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-β-d- and N7-β-d-ribosides are obtained in proportion ~1:3, while calf PNP produced another riboside, tentatively identified as N6-β-d-riboside. The potential application of various forms of PNP for synthesis of the tri-cyclic nucleoside analogs is discussed.


Introduction
Some tri-cyclic analogs of the natural purine bases and their glycosides show intense fluorescence, what enables their application as fluorescent probes in the investigations of structure and function of nucleic acids (DNA, RNA) and enzymes related to nucleic acid metabolism or utilizing nucleotide cofactors [1,2]. The most popular probe of this kind is 1,N 6 -etheno-adenosine (εAdo), easily produced in the reaction of adenosine with chloroacetic aldehyde (CAA) in aqueous environment.
Other etheno-purine derivatives (see Scheme 1) were also prepared but were reported to display rather discouraging emission properties: This refers to the guanine/guanosine derivatives [3][4][5], as well to the products of the analogous reactions of CAA with 2-aminopurine and its riboside [6,7], and 2,6-diaminopurine [7,8], which were found to be only moderately fluorescent in the neutral aqueous derivatives of guanine, its structure similar to one of the known rare t-RNA bases, the so-called 82 Scheme 1.
1,N 2 -ethenoguanine (1) was reported to be nonfluorescent in the neutral aqueous medium [3]. Weak emission was observed from its monoanionic form (Table 1), but no significant emission was detected at low pH (~1), where this compound is protonated. Table 1. Ionization constants (pK a values) and spectral parameters of neutral and ionic forms* of the investigated compounds. The UV spectral data are compiled from refs [1][2][3], and checked in this work, and fluorescence parameters determined in this work, except those for N 2 ,3-ethenoguanine. We found that this compound is an excellent substrate for PNP from E. coli (Figure 1a), with catalytic and Michaelis' constants comparable to that for ribosylation of the parent guanine (Table 2). HPLC analysis of the reaction mixture has shown that there is one main product (>95%), spectrally and chromatographically identical to the nucleoside generated in the chemical way ( Figure 1). The reverse reaction (phosphorolysis of the nucleoside) is also easily observed in the presence of phosphate ions (Figure 1b). These facts may be important in view of a significant mutagenic role of 1,N 2 -ethgenoguanine (1) lesion in many organisms, particularly in bacteria [22].
The mutated form of the E. coli PNP (D204N) reacted in a qualitatively similar way as the wild-type enzyme, with similar spectral changes and comparable rate.
With the calf enzyme (wild-type or the N243D mutant) only very slow reaction was observed under typical conditions (pH 7.3, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, 25 • C). This reaction was ca. 100-fold slower than the ribosylation of guanine. There was also no marked phosphorolysis of the nucleoside by this enzyme in the phosphate buffer (data not shown). We conclude that PNP probably does not play any substantial role in the process of detoxication of etheno-guanosine in mammals, but this kind of degradation can be important in bacteria. buffer, 25 °C). This reaction was ca. 100-fold slower than the ribosylation of guanine. There was also 108 no marked phosphorolysis of the nucleoside by this enzyme in the phosphate buffer (data not 109 shown). We conclude that PNP probably does not play any substantial role in the process of 110 detoxication of etheno-guanosine in mammals, but this kind of degradation can be important in     (2), a non-linear isomer of 1,N 2 -ethgenoguanine (1), is a common and the most persistent lesion in DNA upon treatment with vinyl chloride [12]. Its role in mutagenesis has been addressed in many papers and shown to be significant [23][24][25]. This compound is weakly fluorescent in the neutral aqueous medium and moderately in some organic solvents [3]. The UV absorption of N 2 ,3-ethenoguanine (2) is blue-shifted relative to 1,N 2 -ethenoguanine (1) (Figure 2), but its acido-basic properties are similar to those of the latter compound (Table 1), however, the ionic forms are nonfluorescent.

