Tricyclic nitrogen base 1,N⁶-ethenoadenine and its ribosides as substrates for purine-nucleoside phosphorylases: Spectroscopic and kinetic studies

Abstract

The title compound is an excellent substrate for E. coli PNP, as well as for its D204N mutant. The main product of the synthetic reaction is N9-riboside, but some amount of N7-riboside is also present. Surprisingly, 1,N-ethenoadenine is also ribosylated by both wild-type and mutated (N243D) forms of calf PNP, which catalyze the synthesis of a different riboside, tentatively identified as N6-β-D-ribosyl-1,N-ethenoadenine. All ribosides are susceptible to phosphorolysis by the E. coli PNP (wild type). All the ribosides are fluorescent and can be utilized as analytical probes.

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

“…Kinetic parameters of the synthetic (ribosylation) reaction, catalyzed by the wild-type and mutated forms of PNP, were determined using standard procedures, and are summarized in Table 3. There are some minor differences between wild-type enzymes (E. coli and calf PNP) and forms mutated in the active site, but without qualitative differences, observed previously for some purine analogs [21,22]. Generally, kinetic parameters for ribosylation of 2 by E. coli PNP and its mutated forms do not differ markedly from those determined earlier for natural purines [12], and the K m values are close to 10 µM, hence comparable to those observed for guanine ribosylation under the same conditions.…”

“…The spectra of the linear isomer 1 under neutral conditions (phosphate buffer, pH 7) and in acid are strikingly similar to those of the N 9 -riboside 3, published by Virta et al [23], and are markedly red-shifted relative to analogous spectra of all adenine or guanine derivatives (cf. [21,22]). In basic media, the spectrum is shifted even more, showing maximum absorbance at 367 nm ( Figure 2, Table 1).…”

“…Additionally, PNP's from various sources are utilized as biocatalysts in chemo-enzymatic syntheses of various nucleoside analogs of pharmaceutical and/or analytical significance [16][17][18][19][20]. Our previous investigations have shown that PNP isolated from E. coli, which is known to possess a broad specificity toward various base and nucleoside analogs [16], is also active toward many tri-cyclic nitrogen bases, derived from adenine and guanine [17,21,22], producing in the phosphate free media many potentially useful ribosides, using β-d-ribose-1-phosphate (R1P) as a ribose source. The reverse, phosphorolytic reactions are, in some cases, as rapid as the analogous reactions with natural substrates, like adenosine or guanosine [21,22], and therefore can possess analytical applications.…”

General Principles for Yield Optimization of Nucleoside Phosphorylase‐Catalyzed Transglycosylations

“…Biocatalytic methods offer the efficient and protecting group‐free synthesis of pyrimidine and purine nucleosides. The use of nucleoside phosphorylases (NPases) for the preparation of nucleosides and their analogues in transglycosylation reactions is firmly established and numerous examples of enzymatic or chemoenzymatic syntheses can be found in the literature . NPases catalyze the reversible phosphorolysis of nucleosides to pentose‐1‐phosphates (Scheme , I).…”

General Principles for Yield Optimization of Nucleoside Phosphorylase-Catalyzed Transglycosylations

“…The use of nucleoside phosphorylases (NPases) for the preparation of nucleosides andt heir analogues in transglycosylation reac-tions is firmly established [6] and numerouse xamples of enzymatic or chemoenzymatic syntheses can be found in the literature. [7][8][9][10][11][12][13][14] NPases catalyze the reversible phosphorolysis of nucleosides to pentose-1-phosphates (Scheme 1, I). In transglycosylation reactions, af orward and ar eversen ucleoside phosphorolysis are coupledi nsitu to glycosylate af ree nucleobase with the pentose-1-phosphate generated by the first reaction (Scheme 1, Ia nd II).…”