Enzymatic synthesis and phosphorolysis of 4(2)-thioxo- and 6(5)-azapyrimidine nucleosides by E. coli nucleoside phosphorylases

The trans-2-deoxyribosylation of 4-thiouracil (4SUra) and 2-thiouracil (2SUra), as well as 6-azauracil, 6-azathymine and 6-aza-2-thiothymine was studied using dG and E. coli purine nucleoside phosphorylase (PNP) for the in situ generation of 2-deoxy-α-D-ribofuranose-1-phosphate (dRib-1P) followed by its coupling with the bases catalyzed by either E. coli thymidine (TP) or uridine (UP) phosphorylases. 4SUra revealed satisfactory substrate activity for UP and, unexpectedly, complete inertness for TP; no formation of 2’-deoxy-2-thiouridine (2SUd) was observed under analogous reaction conditions in the presence of UP and TP. On the contrary, 2SU, 2SUd, 4STd and 2STd are good substrates for both UP and TP; moreover, 2SU, 4STd and 2’-deoxy-5-azacytidine (Decitabine) are substrates for PNP and the phosphorolysis of the latter is reversible. Condensation of 2SUra and 5-azacytosine with dRib-1P (Ba salt) catalyzed by the accordant UP and PNP in Tris∙HCl buffer gave 2SUd and 2’-deoxy-5-azacytidine in 27% and 15% yields, respectively. 6-Azauracil and 6-azathymine showed good substrate properties for both TP and UP, whereas only TP recognizes 2-thio-6-azathymine as a substrate. 5-Phenyl and 5-tert-butyl derivatives of 6-azauracil and its 2-thioxo derivative were tested as substrates for UP and TP, and only 5-phenyl- and 5-tert-butyl-6-azauracils displayed very low substrate activity. The role of structural peculiarities and electronic properties in the substrate recognition by E. coli nucleoside phosphorylases is discussed.

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Tricyclic Nucleobase Analogs and Their Ribosides as Substrates and Inhibitors of Purine-Nucleoside Phosphorylases III. Aminopurine Derivatives

Molecules ◽

10.3390/molecules25030681 ◽

2020 ◽

Vol 25 (3) ◽

pp. 681 ◽

Cited By ~ 2

Author(s):

Alicja Stachelska-Wierzchowska ◽

Jacek Wierzchowski ◽

Michał Górka ◽

Agnieszka Bzowska ◽

Ryszard Stolarski ◽

...

Keyword(s):

Enzymatic Synthesis ◽

Purine Nucleoside Phosphorylase ◽

Major Product ◽

Purine Nucleoside ◽

Fluorescent Product ◽

E Coli ◽

Purine Analogs ◽

Mammalian Enzyme ◽

Nucleoside Phosphorylases ◽

Derivatives Of

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2-β-d-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2-β-d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9-β -d-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.

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Facile synthesis of 1-alkoxy-1H-benzo- and 7-azabenzotriazoles from peptide coupling agents, mechanistic studies, and synthetic applications

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.10.200 ◽

2014 ◽

Vol 10 ◽

pp. 1919-1932 ◽

Cited By ~ 5

Author(s):

Mahesh K Lakshman ◽

Manish K Singh ◽

Mukesh Kumar ◽

Raghu Ram Chamala ◽

Vijayender R Yedulla ◽

...

Keyword(s):