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We found that N 2 ,3-ethenoguanine (2) is not a substrate for PNP form E. coli and calf spleen. No reaction traces were observed even with 10-fold higher enzyme concentrations than those applied to the synthesis of 1,N 2 -ethenoguanosine (see previous section). Ribosides of N 2 ,3-ethenoguanine are known to be relatively unstable [26,27], and ribose modifications stabilizing the glycosidic bond were used to study the role of this compound in mutagenesis [25]. Probably this instability of the glycoside bond is the main obstacle in its effective enzymatic ribosylation, although it is not clear why other possible ribosylation sites (like N7 or N 2 ) cannot be utilized by the enzymes.
With guanine as a substrate of the ribosylation reaction, we have found moderate, possibly competitive inhibition of the E. coli PNP by N 2 ,3-ethenoguanine (2), with estimated K i of 38 µM. This value is not far from the K m value for guanine (ca. 54 µM in our conditions, [13]). We conclude that inability of the enzyme to ribosylate (2) is not due to geometric hindrance, but rather should be ascribed to unfavorable energetic factors, in accordance with previous reports about the relative instability of the glycosidic bond in this compound [23].

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We found that N 2 ,3-ethenoguanine (2) is not a substrate for PNP form E. coli and calf spleen. No 133 reaction traces were observed even with 10-fold higher enzyme concentrations than those applied to 134 the synthesis of 1,N 2 -ethenoguanosine (see previous section). Ribosides of N 2 ,3-ethenoguanine are known to be relatively unstable [26,27], and ribose modifications stabilizing the glycosidic bond 136 were used to study the role of this compound in mutagenesis [25]. Probably this instability of the glycoside bond is the main obstacle in its effective enzymatic ribosylation, although it is not clear 138 why other possible ribosylation sites (like N7 or N 2 ) cannot be utilized by the enzymes.

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With guanine as a substrate of the ribosylation reaction, we have found moderate, possibly 140 competitive inhibition of the E. coli PNP by N 2 ,3-ethenoguanine (2), with estimated Ki of 38 μM. This 141 value is not far from the Km value for guanine (ca. 54 μM in our conditions, [13]). We conclude that 142 inability of the enzyme to ribosylate (2) is not due to geometric hindrance, but rather should be 143 ascribed to unfavorable energetic factors, in accordance with previous reports about the relative 144 instability of the glycosidic bond in this compound [23].

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Although the ribosylation rate is slow, the apparent Km value, calculated from the progress 168 curve (see Supplementary Materials), is markedly below 10 μM, so it is somewhat lower than that for 169 the canonical nucleobases [13]. Although the kinetically measured Km cannot be directly used as a   (3) is also a substrate for PNP from E. coli, but the maximal velocity of its ribosylation is rather poor ( Table 2). The product riboside spectrally differs from the substrate base, particularly by its weaker fluorescence, so the ribosylation is easy to follow ( Figure 3). The reaction progress curve initially seems to be linear ( Figure 4b, and Supplementary Materials), pointing to the low K m value. The observed marked differences in the absorption and emission spectra of the product riboside and the parent base is most likely due to prototropic tautomerism (N9H↔N7H) of the latter, not possible for the riboside.

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Fluorescence excitation was taken at 280 nm.

