Synthesis of 6-Aryloxy- and 6-Arylalkoxy-2-chloropurines and Their Interactions with Purine Nucleoside Phosphorylase from Escherichia coli

The phase transfer method was applied to perform the nucleophilic substitution of 2,6- dichloropurines by modified arylalkyl alcohol or phenols. Since under these conditions only the 6-halogen is exchanged, this method gives 2-chloro-6-aryloxy- and 2-chloro-6-arylalkoxypurines. 2-Chloro-6-benzylthiopurine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl bromide. The stereoisomers of 2-chloro-6-(1-phenyl-1-ethoxy)purine were obtained from R- and S-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropurine. All derivatives were tested for inhibition with purified hexameric E. coli purine nucleoside phosphorylase (PNP). For analogues showing IC50 < 10 μm, the type of inhibition and inhibition constants were determined. In all cases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, Kiu, was found to be several-fold lower than the competitive inhibition constant, Kic. This effect seems to be due to the 6-aryloxy- or 6-arylalkoxy substituent, because a natural PNP substrate adenine, as well as 2-chloroadenine, show mixed type inhibition with almost the same inhibition constants Kiu and KiC. The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzyloxy-2-chloro-, 2-chloro-6-(2-phenyl-l-ethoxy), 2-chloro-6-(3-phenyl-l-propoxy)- and 2-chloro-6-ethoxypurines (Kiu = 0.4, 0.6, 1.4, 1.4 and 2.2 μm, respectively). The R-stereoisomer of 2-chloro-6-(1pheny-1-ethoxy)purine has Kiu = 2.0 μm, whereas inhibition of its S counterpart is rather weak (IC50> 12 μm). More rigid (e.g. phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the purine base gave less potent inhibitors (IC50 = 26, 56 and >100 μm, respectively). The derivatives are selective inhibitors of hexameric “high-molecular mass” PNPs because no inhibitory activity vs. trimeric Cellulomonas sp. PNP was detected. By establishing the ligand-dependent stabilization pattern of the E. coli PNP it was shown that the new derivatives, similarly as the natural purine bases, are able to form a dead-end ternary complex with the enzyme and orthophosphate. It was also shown that the derivatives are substrates in the reverse synthetic direction catalyzed by E. coli PNP

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Identification of a Subversive Substrate of Trichomonas vaginalis Purine Nucleoside Phosphorylase and the Crystal Structure of the Enzyme-Substrate Complex

Journal of Biological Chemistry ◽

10.1074/jbc.m501843200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 22318-22325 ◽

Cited By ~ 10

Author(s):

Yang Zang ◽

Wen-Hu Wang ◽

Shaw-Wen Wu ◽

Steven E. Ealick ◽

Ching C. Wang

Keyword(s):

Active Site ◽

Trichomonas Vaginalis ◽

Purine Nucleoside Phosphorylase ◽

Quaternary Structure ◽

Transmitted Disease ◽

Purine Nucleoside ◽

Purine Base ◽

Nucleoside Phosphorylase ◽

E Coli

Trichomonas vaginalis is an anaerobic protozoan parasite that causes trichomoniasis, a common sexually transmitted disease with worldwide impact. One of the pivotal enzymes in its purine salvage pathway, purine nucleoside phosphorylase (PNP), shows physical properties and substrate specificities similar to those of the high molecular mass bacterial PNPs but differing from those of human PNP. While carrying out studies to identify inhibitors of T. vaginalis PNP (TvPNP), we discovered that the nontoxic nucleoside analogue 2-fluoro-2′-deoxyadenosine (F-dAdo) is a “subversive substrate.” Phosphorolysis by TvPNP of F-dAdo, which is not a substrate for human PNP, releases highly cytotoxic 2-fluoroadenine (F-Ade). In vitro studies showed that both F-dAdo and F-Ade exert strong inhibition of T. vaginalis growth with estimated IC50 values of 106 and 84 nm, respectively, suggesting that F-dAdo might be useful as a potential chemotherapeutic agent against T. vaginalis. To understand the basis of TvPNP specificity, the structures of TvPNP complexed with F-dAdo, 2-fluoroadenosine, formycin A, adenosine, inosine, or 2′-deoxyinosine were determined by x-ray crystallography with resolutions ranging from 2.4 to 2.9 Å. These studies showed that the quaternary structure, monomer fold, and active site are similar to those of Escherichia coli PNP. The principal active site difference is at Thr-156, which is alanine in E. coli PNP. In the complex of TvPNP with F-dAdo, Thr-156 causes the purine base to tilt and shift by 0.5 Å as compared with the binding scheme of F-dAdo in E. coli PNP. The structures of the TvPNP complexes suggest opportunities for further improved subversive substrates beyond F-dAdo.

