a non-water acceptor is relevant to the mechanism of the enzymes, to their use as analytical tools, and to their possible use/role in the preparative/in vivo synthesis of nucleotide esters.     

Bacterial Synthesis of Nucleotides

5′-Phosphoribosyl 5-amino-4-imidazole carboxamide was prepared by incubating 5-amino-4-imidazole carboxamide riboside and a phosphate compound with the bacteria characterized to phosphorylate at C_5′ via the phosphoryl transfer reaction. Aromatic phosphate compounds and 5′-nucleotides were able to act as the phosphate donor. This material was isolated chromatographically and its properties were studied. The other bacteria characterized to phosphorylate at C_{3′ (or 2′)} also phosphorylated a little probably at C_{3′ (or 2′)} of 5-amino-4-imidazole carboxamide riboside.

The phosphoryl interconversion between nucleotides and nucleosides was studied to be carried out via the phosphoryl transfer reaction observed in bacteria. The phosphotransferase activity of Ps. trifolii mediated reversibly the phosphoryl transfer between 5′-nucleotides and nucleosides, and its optimal pH was at around 8.5, whereas that of Prot. mirabilis did transfer the phosphoryl radical from 2′- and 3′-nucleotide to nucleoside at its optimal pH, around 5.0.

These donor- and product-isomer specificities of both bacteria were evident to be invariable, regardless of reaction pH and cultural conditions. These reactions, especially using the bacteria characterized to phosphorylate at C_5′ of nucleoside, were demonstrated to catalyze the phosphoryl interconversion between 5′-purine nucleotides and pyrimidine nucleosides or vice versa.     

Synthesis and Evaluation Of 2″-Modified MMI Linked Dimers in Antisense Constructs

Abstract

Synthesis of four methylene(methylimino) (MMI) linked dimers modifed at the 2′-position with fluoro and/or methoxy groups and their incorporation into different sequences has been accomplished. From these dimers, bis 2′-OMe MMI dimer was selected for further studies based on its synthetic accessibility, conformational study by NMR, and Tm analysis. Several chimeric antisense oligomers containing bis 2′-OMe dimers have been synthesized on a 10 μmol scale for in vivo studies.     

Investigation of a Facile Synthetic Method for Phosphorothioate Dimer Synthons In Oligonucleotide Phosphorothioates Synthesis

Abstract

A facile synthetic method of a phosphorothioate dimer block was investigated. Dinucleoside phosphite triester intermediates were obtained in one-pot synthesis by the coupling of a protected nucleoside bearing free 5′-OH and a protected nucleoside bearing free 3′-OH in the presence of phosphorous trichloride (PCl₃) and 1,2,4-triazole. The intermediates were easily sulfurized to afford the desired phosphorothioate dimer blocks in 33-64% overall yields.     

Mechanisms of targeted frameshift mutations: Insertions arising during error-prone or SOS synthesis of DNA containing cis-syn cyclobutane thymine dimers

It is still unclear how frameshift mutations arise at cyclobutane pyrimidine dimers. The polymerase model is commonly used to explain the mechanisms of various mutations. An alternative polymerase-tautomer model was developed for UV-induced mutagenesis. A mechanism was proposed for targeted insertions caused by cis-syn cyclobutane thymine dimers. Targeted insertions are frameshift mutations due to addition of one or more nucleotides in a DNA sequence opposite to a lesion capable of stopping DNA synthesis. Among other factors, cyclobutane pyrimidine dimers can cause targeted insertions. UV irradiation can change the tautomeric form of DNA bases. Five rare tautomeric forms are possible for thymine, and they are stable when the thymine is a component of a cyclobutane dimer. A structural analysis showed that none of the canonical nucleotides can be added opposite to a specific rare thymine tautomer so that hydrogen bonds form between the two bases. A single nucleotide gap is consequently left in the corresponding site of the nascent strand when a specialized or modified DNA polymerase drives SOS or error-prone DNA synthesis on a template containing cis-syn cyclobutane thymine dimers with a base occurring in the rare tautomeric form. If the DNA composition is homogenous within the region, the end of the growing DNA strand may slip to form a complementary pair with the nucleotide adjacent to the dimer according to the Streisinger model, thus producing a loop. A targeted insertion is thereby generated to make the daughter strand longer. Targeted insertions were for the first time assumed to result from the cis-syn cyclobutane thymine dimers wherein one or both of the bases occur in the specific tautomeric form that does not allow the addition and hydrogen bonding of any canonical nucleotide in the opposite position. A model was developed to explain how targeted insertions of one or more nucleotides are caused by cis-syn cyclobutane thymine dimers. Thus, the polymerase-tautomer model can explain the nature and formation of targeted frameshift mutations in addition to hot and cold spots or targeted or untargeted nucleotide substitutions.     

