Excited-State Proton Transfer in 8-Azapurines I: A Kinetic Analysis of 8-Azaxanthine Fluorescence.

A super-continuum white laser with a half-pulse width of ~75 ps was used to observe the kinetics of a postulated excited-state proton transfer in 8-azaxanthine and its 8-methyl derivative. Both compounds exhibited dual emissions in weakly acidified alcoholic media, but only one band was present in aqueous solutions, exhibiting an abnormal Stokes shift (>12,000 cm-1). It was shown that long-wavelength emissions were delayed relative to the excitation pulse within alcoholic media. The rise time was calculated to be 0.4-0.5 ns in both methanol and deuterated methanol. This is equal to the main component of the fluorescence decay in the short-wavelength band (340 nm). Time-resolved emission spectra (TRES) indicated a two-state photo-transformation model in both compounds. Global analysis of the time dependence revealed three exponential components in each compound, one of which had an identical rise-time, with the second attributed to a long-wavelength band decay (6.4 ns for aza-xanthine and 8.3 ns for its 8-methyl derivative). The origin of the third, intermediate decay time (1.41 ns for aza-xanthine and 0.87 ns for 8-methyl-azaxanthine) is uncertain, but decay-associated spectra (DAS) containing both bands suggest the participation of a contact ion pair. These results confirm the model of phototautomerism proposed earlier, but the question of the anomalous isotope effect remains unsolved.

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

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--D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2--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- -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.

Fluorescence of the tri-cyclic adenine and isoguanine derivatives and their ribosides: possible analytical applications.

Fluorescent tri-cyclic purine analogs, derivatives of isoguanine and adenine, were examined as potential substrates of purine-nucleoside phosphorylase. It was found previously that etheno- derivatives of both compounds are ribosylated in phosphate-free media, but ribosylation places in some instances differ from purine N9. New ribosides are examined as potential substrates of human blood PNP and indicators of this enzyme. Of these, N6-riboside of 1,N6-etheno-adenine was found the most promising.

Tri-Cyclic Nucleobase Analogs and their Ribosides as Substrates of Purine-Nucleoside Phosphorylases. II Guanine and Isoguanine Derivatives.

Etheno-derivatives of guanine, O6-methylguanine, and isoguanine were prepared and purified using standard methods. The title compounds were examined as potential substrates of purine-nucleoside phosphorylases from various sources in the reverse (synthetic) pathway. It was found that 1,N2-etheno-guanine and 1,N6-etheno-isoguanine are excellent substrates for purine-nucleoside phosphorylase (PNP) from E. coli, while O6-methyl-N2,3-etheno-guanine exhibited moderate activity vs. this enzyme. The latter two compounds displayed intense fluorescence in neutral aqueous medium, and so did the corresponding ribosylation products. By contrast, PNP from calf spleens exhibited only modest activity towards 1,N6-etheno-isoguanine; the remaining compounds were not ribosylated by this enzyme. The enzymatic ribosylation of 1,N6-etheno-isoguanine using two forms of calf PNP (wild type and N243D) and E. coli PNP (wild type and D204N) gave three different products, which were identified on the basis of NMR analysis and comparison with the product of the isoguanosine reaction with chloroacetic aldehyde, which gave an essentially single compound, identified unequivocally as N9-riboside. With the wild-type E. coli enzyme as a catalyst, N9--d- and N7--d-ribosides are obtained in proportion ~1:3, while calf PNP produced another riboside, tentatively identified as N6--d-riboside. The potential application of various forms of PNP for synthesis of the tri-cyclic nucleoside analogs is discussed.

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

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,N6-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-ß-D-ribosyl-1,N6-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.

1,N6-ethenoadenine and other Fluorescent Nucleobase Analogues as Substrates for Purine-Nucleoside Phosphorylases: Spectroscopic and Kinetic Studies.