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7-Deazapurine 2'-deoxyribofuranosides are noncleavable competitive inhibitors of Escherichia coli purine nucleoside phosphorylase (PNP).

Acta Biochimica Polonica ◽

10.18388/abp.1998_4269 ◽

1998 ◽

Vol 45 (3) ◽

pp. 755-768 ◽

Cited By ~ 5

Author(s):

A Bzowska ◽

Z Kazimierczuk ◽

F Seela

Keyword(s):

Escherichia Coli ◽

Purine Nucleoside Phosphorylase ◽

Modified Nucleosides ◽

Purine Nucleoside ◽

Preliminary Screening ◽

Nucleoside Phosphorylase ◽

E Coli ◽

Potent Inhibition ◽

Competitive Inhibitors ◽

Inhibition Constants

A series of 7-deazapurine 2'-deoxyribofuranosides were synthesized according to already known procedures and their substrate and inhibitor properties with purified E. coli purine nucleoside phosphorylase were examined. In agreement with previous findings, substrate activity was not detected for any of the compounds tested. Most of the nucleosides showed weak inhibition in the preliminary screening, i.e. at a concentration of about 100 microM. However some combinations of 6-chloro, 6-amino or 6-methoxy substituents with bulky hydrophobic groups at position 7 of the base and/or chloro, amino, methoxy or methylthio group at position 2 markedly enhanced affinity of such modified nucleosides for the E. coli enzyme. The most potent inhibition was observed for two nucleosides: 6-chloro- and 2-amino-6-chloro-7-deazapurine 2'-deoxyribofuranosides that show inhibition constants Ki = 2.4 and 2.3 microM, respectively. Several other compounds were also found to be good inhibitors, with inhibition constants in the range 5-50 microM. In all instances the inhibition was competitive vs. the nucleoside substrate 7-methylguanosine. Inhibition constants for 7-deazapurine nucleosides are in general several-fold lower than those observed for their purine counterparts. Therefore 7-deaza modification together with substitutions at positions 2, 6 and 7 of the base is a very promising approach to obtain competitive noncleavable inhibitors of E. coli PNP that may bind to the enzyme with inhibition constants in the microM range.

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Radical Dehalogenation and Purine Nucleoside Phosphorylase E. coli: How Does an Admixture of 2′,3′-Anhydroinosine Hinder 2-fluoro-cordycepin Synthesis

Biomolecules ◽

10.3390/biom11040539 ◽

2021 ◽

Vol 11 (4) ◽

pp. 539

Author(s):

Alexey L. Kayushin ◽

Julia A. Tokunova ◽

Ilja V. Fateev ◽

Alexandra O. Arnautova ◽

Maria Ya. Berzina ◽

...

Keyword(s):

Nmr Spectroscopy ◽

Chemical Synthesis ◽

Purine Nucleoside Phosphorylase ◽

Purine Nucleoside ◽

Preparative Synthesis ◽

Nucleoside Phosphorylase ◽

E Coli ◽

First Time

During the preparative synthesis of 2-fluorocordycepin from 2-fluoroadenosine and 3′-deoxyinosine catalyzed by E. coli purine nucleoside phosphorylase, a slowdown of the reaction and decrease of yield down to 5% were encountered. An unknown nucleoside was found in the reaction mixture and its structure was established. This nucleoside is formed from the admixture of 2′,3′-anhydroinosine, a byproduct in the preparation of 3-′deoxyinosine. Moreover, 2′,3′-anhydroinosine forms during radical dehalogenation of 9-(2′,5′-di-O-acetyl-3′-bromo- -3′-deoxyxylofuranosyl)hypoxanthine, a precursor of 3′-deoxyinosine in chemical synthesis. The products of 2′,3′-anhydroinosine hydrolysis inhibit the formation of 1-phospho-3-deoxyribose during the synthesis of 2-fluorocordycepin. The progress of 2′,3′-anhydroinosine hydrolysis was investigated. The reactions were performed in D2O instead of H2O; this allowed accumulating intermediate substances in sufficient quantities. Two intermediates were isolated and their structures were confirmed by mass and NMR spectroscopy. A mechanism of 2′,3′-anhydroinosine hydrolysis in D2O is fully determined for the first time.

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