Evaluation of purine-nucleoside degrading ability and in vivo uric acid lowering of Streptococcus thermophilus IDCC 2201, a novel antiuricemia strain.

This study evaluated 15 lactic acid bacteria with a focus on their ability to degrade inosine and hypo-xanthine-which are the intermediates in purine metabolism-for the management of hyperuricemia and gout. After a preliminary screening based on HPLC, Lactiplantibacillus plantarum CR1 and Lactiplantibacillus pentosus GZ1 were found to have the highest nucleoside degrading rates, and they were therefore selected for further characterization. S. thermophilus IDCC 2201, which possessed the hpt gene encoding hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and exhibited purine degradation, was also selected for further characterization. These three selected strains were examined in terms of their probiotic effect on lowering serum uric acid in a Sprague-Dawley (SD) rat model of potassium oxonate (PO)-induced hyperuricemia. Among these three strains, the level of serum uric acid was most reduced by S. thermophilus IDCC 2201 (p < 0.05). Further, analysis of the microbiome showed that administration of S. thermophlilus IDCC 2201 led to a significant difference in gut microbiota composition compared to that in the group administered with PO-induced hyperuricemia. Moreover, intestinal short-chain fatty acids (SCFAs) were found to be significantly increased. Altogether, the results of this work indicate that S. thermophilus IDCC 2201 lowers uric acid levels by degrading purine-nucleosides and also restores intestinal flora and SCFAs, ultimately suggesting that S. thermophilus IDCC 2201 is a promising candidate for use as an adjuvant treatment in patients with hyperuricemia.

Probing ligand selectivity in pathogens.

Why does protein kinase A respond to purine nucleosides in certain pathogens, but not to the cyclic nucleotides that activate this kinase in most other organisms?

Putative purine nucleoside interacting residues in the malaria parasite purine uptake transporter PfENT1 are critical for transporter function.

Malaria remains a major public health threat for billions of people worldwide. Infection with obligate intracellular, unicellular parasites from the genus Plasmodium causes malaria. Plasmodium falciparum causes the deadliest form of human malaria. Plasmodium parasites are purine auxotrophic. They rely on purine import from the host red blood cell cytoplasm via equilibrative nucleoside transporters to supply substrates to the purine salvage pathway. We previously developed a high throughput screening assay to identify inhibitors of the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Screening a small molecule library identified PfENT1 inhibitors that blocked proliferation of P. falciparum parasites in in vitro culture. The goal of the current work was to validate a high-resolution model of PfENT1 predicted by the AlphaFold protein structure prediction program. We superimposed the predicted PfENT1 structure on the human homologue structure, hENT1, and developed a structure-based sequence alignment. We mutated the residues in PfENT1 aligned with and flanking the residues in hENT1 that interact with the purine analog, nitrobenzylthioinosine (NBMPR). Mutation of the PfENT1 residues Q135, D287, and R291 that are predicted to form hydrogen bonds to purine nucleosides eliminated purine and pyrimidine transport function in various yeast-based growth and radiolabeled substrate uptake assays. Mutation of two flanking residues, W53 and S290, also resulted in inactive protein. Mutation of L50 that forms hydrophobic interactions with the purine nucleobase reduced transport function. Based on our results the AlphaFold predicted structure for PfENT1 may be useful in guiding medicinal chemistry efforts to improve the potency of our PfENT1 inhibitors.

Structural basis of the substrate recognition and inhibition mechanism of Plasmodium falciparum nucleoside transporter PfENT1.

By lacking de novo purine biosynthesis enzymes, Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates nucleoside uptake in the asexual blood stage. Specific inhibitors of PfENT1 prevent the proliferation of P. falciparum at submicromolar concentrations. However, the substrate recognition and inhibitory mechanism of PfENT1 are still elusive. Here, we report cryo-EM structures of PfENT1 in apo, inosine-bound, and inhibitor-bound states. Together with in vitro binding and uptake assays, we identify that inosine is the primary substrate of PfENT1 and that the inosine-binding site is located in the central cavity of PfENT1. The endofacial inhibitor GSK4 occupies the orthosteric site of PfENT1 and explores the allosteric site to block the conformational change of PfENT1. Furthermore, we propose a general "rocker switch" alternating access cycle for ENT transporters. Understanding the substrate recognition and inhibitory mechanisms of PfENT1 will greatly facilitate future efforts in the rational design of antimalarial drugs.