BACKGROUND: Purine-nucleoside phosphorylase (PNP) is known as a tool for the synthesis of various nucleosides and nucleoside analogues. Mechanism, properties, molecular diversity and inhibitors of PNP, particularly these of pharmacological significance, are briefly characterized. METHODS: UV and fluorescence spectroscopy was used for kinetic experiments, and HPLC chromatography for product analyses. RESULTS: Applications of various forms of PNP to synthesis of selected fluorescent nucleosides, particularly ribosides of 1,N6-ethenoadenine and various 8-azapurines (triazolo[4,5-d]pyrimidines) are reviewed. Different specificity of various PNP forms is described towards nucleobase and analogue substrates as well as variable ribosylation sites observed in some reactions, with a possibility to further modify these features via the site-directed mutagenesis. CONCLUSION: Present and future applications of the fluorescent or fluorogenic ribosides are discussed, with particular emphasis on biochemical and clinical analyses with improved sensitivity.

Site-Selective Ribosylation of Fluorescent Nucleobase Analogs Using Purine-Nucleoside Phosphorylase as a Catalyst: Effects of Point Mutations.

Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-ß-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-ß-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.

Excited-state proton transfer and phototautomerism in nucleobase and nucleoside analogs: a mini-review.

Intermolecular excited-state proton transfer (ESPT) has been observed in several fluorescent nucleobase and/or nucleoside analogs. In the present work, some new examples of ESPT in this class of compounds are presented together with a brief recapitulation of the previously published data. The nucleobases, nucleosides, and their analogs contain many basic and acidic centers and therefore their ESPT behavior may be complex. To interpret the complex data, it is usually necessary to determine the microscopic pK* values for each (or most) of the possible ESPT centers. Typical approach to solve this problem is by analysis of the alkyl derivatives, in which the possibility of the ESPT is reduced. Of particular interest are examples of "phototautomerization via the cation," observed in several systems, which in the neutral media do not undergo ESPT. Protonation of the molecule in the ground state facilitates the two-step phototautomerism in several systems, including formycin A and 2-amino-8-azadenine. Fluorescence of the nucleobase and nucleoside analogs undergoing ESPT is usually solvent-, isotope-, and buffer-ion sensitive, and in some systems the ESPT can be promoted by environmental factors, e.g., the presence of buffer ions. This sensitivity to the microenvironment parameters makes the ESPT systems potentially useful for biological applications.

8-Azapurines as isosteric purine fluorescent probes for nucleic acid and enzymatic research.

The 8-azapurines, and their 7-deaza and 9-deaza congeners, represent a unique class of isosteric (isomorphic) analogues of the natural purines, frequently capable of substituting for the latter in many biochemical processes. Particularly interesting is their propensity to exhibit pH-dependent room-temperature fluorescence in aqueous medium, and in non-polar media. We herein review the physico-chemical properties of this class of compounds, with particular emphasis on the fluorescence emission properties of their neutral and/or ionic species, which has led to their widespread use as fluorescent probes in enzymology, including enzymes involved in purine metabolism, agonists/antagonists of adenosine receptors, mechanisms of catalytic RNAs, RNA editing, etc. They are also exceptionally useful fluorescent probes for analytical and clinical applications in crude cell homogenates.

Two fluorogenic substrates for purine nucleoside phosphorylase, selective for mammalian and bacterial forms of the enzyme.

Two nontypical nucleosides, 7-ß-D-ribosyl-2,6-diamino-8-azapurine and 8-ß-D-ribosyl-2,6-diamino-8-azapurine, have been found to exhibit moderately good, and selective, substrate properties toward calf and bacterial (Escherichia coli) forms of purine nucleoside phosphorylase (PNP). The former compound is effectively phosphorolysed by calf PNP and the latter by PNP from E. coli. Both compounds are fluorescent with λ(max) ∼ 425 to 430 nm, but the reaction product, 2,6-diamino-8-azapurine, emits in a different spectral region (λ(max) ∼ 363 nm) with nearly 40% yield, providing a strong fluorogenic effect at 350 to 360 nm.