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The ribosylation reaction is fully reversible by the addition of a phosphate buffer up to a 183 concentration of ~5 mM (even after 24 h, data not shown). We did not attempt to isolate the pure 184 riboside due to the low efficiency of the synthetic process, but it would be interesting to check the 185 ribosylation site, which may be different from the "canonical" N9, explaining spectral differences 186 between this product and the parent base.  Although the ribosylation rate is slow, the apparent K m value, calculated from the progress curve (see Supplementary Materials), is markedly below 10 µM, so it is somewhat lower than that for the canonical nucleobases [13]. Although the kinetically measured K m cannot be directly used as a measure of binding properties of the purine analog [13], its low value indicates that the geometry of the compound is in this case not unfavorable for the E. coli PNP, and the confirms that the main reason for slow ribosylation and the resistance of the parent N 2 ,3-ethenoguanine (3) to the enzymatic ribosylation is probably unfavorable energetics.
The ribosylation reaction is fully reversible by the addition of a phosphate buffer up to a concentration of~5 mM (even after 24 h, data not shown). We did not attempt to isolate the pure riboside due to the low efficiency of the synthetic process, but it would be interesting to check the ribosylation site, which may be different from the "canonical" N9, explaining spectral differences between this product and the parent base. N 2 ,3-etheno-O 6 -methylguanine (3) effectively competes with guanine in the ribosylation process, acting as quasi inhibitor of the E. coli PNP, with estimated K iapp value 7.5 ± 0.8 µM (competitive model; average of 3 determinations), in agreement with the K m value, estimated from kinetic data (see the previous paragraph). With the calf enzyme, no such inhibition is observed under similar conditions.
The nucleoside of isoGua, isoguanosine, is rather slowly phosphorolysed by the E. coli PNP (V max ca. 40-fold lower than that for guanosine, data not shown), and we did not see any such reaction catalyzed by the calf enzyme, in accordance with the general rule [13], that 6-aminopurine ribosides, not possessing mobile proton at the N1 position, do not react with trimeric forms of PNP, including mammalian enzymes.
Isoguanine reacts readily with CAA to produce a single, and highly fluorescent product, 1,N 6 -etheno-isoguanine (εisoGua, (4)). The etheno-isoguanine fluorescence was long ago proposed as a tool for analytical quantitation of 9-β-d-arabinofuranosyl-2-fluoroadenine (fludarabine, [31]), but, to our knowledge, no systematic examination of the emission properties of εisoGua (4) exists in the literature. Basic spectral data for this compound are given in Table 1.
Spectrophotometric titrations of εisoGua (4) (for details, see the Supplementary Materials) indicate that this compound exists as a neutral species at pH 4.5-7, and above pH 8 undergoes deprotonation, and there is probably a second deprotonation at pH~13. The anionic forms are virtually nonfluorescent, while the neutral form and the cation are strongly fluorescent ( Figure 5 and Table 1). Fluorimetric titrations give results essentially in agreement with the UV data (not shown).
Fluorescence properties of εisoGua (4) strongly suggest a ground-state tautomeric equilibrium. Fluorescence decay, measured at excitation 280 nm at pH 6.5 (neutral form), is clearly nonexponential, but well approximated by a bi-exponential decay function (data not shown), with decay times of 5.1 and 3.4 ns (see Table 1). Similar situations exist in 1,N 6 -adenine, but not its nucleoside [32]. Moreover, the fluorescence excitation spectrum of the neutral form of (4) at pH 6.5, although virtually observation-independent (data not shown), is clearly distinct from the UV absorption ( Figure 6 below), suggesting the existence of a third not-emitting or weakly emitting species. The tautomeric equillibria in εisoGua (4) are even more complex that those of etheno-adenine, where only a N(9)H-N(7)H-N 6 H prototropic tautomerism is likely [18], while in (4) the situation is additionally complicated by possible keto-enol tautomerism (see Scheme 2, below). This problem requires therefore further elucidation.
1,N 6 -etheno-isoguanine (4) is a moderately good substrate for PNP from various sources, but reactions with various types of the enzyme vary not only in rates but also in products obtained, giving at least 3 types of ribosides (Figures 7-9). This situation is somewhat reminiscent of the ribosylation of 1,N 6 -ethenoadenine, where also 3 ribosides were obtained [14,18], all of them fluorescent. In the case of (4), all three ribosylated species are also fluorescent, although with different yields and decay times (Table 1).  Table 1). Fluorimetric titrations give results essentially in agreement with the UV data (not shown).