Amide Bond ◽

Coupling Agents ◽

Cyclic Ketones ◽

Substitution Reactions ◽

Facile Synthesis ◽

Reaction Conditions ◽

Peptide Coupling ◽

Acyclic Nucleoside ◽

Derivatives Of

(1H-Benzo[d][1,2,3]triazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1H-benzo[d][1,2,3]triazol-1-yl 4-methylbenzenesulfonate (Bt-OTs), and 3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl 4-methylbenzenesulfonate (At-OTs) are classically utilized in peptide synthesis for amide-bond formation. However, a previously undescribed reaction of these compounds with alcohols in the presence of a base, leads to 1-alkoxy-1H-benzo- (Bt-OR) and 7-azabenzotriazoles (At-OR). Although BOP undergoes reactions with alcohols to furnish 1-alkoxy-1H-benzotriazoles, Bt-OTs proved to be superior. Both, primary and secondary alcohols undergo reaction under generally mild reaction conditions. Correspondingly, 1-alkoxy-1H-7-azabenzotriazoles were synthesized from At-OTs. Mechanistically, there are three pathways by which these peptide-coupling agents can react with alcohols. From 31P{1H}, [18O]-labeling, and other chemical experiments, phosphonium and tosylate derivatives of alcohols seem to be intermediates. These then react with BtO− and AtO− produced in situ. In order to demonstrate broader utility, this novel reaction has been used to prepare a series of acyclic nucleoside-like compounds. Because BtO− is a nucleofuge, several Bt-OCH2Ar substrates have been evaluated in nucleophilic substitution reactions. Finally, the possible formation of Pd π–allyl complexes by departure of BtO− has been queried. Thus, alpha-allylation of three cyclic ketones was evaluated with 1-(cinnamyloxy)-1H-benzo[d][1,2,3]triazole, via in situ formation of pyrrolidine enamines and Pd catalysis.

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Role of ionization of the phosphate cosubstrate on phosphorolysis by purine nucleoside phosphorylase (PNP) of bacterial (E. coli) and mammalian (human) origin

European Biophysics Journal ◽

10.1007/s00249-007-0205-8 ◽

2007 ◽

Vol 37 (2) ◽

pp. 153-164 ◽

Cited By ~ 8

Author(s):

Anna Modrak-Wójcik ◽

Aneta Kirilenko ◽

David Shugar ◽

Borys Kierdaszuk

Keyword(s):

Purine Nucleoside Phosphorylase ◽

Purine Nucleoside ◽

Human Origin ◽

Nucleoside Phosphorylase ◽

E Coli

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The chemoenzymatic synthesis of clofarabine and related 2′-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.10.173 ◽

2014 ◽

Vol 10 ◽

pp. 1657-1669 ◽

Cited By ~ 17

Author(s):

Ilja V Fateev ◽

Konstantin V Antonov ◽

Irina D Konstantinova ◽

Tatyana I Muravyova ◽

Frank Seela ◽

...

Keyword(s):

Electronic Structure ◽

Chemical Synthesis ◽

Purine Nucleoside Phosphorylase ◽

Substrate Recognition ◽

Chemoenzymatic Synthesis ◽

Similar Reaction ◽

One Pot ◽

E Coli ◽

Enzymatic Transformation ◽

Nucleoside Phosphorylases

Two approaches to the synthesis of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (1, clofarabine) were studied. The first approach consists in the chemical synthesis of 2-deoxy-2-fluoro-α-D-arabinofuranose-1-phosphate (12a, 2FAra-1P) via three step conversion of 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinofuranose (9) into the phosphate 12a without isolation of intermediary products. Condensation of 12a with 2-chloroadenine catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of clofarabine in 67% yield. The reaction was also studied with a number of purine bases (2-aminoadenine and hypoxanthine), their analogues (5-aza-7-deazaguanine and 8-aza-7-deazahypoxanthine) and thymine. The results were compared with those of a similar reaction with α-D-arabinofuranose-1-phosphate (13a, Ara-1P). Differences of the reactivity of various substrates were analyzed by ab initio calculations in terms of the electronic structure (natural purines vs analogues) and stereochemical features (2FAra-1P vs Ara-1P) of the studied compounds to determine the substrate recognition by E. coli nucleoside phosphorylases. The second approach starts with the cascade one-pot enzymatic transformation of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a, followed by its condensation with 2-chloroadenine thereby affording clofarabine in ca. 48% yield in 24 h. The following recombinant E. coli enzymes catalyze the sequential conversion of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a: ribokinase (2-deoxy-2-fluoro-D-arabinofuranose-5-phosphate), phosphopentomutase (PPN; no 1,6-diphosphates of D-hexoses as co-factors required) (12a), and finally PNP. The substrate activities of D-arabinose, D-ribose and D-xylose in the similar cascade syntheses of the relevant 2-chloroadenine nucleosides were studied and compared with the activities of 2-deoxy-2-fluoro-D-arabinose. As expected, D-ribose exhibited the best substrate activity [90% yield of 2-chloroadenosine (8) in 30 min], D-arabinose reached an equilibrium at a concentration of ca. 1:1 of a starting base and the formed 2-chloro-9-(β-D-arabinofuranosyl)adenine (6) in 45 min, the formation of 2-chloro-9-(β-D-xylofuranosyl)adenine (7) proceeded very slowly attaining ca. 8% yield in 48 h.

