Methyltransferases: Functions and Applications.

In this review the current state-of-the-art of S-adenosylmethionine (SAM)-dependent methyltransferases and SAM are evaluated. Their structural classification and diversity is introduced and key mechanistic aspects presented which are then detailed further. Then, catalytic SAM as a target for drugs, and approaches to utilise SAM as a cofactor in synthesis are introduced with different supply and regeneration approaches evaluated. The use of SAM analogues are also described. Finally O-, N-, C- and S-MTs, their synthetic applications and potential for compound diversification is given.

Core modified 8-17 DNAzymes with 2'-deoxy-2'-C-methylpyrimidine nucleosides.

The catalytic core of an 8-17 DNAzyme directed against STAT 3 was modified using (2'R) and (2'S) 2'-deoxy-2'-C-methyluridine and cytidine. While 2'-deoxy-2'-C-methyluridine significantly diminished the catalytic activity, 2'-deoxy-2'-C-methylcytidine replacement was better accepted, being the kact of modified DNAzymes at 8- and 11-positions comparable to the non-modified one. When 2'-O-methyl and phosphorothioate nucleotides were tested in the binding arms together with core modified DNAzymes the kcat was affected in a non predictable way, emphasizing the fact that both chemical substitutions should be considered globally. Finally, 2'-deoxy-2'-C-methyl modified DNAzymes stability was assayed finding that the double 2'-C-methyl modification in the catalytic core enhanced 70% the stability against a T47D cell lysate compared to a non-modified control.

Citrobacter koseri immobilized on agarose beads for nucleoside synthesis: a potential biocatalyst for preparative applications.

The biocatalyzed synthesis of purine nucleosides and their analogs is a case widely studied due to the high pharmaceutical interest of these compounds, providing the whole-cell biocatalysts, a useful tool for this purpose. Vidarabine and fludarabine are commercial examples of expensive bioactive nucleosides that can be prepared using a microbial transglycosylation approach. Citrobacter koseri whole-cells immobilized on agarose beads proved to be an interesting option to transform this biotransformation in a preparative process. The entrapment matrix provided a useful and resistant multipurpose biocatalyst regarding its stability, mechanical strength, microbial viability and reuse. Immobilized biocatalyst retained the initial activity for up to 1 year storage and after 10 years, the biocatalyst did not show cell leaking and still exhibited residual activity. In addition, the biocatalyst could be reused in batch 68 times keeping up to 50% of the initial biocatalytic activity and for at least 124 h in a continuous process.

siRNA Modified with 2'-Deoxy-2'-C-methylpyrimidine Nucleosides.

(2'S)-2'-Deoxy-2'-C-methyluridine and (2'R)-2'-deoxy-2'-C-methyluridine were incorporated in the 3'-overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2'S)-2'-deoxy-2'-C-methylcytidine was incorporated in the 6-position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC50 values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3'-overhang region by (2'S)- or (2'R)-2'-deoxy-2'-C-methyluridine, reducing IC50 to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.

Stabilization of Telomeric I-Motif Structures by (2'S)-2'-Deoxy-2'-C-Methylcytidine Residues.

G-quadruplexes and i-motifs are tetraplex structures present in telomeres and the promoter regions of oncogenes. The possibility of producing nanodevices with pH-sensitive functions has triggered interest in modified oligonucleotides with improved structural properties. We synthesized C-rich oligonucleotides carrying conformationally restricted (2'S)-2'-deoxy-2'-C-methyl-cytidine units. The effect of this modified nucleoside on the stability of intramolecular i-motifs from the vertebrate telomere was investigated by UV, CD, and NMR spectroscopy. The replacement of selected positions of the C-core with C-modified residues induced the formation of stable intercalated tetraplexes at near-neutral pH. This study demonstrates the possibility of enhancing the stability of the i-motif by chemical modification.

A comparative study on phosphotransferase activity of acid phosphatases from Raoultella planticola and Enterobacter aerogenes on nucleosides, sugars, and related compounds.