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Fluorescence decay, measured at excitation 280 nm at pH 6.5 (neutral form), is clearly 220 nonexponential, but well approximated by a bi-exponential decay function (data not shown), with 221 decay times of 5.1 and 3.4 ns (see Table 1). Similar situations exist in 1,N 6 -adenine, but not its 222 nucleoside [32]. Moreover, the fluorescence excitation spectrum of the neutral form of (4) at pH 6.5,

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This problem requires therefore further elucidation.  236 1,N 6 -etheno-isoguanine (4) is a moderately good substrate for PNP from various sources, but 237 reactions with various types of the enzyme vary not only in rates but also in products obtained,

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One chemically from isoguanosine reacting with CAA, and all three enzymatically, using various

Ribosides of 1,N 6 -etheno-isoguanine
As mentioned above, three types of ribosides of 1,N 6 -etheno-isoguanine (4) can be generated. One chemically from isoguanosine reacting with CAA, and all three enzymatically, using various types of PNP as a biocatalyst (Scheme 3). The reaction of isoguanosine with CAA was fairly rapid at pH 4.5 and room temperature and gave essentially a single product, identified as N9-β-d-riboside (5) of εisoGua. Enzymatic ribosylation gave an additional 2 products, clearly not identical with (5). Ribosylation catalyzed by the enzyme from E. coli gave two products: The highly fluorescent N9-riboside (5, minor product), and another riboside with less intense fluorescence shifted to~355 nm (Figure 7 below, lower panels). The analogous reaction catalyzed by the calf PNP gave one main product, very intensely fluorescent, but with UV absorption spectrum markedly shifted to the longer wavelengths (Figure 7, upper panels). The observed ambiguity of the ribosylation sites is analogous to that described earlier for the enzymatic ribosylation of etheno-adenine [18] and some 8-azapurines [33].

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Ribosylation catalyzed by the enzyme from E. coli gave two products: The highly fluorescent 249 N9-riboside (5, minor product), and another riboside with less intense fluorescence shifted to ~355 250 nm (Figure 7 below, lower panels). The analogous reaction catalyzed by the calf PNP gave one main 251 product, very intensely fluorescent, but with UV absorption spectrum markedly shifted to the longer 252 wavelengths (Figure 7, upper panels). The observed ambiguity of the ribosylation sites is analogous 253 to that described earlier for the enzymatic ribosylation of etheno-adenine [18] and some 8-azapurines [33].   NMR spectral analysis was necessary to identify the ribosylation sites (Scheme 3). Identification of the three ribosides of εisoGua (4) was based mainly on connectivities observed in 1 H, 13 C HMBC spectra. 1 H, 1 H COSY spectra allowed for the assignment of all aliphatic sugar protons. Of the three signals of aromatic base protons, the two protons of the etheno group were identified due to the scalar coupling between them, with the εisoGua (4) H8 proton identified by elimination. The presence of C8-H1' and C1'-H8 correlations in the HMBC spectrum allowed for the identification of two of three samples as either N7 (6) or N9-(5) ribosides. The former riboside (6) was identified by the presence of C6-H1' and C6-H8 correlations, where C6 was assigned based on its relatively low chemical shift (typical values and the substituent effects have been reviewed in Reference [34]), compared to the expected chemical shift of C4. By elimination, the remaining sample was identified as the N9-riboside (5), in agreement with chemo-enzymatic identification provided in the previous paragraph. The N 6 -riboside (7) was identified by the presence of the correlation of both etheno protons with C1' and (in the 1 H, 15 N spectrum) N 6 . Selected chemical shifts are given in Table 3, with the full list provided in Supplementary Materials.    *H7 or C7 according to IUPAC notation; **H8 or C8 according to IUPAC notation.