Boron Containing Pyrimidines,Nucleosides, and Oligonucleotides for Neutron Capture Therapy

Abstract

The synthesis and encouraging biological findings with boron-containing nucleosides, such as 5-dihydroxyboryl-2′-deoxyuridine, which could be used for boron neutron capture therapy (BNCT) for the treatment of various malignancies, has provided momentum to synthesize several boron containing nucleosides and oligomers. BNCT is based on the property of the non-radioactive boron-10 isotope to capture low energy neutrons, thereby producing a localized cell-destroying nuclear reaction. Thus, irradiation of tumor cells with neutrons, following incorporation of the boronated nucleoside, would result in the destruction of tumor tissue only. Intracellular phosphorylation by nucleoside kinases, and/or incorporation into the cancer cell DNA as a false nucleotide precursor, followed by irradiation by neutrons, would lead primarily to tumor cell death. The synthetic and biological approaches for boronated pyrimidines, nucleosides, and oligonucleotides for BNCT are reviewed.     

Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V

The ability of HeLa DNA polymerases to carry out DNA synthesis from incisions made by various endodeoxyribonucleases which recognize or form baseless sites in DNA was examined. DNA polymerase beta carried out limited strand displacement synthesis from 3'-hydroxyl nucleotide termini made by HeLa apurinic/apyrimidinic (AP) endonuclease II at the 5'-side of apurinic sites. Escherichia coli endonuclease III incises at the 3'-side of apurinic sites to produce nicks with 3'-deoxyribose termini which did not efficiently support DNA synthesis with beta-polymerase. However, these nicks could be activated to support limited DNA synthesis by HeLa AP endonuclease II, an enzyme which removes the baseless sugar phosphate from the 3'-termini, thus creating a one-nucleotide gap. With dGTP as the only nucleoside triphosphate present, the beta-polymerase catalyzed one-nucleotide DNA repair synthesis from those gaps which lacked dGMP. In contrast, HeLa DNA polymerase alpha was unreactive with all of the above incised DNA substrates. Larger patches of DNA synthesis were produced by nick translation from one-nucleotide gaps with HeLa DNA polymerase beta and HeLa DNase V. Moreover, incisions made by E. coli endonuclease III were activated to support DNA synthesis by the DNase V which removed the 3'-deoxyribose termini. HeLa DNase V also stimulated both the rate and extent of DNA synthesis by DNA polymerase beta from AP endonuclease II incisions. In this case the baseless sugar phosphate was removed from the 5'-termini, and nick translational synthesis occurred. Complete DNA excision repair of pyrimidine dimers was achieved with the beta-polymerase, DNase V, and DNA ligase from incisions made in UV-irradiated DNA by T4 UV endonuclease and HeLa AP endonuclease II. Such incisions produce a one-nucleotide gap containing 3'-hydroxyl nucleotide and 5'-thymine: thymidylate cyclobutane dimer termini. DNase V removes pyrimidine dimers primarily as a dinucleotide and then promotes nick translational DNA synthesis.     

CHIMERIC RNA WITH MODIFIED BACKBONES: ALTERNATING METHYLENE(METHYLIMINO) LINKED PHOSPHODIESTER BACKBONE OLIGONUCLEOTIDES WITH 2′-OH AND 2′-OMe GROUPS

Synthesis of a novel ribo-MMI dimer with 2′-OH and 2′-OMe in 5′- and 3′-nucleosides, respectively is presented. The synthesis was accomplished by reductive coupling of 3′-deoxy-3′-C-formyluridine and 2′-O-methyl-5′-O-methylaminouridine via a thioacetal as the key intermediate for the top part of the dimer. Incorporation of ribo- MMI dimers into oligonucleotides increased binding affinity for target RNA.     