Purine Nucleosides Interfere with c-di-AMP Levels and Act as Adjuvants To Re-Sensitize MRSA To ß-Lactam Antibiotics.

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA ß-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased ß-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce ß-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on ß-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced ß-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as ß-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level ß-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the ß-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other ß-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for ß-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of ß-lactams against MRSA and potentially other AMR pathogens.

Adamantane-Substituted Purine Nucleosides: Synthesis, Host-Guest Complexes with ß-Cyclodextrin and Biological Activity.

Purine nucleosides represent an interesting group of nitrogen heterocycles, showing a wide range of biological effects. In this study, we designed and synthesized a series of 6,9-disubstituted and 2,6,9-trisubstituted purine ribonucleosides via consecutive nucleophilic aromatic substitution, glycosylation, and deprotection of the ribofuranose unit. We prepared eight new purine nucleosides bearing unique adamantylated aromatic amines at position 6. Additionally, the ability of the synthesized purine nucleosides to form stable host-guest complexes with ß-cyclodextrin (ß-CD) was confirmed using nuclear magnetic resonance (NMR) and mass spectrometry (ESI-MS) experiments. The in vitro antiproliferative activity of purine nucleosides and their equimolar mixtures with ß-CD was tested against two types of human tumor cell line. Six adamantane-based purine nucleosides showed an antiproliferative activity in the micromolar range. Moreover, their effect was only slightly suppressed by the presence of ß-CD, which was probably due to the competitive binding of the corresponding purine nucleoside inside the ß-CD cavity.

Concentrative Nucleoside Transporter, CNT, Results in Selective Toxicity of Toyocamycin against Candida albicans.

Toyocamycin (TM) is an adenosine-analog antibiotic isolated from Streptomyces toyocaensis. It inhibits Candida albicans, several plant fungal pathogens, and human cells, but many fungi, including Saccharomyces cerevisiae, are much less susceptible to TM. Aiming to clarify why TM and its analogs tubercidin and 5-iodotubercidin are active against C. albicans but not S. cerevisiae, this study focused on the absence of purine nucleoside transport activity from S. cerevisiae. When the concentrative nucleoside transporter (CNT) of C. albicans was expressed in S. cerevisiae, the recombinant strain became sensitive to TM and its analogs. The expression of C. albicans purine nucleoside permease in S. cerevisiae did not result in sensitivity to TM. Clustered regularly interspaced short palindromic repeat-mediated disruption of CNT was performed in C. albicans. The CNTΔ strain of C. albicans became insensitive to TM and its analogs. These data suggest that the toxicity of TM and its analogs toward C. albicans results from their transport via CNT. Interestingly, S. cerevisiae also became sensitive to TM and its analogs if human CNT3 was introduced into cells. These findings enhance our understanding of the mechanisms of action of adenosine analogs toward Candida pathogens and human cells. IMPORTANCE We investigated the mechanism of toxicity of TM and its analogs to C. albicans. Inspired by the effect of the copresence of TM and purine nucleosides on cell growth of C. albicans, we investigated the involvement of CNT in the toxicity mechanism by expressing CNT of C. albicans (CaCNT) in S. cerevisiae and deleting CaCNT in C. albicans. Our examinations clearly demonstrated that CaCNT is responsible for the toxicity of TM to C. albicans. S. cerevisiae expressing the human ortholog of CaCNT also became sensitive to TM and its analogs, and the order of effects of the TM analogs was a little different between CaCNT- and hCNT3-expressing S. cerevisiae. These findings are beneficial for an understanding of the mechanisms of action of adenosine analogs toward Candida pathogens and human cells and also the development of new antifungal drugs.

Lactobacillus gasseri PA-3 directly incorporates purine mononucleotides and utilizes them for growth.