Enzymatic synthesis of highly fluorescent 8-azapurine ribosides using a purine nucleoside phosphorylase reverse reaction: variable ribosylation sites.

Various forms of purine-nucleoside phosphorylase (PNP) were used as catalysts of enzymatic ribosylation of selected fluorescent 8-azapurines. It was found that the recombinant calf PNP catalyzes ribosylation of 2,6-diamino-8-azapurine in a phosphate-free medium, with ribose-1-phosphate as ribose donor, but the ribosylation site is predominantly N7 and N8, with the proportion of N8/N7 ribosylated products markedly dependent on the reaction conditions. Both products are fluorescent. Application of the E. coli PNP gave a mixture of N8 and N9-substituted ribosides. Fluorescence of the ribosylated 2,6-diamino-8-azapurine has been briefly characterized. The highest quantum yield, ~0.9, was obtained for N9-ß-d-riboside (λmax 365 nm), while for N8-ß-d-riboside, emitting at ~430 nm, the fluorescence quantum yield was found to be close to 0.4. Ribosylation of 8-azaguanine with calf PNP as a catalyst goes exclusively to N9. By contrast, the E. coli PNP ribosylates 8-azaGua predominantly at N9, with minor, but highly fluorescent products ribosylated at N8/N7.

The hydrolysis of 5'-CAP dinucleotide analogs: catalysis by bi- and terpyridine complexes of Cu²âº and Zn²âº ions.

The kinetics of the hydrolysis of P(1)-(7-methylguanosinyl-5') P(3)-(guanosinyl-5') triphosphate (m(7)GpppG), P(1)-(7-methylguanosinyl-5') P(4)-(guanosinyl-5') tetraphosphate (m(7)GppppG), and diadenosine 5', 5'( ')-P(1),P(3) -triphosphate (ApppA) in the presence of several Cu(2+) or Zn(2+) ions complexed with bi- or terpyridine has been studied at pH 8.0 and 60 °C. Time-dependent product distributions at various metal complex concentrations have been determined by capillary zone electrophoresis and reversed-phase high performance liquid chromatography. The results show that the predominant hydrolytic reaction is the cleavage of 5',5'-oligophosphate bridge, with Cu(2+) complexes being approximately 15-fold more efficient catalysts than Zn(2+) chelates. In addition, the effect of metal ions complexes at pH 7.0 and 8.0 on the imidazole ring opening in m(7)Gua mononucleotides has been studied. The influence of Cu(2+) complexes on imidazole ring cleavage of mononucleotides is modest, whereas Zn(2+) complexes are almost inactive.

Salivary aldehyde dehydrogenase - temporal and population variability, correlations with drinking and smoking habits and activity towards aldehydes contained in food.

Fluorimetric method based on oxidation of the fluorogenic 6-methoxy-2-naphthaldehyde was applied to evaluate temporal and population variability of the specific activity of salivary aldehyde dehydrogenase (ALDH) and the degree of its inactivation in healthy human population. Analyzed was also its dependence on drinking and smoking habits, coffee consumption, and its sensitivity to N-acetylcysteine. Both the specific activity of salivary ALDH and the degree of its inactivation were highly variable during the day, with the highest activities recorded in the morning hours. The activities were also highly variable both intra- and interpersonally, and negatively correlated with age, and this correlation was stronger for the subgroup of volunteers declaring abstinence from alcohol and tobacco. Moderately positive correlations of salivary ALDH specific activity with alcohol consumption and tobacco smoking were also recorded (r(s) ~0.27; p=0.004 and r(s) =0.30; p=0.001, respectively). Moderate coffee consumption correlated positively with the inactivation of salivary ALDH, particularly in the subgroup of non-drinking and non-smoking volunteers. It was found that mechanical stimulation of the saliva flow increases the specific activity of salivary ALDH. The specific activity of the salivary ALDH was strongly and positively correlated with that of superoxide dismutase, and somewhat less with salivary peroxidase. The antioxidant-containing drug N-acetylcysteine increased activity of salivary ALDH presumably by preventing its inactivation in the oral cavity. Some food-related aldehydes, mainly cinnamic aldehyde and anisaldehyde, were excellent substrates of the salivary ALDH3A1 enzyme, while alkenals, particularly those with short chain, were characterized by lower affinity towards this enzyme but high catalytic constants. The protective role of salivary ALDH against aldehydes in food and those found in the cigarette smoke is discussed, as well as its participation in diminishing the effects of alcohol- and smoking-related oxidative stress.