Natural and modified nucleoside-5'-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.

Influence of conformationally restricted pyrimidines on the activity of 10-23 DNAzymes.

The catalytic core of a 10-23 DNAzyme was modified introducing conformationally restricted nucleosides such as (2'R)-, (2'S)-2'-deoxy-2'-C-methyluridine, (2'R)-, (2'S)-2'-deoxy-2'-C-methylcytidine, 2,2'-anhydrouridine and LNA-C, in one, two or three positions. Catalytic activities under pseudo first order conditions were compared at different Mg(2+) concentrations using a short RNA substrate. At low Mg(2+) concentrations, triple modified DNAzymes with similar kinetic performance to that displayed by the non-modified control were identified. In the search for a partial explanation of the obtained results, in silico studies were carried out in order to explore the conformational behavior of 2'-deoxy-2'-C-methylpyrimidines in the context of a loop structure, suggesting that at least partial flexibility is needed for the maintenance of activity. Finally, the modified 2'-C-methyl DNAzyme activity was tested assessing the inhibition of Stat3 expression and the decrease in cell proliferation using the human breast cancer cell line T47D.

Streptomyces griseus: A new biocatalyst with N-oxygenase activity.

Aromatic nitro compounds are key building blocks for many industrial syntheses and are also components of explosives, drugs and pesticides. Due to the environmentally unfriendly experimental conditions involved in their chemical syntheses, industrial processes would benefit from the use of biocatalysts. Among potentially useful enzymes, N-oxygenases, whose role is to oxygenate primary amines, are becoming relevant. These enzymes are involved in different secondary metabolic pathways in Streptomyces and in few other bacteria, forming part of the enzyme pools implicated in antibiotic synthesis. In this work, a group of Streptomyces strains, whose biomass was obtained from simple and novel culture media, were identified as new sources of N-oxygenase activity. Furthermore, the use of unspecific metabolic stimulation strategies allowed substantial improvements in the activity of whole cells as biocatalysts. It is remarkable the 6 to 50-fold increase in nitro compound yields compared to the biotransformation under standard conditions when Streptomyces griseus was the biocatalyst. In addition, biocatalyst substrate acceptance was studied in order to determine the biocatalytic potential of this enzyme.

N-oxygenation of amino compounds: Early stages in its application to the biocatalyzed preparation of bioactive compounds.

Among the compounds that contain unusual functional groups, nitro is perhaps one of the most interesting due to the valuable properties it confers on pharmaceuticals and explosives. Traditional chemistry has for many years used environmentally unfriendly strategies; in contrast, the biocatalyzed production of this type of products offers a promising alternative. The small family of enzymes formed by N-oxygenases allows the conversion of an amino group to a nitro through the sequential addition of oxygen. These enzymes also make it possible to obtain other less oxidized N-O functions, such as hydroxylamine or nitroso, present in intermediate or final products. The current substrates on which these enzymes are reported to work encompass a few aromatic molecules and sugars. The unique characteristics of N-oxygenases and the great economic value of the products that they could generate, place them in a position of very high scientific and industrial interest. The most important and best studied N-oxygenases will be presented here.

10-23 DNAzyme modified with (2'R)- and (2'S)-2'-deoxy-2'-C-methyluridine in the catalytic core.

The catalytic core of a 10-23 DNAzyme was modified using (2'R), (2'S)-2'-deoxy-2'-C-methyluridine and LNA-T. Catalytic activities under pseudo first order conditions were compared at different Mg(2+) concentrations, indicating that certain 2'-C-methyl modified DNAzymes have significant activities. Resistance against MCF-7 cell lysate and endonuclease RQ1 was also measured, showing that the introduction of 2'-C-methyl-2'-deoxynucleosides increased the stability.

2-C-methyluridine modified hammerhead ribozyme against the estrogen receptor.