Lactococcus lactis has been reported unable to directly incorporate mononucleotides but instead requires their external dephosphorylation by nucleotidases to the corresponding nucleosides prior to their incorporation. Although Lactobacillus gasseri PA-3 (PA-3), a strain of lactic acid bacteria, has been found to incorporate purine mononucleotides such as adenosine 5'-monophosphate (AMP), it remains unclear whether these bacteria directly incorporate these mononucleotides or incorporate them after dephosphorylation to the corresponding nucleosides. This study evaluated whether PA-3 incorporated radioactively-labeled mononucleotides in the presence or absence of the 5'-nucleotidase inhibitor α,ß-methylene ADP (APCP). PA-3 took up 14C-AMP in the presence of APCP, as well as incorporating 32P-AMP. Furthermore, radioactivity was detected in the RNA/DNA of bacterial cells cultured in the presence of 32P-AMP. Taken together, these findings indicated that PA-3 incorporated purine mononucleotides directly rather than after their dephosphorylation to purine nucleosides and that PA-3 utilizes these purine mononucleotides in the synthesis of RNA and DNA. Although additional studies are required to identify purine mononucleotide transporters in PA-3, this study is the first to show that some lactic acid bacteria directly incorporate purine mononucleotides and use them for growth.

The purine nucleoside phosphorylase pnp-1 regulates epithelial cell resistance to infection in C. elegans.

Intestinal epithelial cells are subject to attack by a diverse array of microbes, including intracellular as well as extracellular pathogens. While defense in epithelial cells can be triggered by pattern recognition receptor-mediated detection of microbe-associated molecular patterns, there is much to be learned about how they sense infection via perturbations of host physiology, which often occur during infection. A recently described host defense response in the nematode C. elegans called the Intracellular Pathogen Response (IPR) can be triggered by infection with diverse natural intracellular pathogens, as well as by perturbations to protein homeostasis. From a forward genetic screen, we identified the C. elegans ortholog of purine nucleoside phosphorylase pnp-1 as a negative regulator of IPR gene expression, as well as a negative regulator of genes induced by extracellular pathogens. Accordingly, pnp-1 mutants have resistance to both intracellular and extracellular pathogens. Metabolomics analysis indicates that C. elegans pnp-1 likely has enzymatic activity similar to its human ortholog, serving to convert purine nucleosides into free bases. Classic genetic studies have shown how mutations in human purine nucleoside phosphorylase cause immunodeficiency due to T-cell dysfunction. Here we show that C. elegans pnp-1 acts in intestinal epithelial cells to regulate defense. Altogether, these results indicate that perturbations in purine metabolism are likely monitored as a cue to promote defense against epithelial infection in the nematode C. elegans.

NUDT15: A bench to bedside success story.

Recently, the enzyme nudix hydrolase 15 (NUDT15) has been identified as an additional component of the thiopurine metabolism pathway. NUDT15 (also known as MTH2) catalyzes the dephosphorylation of 6-thioguanosine triphosphate (6-TGTP) and 6-thio-deoxyguanosine triphosphate (6-TdGTP), which is the active metabolite of thiopurine medications. Thiopurine compounds, which were first synthesized in the 1950s, are widely used in the treatment of childhood leukemia, inflammatory bowel disease, and autoimmune disorders. For many years, TPMT has been recognized as an enzyme that is involved in thiopurine metabolism, and interindividual variation in TPMT activity has been known to contribute to differences in risk of thiopurine toxicity. Genetic variation that leads to decreased NUDT15 activity has been recognized as an additional contributor, beyond TPMT, to thiopurine toxicity. In some populations, including Asian and Latino populations, NUDT15 genetic variants are more common than TPMT variants, making this a significant biomarker of toxicity. Clinical genetic testing is now available for a subset of NUDT15 variants, representing a remarkably fast translation from bench to bedside. This review will focus on NUDT15 - from discovery to clinical implementation.

Unnatural bases for recognition of noncoding nucleic acid interfaces.