Salivary aldehyde dehydrogenase activity--influence of drugs intake, preliminary research.

The salivary aldehyde dehydrogenase (ALDH3A1) oxidizes mainly aromatic and long chain aliphatic aldehydes. This enzyme protects organisms from aldehydes originating from food and air pollution, and can also be an important factor in chemical carcinogenesis prevention. In the majority of saliva samples the ALDH3A1 is more than in 60% inactive due to sulfhydryl groups oxidation in the active site. Our previous studies showed that the ALDH3A1 activity (the total activity and the inactivation degree) is strongly variable during a day and within the healthy population. The aim of the present study was to describe the influence of drugs intake on ALDH3A1 activity. Hierarchical clustering grouped two dimensional data (the total ALDH activity, the inactivation degree) derived from a group of 124 subjects into four clusters. Clusters were analyzed by a correspondence analysis. The total ALDH3A1 activity and an inactivation degree vary in healthy subjects depending on many factors including cigarette smoking, alcohol and coffee consumption and age. This work demonstrates that treated hypertension and acute pain/infection as well as hormonal contraceptive drugs intake also significantly affect the salivary ALDH activity. The result is significant for food safety and nutrition research.

The oxidation status of ALDH3A1 in human saliva and its correlation with antioxidant capacity measured by ORAC method.

Oxidation status of the salivary aldehyde dehydrogenase (ALDH) was measured in healthy human population using two-assay fluorimetric method and compared with antioxidant capacity (ORAC) in non-smoking and heavy smokers group. Influence of high or low antioxidant diet was also examined. Except for the group of smokers, the salivary ALDH oxidation degree in human saliva was not correlated with antioxidant capacity. Simultaneously direct administration of the antioxidant-containing drug, Fluimucil, resulted in short-term, but statistically significant increase of the reduced (active) form of the enzyme, presumably due to a radical-scavenging activity of the drug.

Salivary aldehyde dehydrogenase: activity towards aromatic aldehydes and comparison with recombinant ALDH3A1.

A series of aromatic aldehydes was examined as substrates for salivary aldehyde dehydrogenase (sALDH) and the recombinant ALDH3A1. Para-substituted benzaldehydes, cinnamic aldehyde and 2-naphthaldehydes were found to be excellent substrates, and kinetic parameters for both salivary and recombinant ALDH were nearly identical. It was demonstrated that for the fluorogenic naphthaldehydes the only produced reaction product after incubation in saliva is the carboxylate.

Fluorescence emission properties of 8-aza analogues of caffeine, theophylline, and related N-alkyl xanthines.

Fluorescence emission properties of 8-azacaffeine, 8-azatheophylline and other N-alkylated 8-azaxanthines (8-azaXan) have been examined. It is shown that N-methylated 8-azaxanthines, as well as 8-azatheophylline, are highly fluorescent in aqueous medium as the neutral, and, in some instances, also as the monoanionic, forms. 8-Azacaffeine exhibits moderate emission, but its isomer, 1,3,8-trimethyl-8-azaXan, is highly fluorescent. All three 8-azaxanthines monomethylated on the triazole ring, as well as 8-azaxanthosine, exhibit increased acidity in the excited state. Some fluorescent pyrazolo[4,3-d]pyrimidine-5,7-diones, xanthine congeners of pyrazolo[4,3-d]pyrimidines, are also reported. Many of these are good fluorescent probes in enzymatic, receptor binding, and nucleic acid systems, some examples of which are presented. In particular, 8-azaXan is an excellent fluorescent probe for purine nucleoside phosphorylases, as a fluorogenic substrate in the reverse, synthetic pathway.