A new synthesis of 2'-C-methyluridine phosphoramidite is presented. Special emphasis is dedicated to the improvement of the protection of the tertiary 2'-hydroxyl group. Comparison to previous protecting strategies and analysis of stability under 5'-DMTr removing conditions are discussed. The synthetic incorporation of this modified nucleoside into the catalytic core of a hammerhead ribozyme against the estrogen receptor alpha protein (ER-alpha), and transfection experiments in MCF-7 cell line are also presented.

Synthesis of 9-beta-d-arabinofuranosylguanine by combined use of two whole cell biocatalysts.

Unlike the preparation of other purine nucleosides, transglycosylation from a pyrimidine nucleoside and guanine is difficult because of the low solubility of this base. Thus, another strategy, based on the coupled action of two whole cell biocatalyzed reactions, transglycosylation and deamination, was used. Enterobacter gergoviae and Arthrobacter oxydans were employed to synthesize 9-beta-d-arabinofuranosylguanine (AraG), an efficient anti leukemic drug.

Arthrobacter oxydans as a biocatalyst for purine deamination.

Deaminases are enzymes that catalyze the hydrolysis of amino groups of nucleosides or their bases. Because these enzymes play important roles in nucleotide metabolism, they are relevant targets in anticancer and antibacterial therapies. Mammalian deaminases are commercially available but the use of bacterial whole cells, especially as biocatalysts, is continuously growing because of their economical benefits. Moreover, deaminases are useful for the preparative chemoenzymatic transformation of nucleoside and base analogues into a variety of derivatives. The purine deaminase activities of Arthrobacter oxydans, a gram-positive bacterium utilized widely in bioremediation, were studied. The presence of adenosine, adenine and guanine deaminases was demonstrated and some purine bases and nucleosides were analyzed as substrates. Using A. oxydans whole cells as the biocatalyst, different purine compounds such as the anti-HIV, 2',3'-dideoxyinosine (73%, 2 h) were obtained.

An enzymatic alternative for the synthesis of nucleoside 5'-monophosphates.

A new procedure was carried out for the synthesis of nucleoside 5'-monophosphates, involving the use of two enzymes. The first step applied phospholipase D from Streptomyces netropsis and phosphatidylcholine as phosphatidyl donor, to give 5'-(3-sn-phosphatidyl) nucleosides (C, U, A, I). These were selectively hydrolysed in the second step by the action of phospholipase C from Bacillus cereus to produce the respective 5'-nucleotides. Application of this methodology on a preparative scale conducted to 5'-adenosine monophosphate in 63% overall yield from adenosine. The regioselectivity of these enzymes avoids protection steps, the overall synthesis is performed under mild reaction conditions and product isolation is easily achieved.

Whole cell biocatalysts for the preparation of nucleosides and their derivatives.

Nucleosides constitute an extensive group of natural and chemically modified compounds that display a wide range of structures and activities. Different biocatalysts have been developed for their preparation, but the choice of commercially available enzymes is limited. Therefore, the search of new biocatalysts is particularly attractive. In this sense, microorganisms are a vast source of enzymatic diversity that can be directly used as a whole cell biocatalysts providing a potential cheaper and suitable route for industrial applications. METHODS: This work makes particular emphasis on the following methods: the biocatalyzed whole cell synthesis of nucleosides mediated by phosphorylases, key biocatalyzed steps involved in other chemoenzymatic routes to prepare nucleoside analogues and the transformation of nucleosides in derivatives with particular properties. RESULTS: The literature covered in this work confirms that biocatalytic procedures that make use of whole cell systems can be successfully applied to obtain a wide variety of nucleoside analogues and their derivatives, providing alternative and complementary routes to traditional chemistry. The direct use of microbial whole cells as biocatalysts affords competitive results since it avoids the cumbersome procedures involved in enzyme isolation and facilitates multienzymatic processes. These biocatalysts also maintain the enzymes in their natural environment, protecting their activities from reaction conditions. CONCLUSION: Although the information presented herein shows that these methodologies have reached a high degree of development, it is expected that future contributions of protein engineering and nucleoside metabolism knowledge, among other disciplines, will expand the already wide range of applications in nucleoside chemistry of whole cell biocatalysis.