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In the 50 mM phosphate buffer, pH 6.5, the N9-riboside (5) Table 3. Selected chemical shifts of the three ribosides of εisoGua. Chemical shift labels follow the naming convention of IUPAC-IUBMB-IUPAB [35], extended for the etheno protons (see Scheme 1). NA -resonance not assigned. For the N7and N9-ribosides, atoms in the positions 10 and 11 could not be unequivocally differentiated, and the two possible values are slash-separated. In the 50 mM phosphate buffer, pH 6.5, the N9-riboside (5) is not phosphorolysed by the calf PNP, but the reaction with the E. coli enzyme is fairly rapid (see Supplementary Materials). Even more rapid is phosphorolysis of the N7-riboside (6) by the same type of PNP, while the N 6 -riboside (7) is only slowly deribosylated by the calf enzyme, but much more rapidly by the E. coli PNP (data not shown). This is to be compared with 1,N 6 -ethenoadenosine, resistant to the calf enzyme, but rapidly phosphorolysed by the E. coli PNP [18]. All three ribosides are fairly stable in neutral aqueous environment, and no significant instability was seen at pH 11 (data not shown). The respective emission yields of the neutral forms are comparable to those of the best-known fluorescent probes [1, 18,33,36]. For these reasons, they seem to be more universal as probes than the somewhat unstable 1,N 6 -ethenoadenosine [1].  (7) is only slowly deribosylated by the calf enzyme, but much more rapidly by the E. coli PNP (data 291 not shown). This is to be compared with 1,N 6 -ethenoadenosine, resistant to the calf enzyme, but 292 rapidly phosphorolysed by the E. coli PNP [18]. All three ribosides are fairly stable in neutral 293 aqueous environment, and no significant instability was seen at pH 11 (data not shown). The 294 respective emission yields of the neutral forms are comparable to those of the best-known 295 fluorescent probes [1, 18,33,36]. For these reasons, they seem to be more universal as probes than the

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Various types of PNP mutated at the active site are known, some of them with qualitatively 305 different catalytic characteristics [33,37,38]. We have therefore checked two of such mutants, namely 306 the D204N mutant of the E. coli PNP and the "complementing" N243D mutant of the calf enzyme.

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In the case of εisoGua (4), the mutation effect is not very pronounced, with the exception of the 310 E. coli PNP, where emission spectrum of the product mixture was shifted to the red, relative to the 311 wild-type PNP reaction (Figure 7 and 8). This latter fact suggests a larger contribution by the rates (catalytic constants) were comparable to those obtained for the wild-type enzymes. These 314 results are analogous to those reported for 1,N 6 -ethenoadenine as a substrate [18]. Various types of PNP mutated at the active site are known, some of them with qualitatively different catalytic characteristics [33,37,38]. We have therefore checked two of such mutants, namely the D204N mutant of the E. coli PNP and the "complementing" N243D mutant of the calf enzyme. Both mutations were previously shown to alter ribosylation specificity of PNP with 6-amino-purines [37] and 8-azapurines [33,38] as substrates.
In the case of εisoGua (4), the mutation effect is not very pronounced, with the exception of the E. coli PNP, where emission spectrum of the product mixture was shifted to the red, relative to the wild-type PNP reaction (Figures 7 and 8). This latter fact suggests a larger contribution by the N9-riboside (5) as a ribosylation product, with a possible contribution by other product(s). Reaction rates (catalytic constants) were comparable to those obtained for the wild-type enzymes. These results are analogous to those reported for 1,N 6 -ethenoadenine as a substrate [18].   . Electronic absorption spectra of three ribosides of 1,N 6 -etheno-isoguanine, purified by HPLC. Spectra of N9-(red color) and N7-β-d-riboside (blue) were measured in 50 mM phosphate buffer, pH 6.5, and that of N 6 -riboside (yellow), at pH 7.
Etheno-derivatives of guanine and guanosine were obtained analogously to Hořejši et al. [6], from the reaction of 2-amino-6-chloropurine riboside with ca 7-fold molar excess of chloroacetaldehyde (CAA, [5,6]). The reaction was carried out in aqueous environment, at room temperature and pH 4-4.5, controlled by addition of sodium bicarbonate. After 3 days, the reaction mixture was acidified to pH 1 and boiled by~3 min. HPLC analysis ( Figure 10) revealed a significant degree of hydrolysis of chloride, as well as of ribosyl moiety.
The main product was identified as 1,N 2 -ethenoguanine (1) on the basis of UV spectra in various conditions and lack of fluorescence. The second isomer, N 2 ,3-ethenoguanine (2), was identified on the basis of its fluorescence and UV spectra shifted to~260 nm. The third major product was 1,N 2 -ethenoguanosine, which was proved by its UV absorption and ready enzymatic phosphorolysis to 1,N 2 -ethenoguanine (1, see Section 2.1). Smaller amounts of guanine and the starting 2-amino-6-chloropurine riboside were also found. The main products were purified via the semi-preparative HPLC (C-18 column), eluted with water (10-12 min), followed by water-methanol gradient (10-30%, 40 min). The analogous method, with various reaction times, was used for other compounds (see below). The progress of the reactions was monitored spectrally or, wherever possible, also fluorimetrically.