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Fischer-Tropsch synthesis nanostructured catalysts: understanding structural characteristics and catalytic reaction

Nanotechnology Reviews ◽

10.1515/ntrev-2013-0025 ◽

2013 ◽

Vol 2 (5) ◽

pp. 547-576 ◽

Cited By ~ 10

Author(s):

Peng Zhai ◽

Geng Sun ◽

Qingjun Zhu ◽

Ding Ma

Keyword(s):

Structural Characteristics ◽

Nanostructured Catalysts ◽

Catalytic Performance ◽

Tropsch Synthesis ◽

Fischer Tropsch ◽

Reaction Conditions ◽

Catalyst Structure ◽

Fischer Tropsch Synthesis

AbstractOne key goal of heterogeneous catalysis study is to understand the correlation between the catalyst structure and its corresponding catalytic activity. In this review, we focus on recent strategies to synthesize well-defined Fischer-Tropsch synthesis (FTS) nanostructured catalysts and their catalytic performance in FTS. The development of those promising catalysts highlights the potentials of nanostructured materials to unravel the complex and dynamic reaction mechanism, particularly under the in situ reaction conditions. The crucial factors associated with the catalyst compositions and structures and their effects on the FTS activities are discussed with an emphasis on the role of theoretical modeling and experimental results.

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A Multi-Enzymatic Cascade Reaction for the Synthesis of Vidarabine 5′-Monophosphate

Catalysts ◽

10.3390/catal10010060 ◽

2020 ◽

Vol 10 (1) ◽

pp. 60 ◽

Cited By ~ 2

Author(s):

Marina Simona Robescu ◽

Immacolata Serra ◽

Marco Terreni ◽

Daniela Ubiali ◽

Teodora Bavaro

Keyword(s):

Purine Nucleoside Phosphorylase ◽

Enzymatic Reaction ◽

Antiviral Drug ◽

Product Formation ◽

Reaction Conditions ◽

One Pot ◽

Medium Temperature ◽

Phosphate Donor ◽

The One ◽

Nucleoside Phosphorylases

We here described a three-step multi-enzymatic reaction for the one-pot synthesis of vidarabine 5′-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade), and adenosine triphosphate (ATP) as precursors. To this aim, three enzymes involved in the biosynthesis of nucleosides and nucleotides were used in a cascade mode after immobilization: uridine phosphorylase from Clostridium perfringens (CpUP), a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), and deoxyadenosine kinase from Dictyostelium discoideum (DddAK). Specifically, CpUP catalyzes the phosphorolysis of araU thus generating uracil and α-d-arabinose-1-phosphate. AhPNP catalyzes the coupling between this latter compound and Ade to form araA (vidarabine). This nucleoside becomes the substrate of DddAK, which produces the 5′-mononucleotide counterpart (araA-MP) using ATP as the phosphate donor. Reaction conditions (i.e., medium, temperature, immobilization carriers) and biocatalyst stability have been balanced to achieve the highest conversion of vidarabine 5′-monophosphate (≥95.5%). The combination of the nucleoside phosphorylases twosome with deoxyadenosine kinase in a one-pot cascade allowed (i) a complete shift in the equilibrium-controlled synthesis of the nucleoside towards the product formation; and (ii) to overcome the solubility constraints of araA in aqueous medium, thus providing a new route to the highly productive synthesis of araA-MP.

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Iminopyridine Ni(II) Catalysts Affording Oily Hyperbranched Oligoethylenes and/or Crystalline Polyethylenes Depending on the Reaction Conditions: Possible Role of In Situ Catalyst Structure Modifications

10.20944/preprints202103.0600.v1 ◽

2021 ◽

Author(s):

Ilaria D'Auria ◽

Zeinab Saki ◽

Claudio Pellecchia

Keyword(s):

Ethylene Polymerization ◽

Active Species ◽

Polymerization Catalysts ◽

Reaction Conditions ◽

Catalyst Structure ◽

Variable Content ◽

Peculiar Behavior ◽

Number Of Branches

Nickel-based ethylene polymerization catalysts have unique features, being able to produce macromolecules with a variable content of branches, resulting in polymers ranging from semicrystalline plastics to elastomers to hyperbranched amorphous waxes and oils. In addition to Brookhart's α-diimine catalysts, iminopyridine Ni(II) complexes are among the most investigated systems. We report that Ni(II) complexes bearing aryliminopyridine ligands with bulky substituents both at the imino moiety and in the 6-position of pyridine afford either hyperbranched low molecular weight polyethylene oils or prevailingly linear crystalline polyethylenes or both depending on the ligand structure and the reaction conditions. The formation of multiple active species in situ is suggested by analysis of the post-polymerization catalyst residues, showing the partial reduction of the imino function. Some related arylaminopyridine Ni(II) complexes were also synthesized and tested, showing a peculiar behavior, i.e. the number of branches of the produced polyethylenes increases while ethylene pressure increases.