Modified Nucleoside Triphosphates for In-vitro Selection Techniques.

The development of SELEX (Selective Enhancement of Ligands by Exponential Enrichment) provides a powerful tool for the search of functional oligonucleotides with the ability to bind ligands with high affinity and selectivity (aptamers) and for the discovery of nucleic acid sequences with diverse enzymatic activities (ribozymes and DNAzymes). This technique has been extensively applied to the selection of natural DNA or RNA molecules but, in order to improve chemical and structural diversity as well as for particular applications where further chemical or biological stability is necessary, the extension of this strategy to modified oligonucleotides is desirable. Taking into account these needs, this review intends to collect the research carried out during the past years, focusing mainly on the use of modified nucleotides in SELEX and the development of mutant enzymes for broadening nucleoside triphosphates acceptance. In addition, comments regarding the synthesis of modified nucleoside triphosphate will be briefly discussed.

New and highly active microbial phosphotriesterase sources.

Many toxic insecticides used worldwide as well as some chemical warfare agents are phosphotriester derivatives. Therefore, detoxification of organophosphorus compounds has become the subject of many studies and in particular bioremediation, based on the phosphotriesterase catalysed hydrolysis of these compounds, has shown to be an effective and ecological methodology. In order to identify new bacterial phosphotriesterases, a simple and sensitive fluorimetric screening method on solid media was employed that allowed the selection of six strains with phosphotriesterase activity. Since pH and temperature are important parameters for bioremediation of contaminated soils and waters, the influence of these variables on the rate of the enzymatic hydrolysis was assessed. This study afforded notable results, being the most remarkable one the increased activity exhibited by Nocardia asteroides and Streptomyces setonii strains at 50°C, 7 and 30 times higher than at 30°C, respectively. Compared with the results obtained with Brevundimonas diminuta, whose activity is usually considered as reference, an increase of 26 and 75 times is observed, respectively.

Activity and stability of hammerhead ribozymes containing 2'-C-methyluridine: a new RNA mimic.

We propose 2'-C-methylnucleotides as a new class of 2'-modified RNA mimics. These analogues are expected to provide 2'-OH groups capable of reproducing the interactions observed in natural RNA and, due to the presence of the Me group, to possess increased stability towards nucleases. In this work, we investigate the catalytic activity and nuclease resistance of hammerhead ribozymes carrying 2'-C-methyluridines in positions 4 and 7 of the catalytic core. We describe the in vitro activity of these chimeric molecules and their stability in cell lysate, fetal calf serum, and cell culture medium. The data show that, when only position 4 is modified, activity decreases twofold; while, when both 4 and 7 positions are substituted, a sevenfold drop in activity is observed. Regarding biological stability, the main increase of the half-life time is observed when position 7 is modified. These results suggest that 2'-C-methylnucleotides may be useful in the design of chemically synthesized RNA mimics with biological activity.

Exploring the Use of a Guanine-Rich Catalytic DNA for Sulfoxide Preparation.

A guanine-rich DNA oligonucleotide complexed with hemin was used to catalyze controlled oxygen transfer reactions to different sulfides for sulfoxide preparation in the presence of H2O2. Comparable activities were obtained when using fully modified L-DNA. In addition, oligonucleotide immobilization led to an active catalyst which could be successfully recovered and reused without loss of activity.

Biocatalytic approaches applied to the synthesis of nucleoside prodrugs.

Nucleosides are valuable bioactive molecules, which display antiviral and antitumour activities. Diverse types of prodrugs are designed to enhance their therapeutic efficacy, however this strategy faces the troublesome selectivity issues of nucleoside chemistry. In this context, the aim of this review is to give an overview of the opportunities provided by biocatalytic procedures in the preparation of nucleoside prodrugs. The potential of biocatalysis in this research area will be presented through examples covering the different types of nucleoside prodrugs: nucleoside analogues as prodrugs, nucleoside lipophilic prodrugs and nucleoside hydrophilic prodrugs.