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.     

Solution structure of the Mesorhizobium loti K1 channel cyclic nucleotide‐binding domain in complex with cAMP

Cyclic nucleotide‐sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide‐binding domain (CNBD). However, the mechanism by which the binding of cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a cyclic nucleotide‐sensitive K⁺ channel from Mesorhizobium loti. The protein consists of a wide anti‐parallel β‐roll topped by a helical bundle comprising five α‐helices and a short 3₁₀‐helix. In contrast to the dimeric arrangement (‘dimer‐of‐dimers’) in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other.     

The Synthesis of the Sixteen Possible 2′-O-Methyl MMI Dimer Phosphoramidites: Building Blocks for the Synthesis of Novel Antisense Oligonucleotides

Abstract

The synthesis of Methylene(methylimino) or MMI linked nucleoside dimers in all sixteen possible configurations has been accomplished via a reductive coupling of a nucleosidic aldehyde with an hydroxylamine. This has allowed us to prepare all of the necessary 2′-O-methyl MMI dimer building blocks necessary for use in an antisense motif.     

Significance of Mineral Salts in Prebiotic RNA Synthesis Catalyzed by Montmorillonite

The montmorillonite-catalyzed reactions of the 5′-phosphorimidazolide of adenosine used as a model generated RNA type oligomers. These reactions were found to be dependent on the presence of mineral salts. Whereas montmorillonite (pH 7) produced only dimers and traces of trimer in water, addition of sodium chloride (0.1–2.0 M) enhanced the chain length of oligomers to 10-mers as detected by HPLC. Maximum catalytic activity was observed with sodium chloride at a concentration between 0.8 and 1.2 M. This concentration of sodium chloride resembled its abundance in the ancient oceans (0.9–1.2 M). Magnesium chloride produced a similar effect but its joint action with sodium chloride did not produce any difference in the oligomer chain length. Therefore, Mg²⁺ was not deemed necessary for generating longer oligomers. The effect of monovalent cations upon RNA chain length was: Li⁺ > Na⁺ > K⁺. A similar effect was observed with the anions with enhanced oligomer length in the following order: Cl^? > Br^? > I^?. Thus, the smaller ions facilitated the formation of the longest oligomers. Inorganic salts that tend to salt out organic compounds from water and salts which show salt-in effects had no influence on the oligomerization process indicating that the montmorillonite-catalyzed RNA synthesis is not affected by either of these hydrophobic or hydrophilic interactions. A 2.3-fold decrease in the yield of cyclic dimer was observed upon increasing the sodium chloride concentration from 0.2 to 2.0 M. Inhibition of cyclic dimer formation is vital for increasing the yield of linear dimers and longer oligomers. In summary, sodium chloride is likely to have played an essential role in any clay mineral-catalyzed prebiotic RNA synthesis.     

Mechanism of Action of Showdomycin

Among the syntheses of DNA, RNA and protein in Escherichia coli cells, the DNA synthesis was found to be preferentially inhibited at lower concentrations of showdomycin. At such lower concentrations of this antibiotic, serious decreases in the synthesis of deoxycytidine phosphates and in de novo synthesis of deoxythymidine phosphates were found in parallel with the decrease in the synthesis of DNA, although the syntheses of other pyrimidine nucleotides were not significantly diminished. The salvage synthesis of deoxythymidine phosphates was very resistant to this antibiotic. The inhibitory action of this antibiotic on DNA synthesis could be reversed by the concomitant addition of a thiol compound or a nucleoside. When a nucleoside was added after the completion of the inhibition by showdomycin, the recovery of the DNA synthesis from the inhibition was detected only after the recovery of the syntheses of pyrimidine ribotides, pyrimidine deoxyribotides and RNA have become distinct.