Fluorimetric detection of aldehyde dehydrogenase activity in human tissues in diagnostic of cancers of oral cavity.

Aldehyde dehydrogenase (ALDH) is known to be susceptible to oxidation, and its assays require stabilization of the enzyme by thiols. Application of the fluorimetric method to assay the ALDH activity in human saliva demonstrated significant differences between procedures utilizing glutathione (GSH) and dithiothreitol (DTT) as stabilizing agents. It has been recently shown that average aldehyde dehydrogenase (ALDH3A1) activity in cancerous (oral cavity cancer) patients' tissues was higher than that found in the control group, what may indicate induction of tumor-specific ALDH.

Salivary aldehyde dehydrogenase--reversible oxidation of the enzyme and its inhibition by caffeine, investigated using fluorimetric method.

OBJECTIVE: We have applied fluorimetric method to monitor aldehyde dehydrogenase (ALDH*) activity in human saliva samples to study inactivation, reactivation and inhibition of the enzyme. DESIGN: Saliva samples were collected to buffer stock solution, containing various thiols, and assayed in the presence of the fluorogenic substrate 6-dimethylamino-2-naphthaldehyde and NAD(+). Fluorescence of the produced 6-dimethylamino-2-naphthalene carboxylate was used to measure the reaction rate. RESULTS: Kinetic parameters for the highly fluorogenic substrate, 6-dimethylamino-2-naphthaldehyde were measured, with apparent K(m) of 7.9 microM at pH 7.3. The apparent K(m) for NAD(+) was 1.2 microM. The observed ALDH activity is unstable in the absence of thiols, but can be stabilized by 1mM glutathione, and inactivated enzyme can be re-activated within 10 min by treatment of 0.5 mM DTT. Two-assay procedure was applied to measure degree of inactivation of ALDH in saliva samples. It was found that degree of ALDH inactivation in fresh samples, stabilized by glutathione, is between 0% and 90%, with average value ca. 40%. Caffeine and theophylline were shown to be moderate inhibitors of salivary ALDH. CONCLUSIONS: Oxidation of the salivary ALDH in fresh saliva may be reliably measured using fluorimetric two-assay procedure. Preliminary statistics indicate that in most individuals this enzyme is partially inactive. Inhibition of the salivary ALDH by caffeine may have consequences for nutrition safety.

Fluorescence studies of calf spleen purine nucleoside phosphorylase (PNP) complexes with guanine and 9-deazaguanine.

Interactions of trimeric calf spleen purine nucleoside phosphorylase (PNP) with guanine (Gua) and its analogue, 9-deazaguanine (9-deaza-Gua), were studied by means of the steady-state fluorescence. The aim was to test the hypothesis that the enzyme stabilizes the anionic form of purine, inferred previously from the unusual increase of fluorescence observed after binding of guanine by calf spleen PNP. We have found that the dissociation constants obtained form titration experiments are in fact pH-independent in the range 7.0-10.25 for both PNP/Gua and PNP/9-deaza-Gua complexes. In particular, at pH 7.0 we found Kd = 0.12 +/- 0.02 micro M for Gua and 0.16 +/- 0.01 micro M for 9-deaza-Gua, while at the conditions where there is more than 40% of the anionic form the respective values were Kd = 0.15 +/- 0.01 micro M for Gua (pH 9.0) and 0.25 +/- 0.02 micro M for 9-deaza-Gua (pH 10.25). Hence, the enzyme does not prefer binding of anionic forms of these ligands in respect to the neutral ones. This result questions the involvement of the anionic forms in the reaction catalyzed by trimeric PNPs, and contradicts the hypothesis of a strong hydrogen bond formation between the enzyme Asn 243 residue and the purine N7 position.