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Etheno derivatives of isoguanine (2-hydroxy-6-aminopurine) and isoguanosine were prepared 354 in analogous ways, but the nucleoside was somewhat more reactive (reaction time was 2-3 days) 355 then the base (7 days). In both cases only a single product, easily identified as the 1,N 6 -etheno 356 derivative, was obtained and purified using semi-preparative HPLC with methanol gradient as the     [3] led to N 2 ,3-ethenoguanine (2), identical to the previously obtained sample (see above).
Etheno derivatives of isoguanine (2-hydroxy-6-aminopurine) and isoguanosine were prepared in analogous ways, but the nucleoside was somewhat more reactive (reaction time was 2-3 days) then the base (7 days). In both cases only a single product, easily identified as the 1,N 6 -etheno derivative, was obtained and purified using semi-preparative HPLC with methanol gradient as the main eluent (see Supplementary Materials).
The detailed procedure for (4): Isoguanine (200 mg) dissolved initially in~10 mL of 1 M HCl, added 1 mL of aqueous CAA, reaction pH elevated to~2 by addition of bicarbonate, and left overnight. The next day, the reaction pH elevated to~3, and after 24 h to 4-4.5 and was maintained by bicarbonate and/or acetic acid. After 7 days the crude precipitate was collected and dried. HPLC analysis showed 70% progress of the reaction. The crude precipitate was used as such for the ribosylation experiments and aliquot purified using HPLC.
General procedure for milligram-scale enzymatic syntheses of εisoGua (4) ribosides is as follows: 10 mg of substrate dissolved in diluted aqueous ammonia (ca. 10 mL) and stepwise added to ca. 5 mL of 100 mM HEPES buffer, pH~7, containing ca. 10 mM solution of R1P and 20 µL of concentrated (~2.6 mg/mL) E. coli PNP. The reaction was carried out at 30 • C for 3 days, the mixture concentrated, and products separated as described below. Typical HPLC profiles are shown on Figure 11.   Product separation and purification used HPLC on a UFLC system from Shimadzu (Kyoto, Japan) equipped with UV (diode-array) detection at 260, 280 and 315 nm, and a fluorescence detector. The column used was a Kromasil reversed-phase a semi-preparative C-18 column (250 × 10 mm, 5-µm particle size). Elution was initially (10-15 min) isocratic, followed by water-methanol gradient (usually 10-30% methanol for 35-40 min).
For kinetic analyses, enzymatic ribosylation reactions were carried out in 1 mL cuvettes (pathlength 4 mm) in~50 mM HEPES buffer, pH 7.3, using ca. 0.5 mM R1P as a ribose source. On a larger scale, reactions were run in Eppendorf tubes, volume 2-3 mL, using either R1P or 7-methylguanosine as ribose sources. Phosphorolysis reactions were run in 50 mM phosphate buffer, pH 7.0 or 6.5. Data were analyzed by standard methods (see Supplementary Materials).
Fluorescence spectra were measured on a Varian Eclipse instrument (Varian Corp., Palo Alto, CA, USA), and UV absorption kinetic experiments were performed on a Cary 5000 (Varian) thermostated spectrophotometer. Fluorescence yields were determined relative to tryptophan (0.15) or 1,N 6 -ethenoadenosine in water (0.56, [2]). Spectra were measured in semi-micro cuvettes, pathlength 4 mm, to diminish the inner-filter effect. Fluorescence decay was measured and analyzed using a FluoTime 200 lifetime fluorometer (PicoQuant GmbH, Germany), equipped with an R3809U-50 microchannel-plate photomultiplier (MCP-PMT, Hamamatsu, Japan), with 280 nm excitation by sub-nanosecond pulsed LED, as previously described [39]. All buffers were of analytical grade and show no fluorescence background.
NMR measurements were performed on a Bruker Avance III HD 800 MHz spectrometer equipped with a cryogenically-cooled H-C/N-D TCI probe at 25 • C (sample identified as N 7 riboside) or 50 • C (the other two samples). For all samples the following spectra were acquired: A standard proton spectrum, a 1 H, 1 H gradient selected COSY, a gradient selected 1 H, 13 C HSQC and a gradient selected 1 H, 13 C HMBC tuned for 7 Hz J coupling with a double low pass J filter. For the sampled identified as the N7-riboside a gradient selected 1 H, the 15 N HMBC experiment tuned for 15 Hz J coupling was also acquired. 1 H chemical shift was referenced by the field-locked substitution method using a <1% sample of TMS in DMSO-d6, 13 C and 15 N chemical shifts were referenced using the unified chemical shift scale [40]. 2D spectra were processed using the TopSpin 3.5 pl7 software package (Bruker) and inspected using the Sparky program [41] with manual peak-picking. Recombinant E. coli and calf spleen PNPs as well as their mutated forms were expressed in E. coli and purified according to the procedures described earlier [42,43].