The notion of using synthetic heterocycles instead of the native bases to interface with DNA and RNA has been explored for nearly 60 years. Unnatural bases compatible with the DNA/RNA coding interface have the potential to expand the genetic code and co-opt the machinery of biology to access new macromolecular function; accordingly, this body of research is core to synthetic biology. While much of the literature on artificial bases focuses on code expansion, there is a significant and growing effort on docking synthetic heterocycles to noncoding nucleic acid interfaces; this approach seeks to illuminate major processes of nucleic acids, including regulation of transcription, translation, transport, and transcript lifetimes. These major avenues of research at the coding and noncoding interfaces have in common fundamental principles in molecular recognition. Herein, we provide an overview of foundational literature in biophysics of base recognition and unnatural bases in coding to provide context for the developing area of targeting noncoding nucleic acid interfaces with synthetic bases, with a focus on systems developed through iterative design and biophysical study.

Purine metabolism regulates DNA repair and therapy resistance in glioblastoma.

Intratumoral genomic heterogeneity in glioblastoma (GBM) is a barrier to overcoming therapy resistance. Treatments that are effective independent of genotype are urgently needed. By correlating intracellular metabolite levels with radiation resistance across dozens of genomically-distinct models of GBM, we find that purine metabolites, especially guanylates, strongly correlate with radiation resistance. Inhibiting GTP synthesis radiosensitizes GBM cells and patient-derived neurospheres by impairing DNA repair. Likewise, administration of exogenous purine nucleosides protects sensitive GBM models from radiation by promoting DNA repair. Neither modulating pyrimidine metabolism nor purine salvage has similar effects. An FDA-approved inhibitor of GTP synthesis potentiates the effects of radiation in flank and orthotopic patient-derived xenograft models of GBM. High expression of the rate-limiting enzyme of de novo GTP synthesis is associated with shorter survival in GBM patients. These findings indicate that inhibiting purine synthesis may be a promising strategy to overcome therapy resistance in this genomically heterogeneous disease.

An Enzymatic Flow-Based Preparative Route to Vidarabine.

The bi-enzymatic synthesis of the antiviral drug vidarabine (arabinosyladenine, ara-A), catalyzed by uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), was re-designed under continuous-flow conditions. Glyoxyl-agarose and EziGTM1 (Opal) were used as immobilization carriers for carrying out this preparative biotransformation. Upon setting-up reaction parameters (substrate concentration and molar ratio, temperature, pressure, residence time), 1 g of vidarabine was obtained in 55% isolated yield and >99% purity by simply running the flow reactor for 1 week and then collecting (by filtration) the nucleoside precipitated out of the exiting flow. Taking into account the substrate specificity of CpUP and AhPNP, the results obtained pave the way to the use of the CpUP/AhPNP-based bioreactor for the preparation of other purine nucleosides.

Lambda-cyhalothrin exposure alters purine nucleotide hydrolysis and nucleotidase gene expression pattern in platelets and liver of rats.

Lambda-cyhalothrin (LCT) is a broad-spectrum pesticide widely used in agriculture throughout the world. This pesticide is considered a potential contaminant of surface and underground water as well as food, posing a risk to ecosystems and humans. In this sense, we decided to evaluate the activity of enzymes belonging to the purinergic system, which is linked with regulation of extracellular nucleotides and nucleosides, such as adenosine triphosphate (ATP) and adenosine (Ado) molecules involved in the regulation of inflammatory response. However, there are no data concerning the effects of LCT exposure on the purinergic system, where extracellular nucleotides act as signaling molecules. The aim of this study was to evaluate nucleotide hydrolysis by E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase), Ecto-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase), ecto-5'-nucleotidase and ecto-adenosine deaminase (E-ADA) in platelets and liver of adult rats on days 7, 30, 45 and 60 after daily gavage with 6.2 and 31.1 mg/kg bw of LCT. Gene expression patterns of NTPDases1-3 and 5'-nucleotidase were also determined in those tissues. In parallel, lambda-cyhalothrin metabolites [3-(2-chloro-3,3,3- trifluoroprop-1-enyl)-2,2-dimethyl-cyclopropane carboxylic acid (CFMP), 4-hydroxyphenoxybenzoic acid (4-OH-3-PBA), and 3-phenoxybenzoic acid (3-PBA)] were measured in plasma. Results showed that exposure rats to LCT caused a significant increase in the assessed enzymes activities. Gene expression pattern of ectonucleotidases further revealed a significant increase in E-NTPDase1, E-NTPDase2, and E-NTPDase3 mRNA levels after LCT administration at all times. A dose-dependent increase in LCT metabolite levels was also observed but there no significant variations in levels from weeks to week, suggesting steady-steady equilibrium. Correlation analyses revealed that LCT metabolites in the liver and plasma were positively correlated with the adenine nucleotides hydrolyzing enzyme, E-ADA and E-NPP activities in platelets and liver of rats exposed to lambda-cyhalothin. Our results show that LCT and its metabolites may affect purinergic enzymatic cascade and cause alterations in energy metabolism.