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Understanding the Role of Agricultural Practices in the Potential Colonization and Contamination by Escherichia coli in the Rhizospheres of Fresh Produce

Journal of Food Protection ◽

10.4315/0362-028x-73.11.2001 ◽

2010 ◽

Vol 73 (11) ◽

pp. 2001-2009 ◽

Cited By ~ 13

Author(s):

MUSSIE Y. HABTESELASSIE ◽

MARIANNE BISCHOFF ◽

BRUCE APPLEGATE ◽

BRADLEY REUHS ◽

RONALD F. TURCO

Keyword(s):

Escherichia Coli ◽

Irrigation Water ◽

Bacterial Colonization ◽

Agricultural Practices ◽

E Coli ◽

Lettuce Seeds ◽

Growth System ◽

Low Levels

To better protect consumers from exposure to produce contaminated with Escherichia coli, the potential transfer of E. coli from manure or irrigation water to plants must be better understood. We used E. coli strains expressing bioluminescence (E. coli O157:H7 lux) or multiantibiotic resistance (E. coli2+) in this study. These marked strains enabled us to visualize in situ rhizosphere colonization and metabolic activity and to track the occurrence and survival of E. coli in soil, rhizosphere, and phyllosphere. When radish and lettuce seeds were treated with E. coli O157:H7 lux and grown in an agar-based growth system, rapid bacterial colonization of the germinating seedlings and high levels of microbial activity were seen. Introduction of E. coli2+ to soil via manure or via manure in irrigation water showed that E. coli could establish itself in the lettuce rhizosphere. Regardless of introduction method, 15 days subsequent to its establishment in the rhizosphere, E. coli2+ was detected on the phyllosphere of lettuce at an average number of 2.5 log CFU/g. When E. coli2+ was introduced 17 and 32 days postseeding to untreated soil (rather than the plant surface) via irrigation, it was detected at low levels (1.4 log CFU/g) on the lettuce phyllosphere 10 days later. While E. coli2+ persisted in the bulk and rhizosphere soil throughout the study period (day 41), it was not detected on the external portions of the phyllosphere after 27 days. Overall, we find that E. coli is mobile in the plant system and responds to the rhizosphere like other bacteria.

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The Large Resolvase TndX Is Required and Sufficient for Integration and Excision of Derivatives of the Novel Conjugative Transposon Tn5397

Journal of Bacteriology ◽

10.1128/jb.182.23.6577-6583.2000 ◽

2000 ◽

Vol 182 (23) ◽

pp. 6577-6583 ◽

Cited By ~ 43

Author(s):

Hongmei Wang ◽

Peter Mullany

Keyword(s):

Hot Spots ◽

Activity Analysis ◽

The Novel ◽

Conjugation System ◽

Conjugative Transposon ◽

Site Specific ◽

E Coli ◽

Derivatives Of ◽

Conserved Amino Acids

ABSTRACT Tn5397 is a novel conjugative transposon, originally isolated from Clostridium difficile. This element can transfer between C. difficile strains and to and fromBacillus subtilis. It encodes a conjugation system that is very similar to that of Tn916. However, insertion and excision of Tn5397 appears to be dependent on the product of the element encoded gene tndX, a member of the large resolvase family of site-specific recombinases. To test the role oftndX, the gene was cloned and the protein was expressed inEscherichia coli. The ability of TndX to catalyze the insertion and excision of derivatives (minitransposons) of Tn5397 representing the putative circular and integrated forms, respectively, was investigated. TndX was required for both insertion and excision. Mutagenesis studies showed that some of the highly conserved amino acids at the N-terminal resolvase domain and the C-terminal nonconserved region of TndX are essential for activity. Analysis of the target site choices showed that the cloned Tn5397 targets from C. difficile and B. subtilis were still hot spots for the minitransposon insertion inE. coli.

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