Conclusions
A known chemical mutagen and carcinogen, vinyl chloride, acts as a modifier of nucleobases, in particular, adenine and guanine moieties [12], which upon extending the heterocyclic system change the respective coding properties [21, 25,44]. We have found that at least in bacteria the modified nucleosides can be further degraded by the bacterial PNP, the fact that may account for bacteria's resistance to mutagenesis, together with the confirmed role of the bacterial glycosylases [45] and deoxyribosyl-transferases [45,46] in this process. PNP may have also some role in the metabolism of the rare t-RNA component, the Y-base and its nucleoside, wyosine.
Another important application of PNP is in chemo-enzymatic synthesis of bioactive nucleoside analogs, utilizing, among others, various types of PNP [14,16,17,46,47]. This application may be extended to tri-cyclic nucleobase analogs, particularly to adenine, isoguanine, and guanine derivatives. As shown in this and the previous [18] works, various forms of PNP are able to synthesize ribosides of such extended nucleobase analogs. In particular, N9-β-d-riboside of (1) can be obtained quantitatively from (1), and N9-β-dand N7-β-d-ribosides of (4) as a mixture, using the E. coli PNP as a biocatalyst. Additionally, a non-typical N 6 -β-d-riboside (7) of εisoGua (4) can be nearly quantitatively obtained from (4) using the calf enzyme.
The next work (in preparation) shows that also various etheno-derivatives of the amino-purines, including etheno-2-aminopurine isomers, readily react with PNP to give various ribosides. The above results indicate need for further investigations on the enzymatic ribosylation of various nucleobase analogs to improve specificity and yields. Since the number of the known molecular forms of PNP (natural and mutated) is large [13,16], there is a good chance to obtain specific and efficient biocatalysts for a given task.
Finally, attention should be drawn to the new, highly fluorescent nucleoside analogs, derived from mutagenic O 6 -methylguanine and isoguanine. These compounds may be good candidates for the study of ligand-binding equillibria using various PNP forms and for analytical applications.