The clinical application of purine nucleosides as biomarkers of tissue Ischemia and hypoxia in humans in vivo.

During periods of ischemia and hypoxia, intracellular adenosine triphosphate stores are rapidly depleted. Its metabolism results in release of purine nucleosides into the systemic circulation. While the potential of purine nucleosides as a biomarker of ischemia has long been recognized, this has been limited by their complex physiological role and inherent instability leading to problematic sampling and prolonged, complex analysis procedures. Purine release has been demonstrated from cerebral tissue in patients undergoing carotid endarterectomy and patients presenting to hospital with stroke and transient ischemic attack. Rises in purine nucleosides have also been demonstrated in patients with angina and myocardial infarction, during systemic hypoxia, exercise, in patients with peripheral arterial disease and during surgery. This article reviews purine nucleoside production in ischemia, the development of purine analysis technology and details results of the studies investigating purine nucleosides as a biomarker of ischemia with suggestions for areas of future research.

Amino acid-linked platinum(II) compounds: non-canonical nucleoside preferences and influence on glycosidic bond stabilities.

Nucleobases serve as ideal targets where drugs bind and exert their anticancer activities. Cisplatin (cisPt) preferentially coordinates to 2'-deoxyguanosine (dGuo) residues within DNA. The dGuo adducts that are formed alter the DNA structure, contributing to inhibition of function and ultimately cancer cell death. Despite its success as an anticancer drug, cisPt has a number of drawbacks that reduce its efficacy, including repair of adducts and drug resistance. Some approaches to overcome this problem involve development of compounds that coordinate to other purine nucleobases, including those found in RNA. In this work, amino acid-linked platinum(II) (AAPt) compounds of alanine and ornithine (AlaPt and OrnPt, respectively) were studied. Their reactivity preferences for DNA and RNA purine nucleosides (i.e., 2'-deoxyadenosine (dAdo), adenosine (Ado), dGuo, and guanosine (Guo)) were determined. The chosen compounds form predominantly monofunctional adducts by reacting at the N1, N3, or N7 positions of purine nucleobases. In addition, features of AAPt compounds that impact the glycosidic bond stability of Ado residues were explored. The glycosidic bond cleavage is activated differentially for AlaPt-Ado and OrnPt-Ado isomers. Formation of unique adducts at non-canonical residues and subsequent destabilization of the glycosidic bonds are important features that could circumvent platinum-based drug resistance.

Fructose-1,6-Bisphosphate Protects Hippocampal Rat Slices from NMDA Excitotoxicity.

Effects of fructose 1,6-bisphosphate (F-1,6-P2) towards N-methyl-d-aspartate NMDA excitotoxicity were evaluated in rat organotypic hippocampal brain slice cultures (OHSC) challenged for 3 h with 30 µM NMDA, followed by incubations (24, 48, and 72 h) without (controls) and with F-1,6-P2 (0.5, 1 or 1.5 mM). At each time, cell necrosis was determined by measuring LDH in the medium. Energy metabolism was evaluated by measuring ATP, GTP, ADP, AMP, and ATP catabolites (nucleosides and oxypurines) in deproteinized OHSC extracts. Gene expressions of phosphofructokinase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase were also measured. F-1,6-P2 dose-dependently decreased NMDA excitotoxicity, abolishing cell necrosis at the highest concentration tested (1.5 mM). Additionally, F-1,6-P2 attenuated cell energy imbalance caused by NMDA, ameliorating the mitochondrial phosphorylating capacity (increase in ATP/ADP ratio) Metabolism normalization occurred when using 1.5 mM F-1,6-P2. Remarkable increase in expressions of phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase (up to 25 times over the values of controls) was also observed. Since this phenomenon was recorded even in OHSC treated with F-1,6-P2 with no prior challenge with NMDA, it is highly conceivable that F-1,6-P2 can enter into intact cerebral cells producing significant benefits on energy metabolism. These effects are possibly mediated by changes occurring at the gene level, thus opening new perspectives for F-1,6-P2 application as a useful adjuvant to rescue mitochondrial metabolism of cerebral cells under stressing conditions.

Efficient Synthesis of Purine Nucleoside Analogs by a New Trimeric Purine Nucleoside Phosphorylase from Aneurinibacillus migulanus AM007.

Purine nucleoside phosphorylases (PNPs) are promising biocatalysts for the synthesis of purine nucleoside analogs. Although a number of PNPs have been reported, the development of highly efficient enzymes for industrial applications is still in high demand. Herein, a new trimeric purine nucleoside phosphorylase (AmPNP) from Aneurinibacillus migulanus AM007 was cloned and heterologously expressed in Escherichia coli BL21(DE3). The AmPNP showed good thermostability and a broad range of pH stability. The enzyme was thermostable below 55 °C for 12 h (retaining nearly 100% of its initial activity), and retained nearly 100% of the initial activity in alkaline buffer systems (pH 7.0-9.0) at 60 °C for 2 h. Then, a one-pot, two-enzyme mode of transglycosylation reaction was successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP) derived from Brevibacillus borstelensis LK01 and AmPNP for the production of purine nucleoside analogs. Conversions of 2,6-diaminopurine ribonucleoside (1), 2-amino-6-chloropurine ribonucleoside (2), and 6-thioguanine ribonucleoside (3) synthesized still reached >90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of BbPyNP: AmPNP (2:20 µg/mL). Thus, the new trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.

Synthesis of Cytokinins via Enzymatic Arsenolysis of Purine Nucleosides.

This unit describes an effective method for the preparation of natural cytokinins and their synthetic derivatives based on enzymatic cleavage of the N-glycosidic bond of N6 -substituted adenosine or O6 -substituted inosine derivatives in the presence of purine nucleoside phosphorylase (PNP) and Na2 HAsO4 . The arsenolysis reaction is irreversible due to the hydrolysis of the resulting α-D-ribose-1-arsenate. As a result, the desired products are formed in near-quantitative yields, as indicated by high-performance liquid chromatography (HPLC) analysis, and can easily be isolated. In the strategy used here, the ribose residue acts as a protective group. © 2018 by John Wiley & Sons, Inc.

Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates.

We synthesized a small library of eighteen 5-substituted pyrimidine or 7-substituted 7-deazapurine nucleoside triphosphates bearing methyl, ethynyl, phenyl, benzofuryl or dibenzofuryl groups through cross-coupling reactions of nucleosides followed by triphosphorylation or through direct cross-coupling reactions of halogenated nucleoside triphosphates. We systematically studied the influence of the modification on the efficiency of T7 RNA polymerase catalyzed synthesis of modified RNA and found that modified ATP, UTP and CTP analogues bearing smaller modifications were good substrates and building blocks for the RNA synthesis even in difficult sequences incorporating multiple modified nucleotides. Bulky dibenzofuryl derivatives of ATP and GTP were not substrates for the RNA polymerase. In the case of modified GTP analogues, a modified procedure using a special promoter and GMP as initiator needed to be used to obtain efficient RNA synthesis. The T7 RNA polymerase synthesis of modified RNA can be very efficiently used for synthesis of modified RNA but the method has constraints in the sequence of the first three nucleotides of the transcript, which must contain a non-modified G in the +1 position.