Multi-Enzymatic Cascades in the Synthesis of Modified Nucleosides: Comparison of the Thermophilic and Mesophilic Pathways

A comparative study of the possibilities of using ribokinase → phosphopentomutase → nucleoside phosphorylase cascades in the synthesis of modified nucleosides was carried out. Recombinant phosphopentomutase from Thermus thermophilus HB27 was obtained for the first time: a strain producing a soluble form of the enzyme was created, and a method for its isolation and chromatographic purification was developed. It was shown that cascade syntheses of modified nucleosides can be carried out both by the mesophilic and thermophilic routes from D-pentoses: ribose, 2-deoxyribose, arabinose, xylose, and 2-deoxy-2-fluoroarabinose. The efficiency of 2-chloradenine nucleoside synthesis decreases in the following order: Rib (92), dRib (74), Ara (66), F-Ara (8), and Xyl (2%) in 30 min for mesophilic enzymes. For thermophilic enzymes: Rib (76), dRib (62), Ara (32), F-Ara (<1), and Xyl (2%) in 30 min. Upon incubation of the reaction mixtures for a day, the amounts of 2-chloroadenine riboside (thermophilic cascade), 2-deoxyribosides (both cascades), and arabinoside (mesophilic cascade) decreased roughly by half. The conversion of the base to 2-fluoroarabinosides and xylosides continued to increase in both cases and reached 20-40%. Four nucleosides were quantitatively produced by a cascade of enzymes from D-ribose and D-arabinose. The ribosides of 8-azaguanine (thermophilic cascade) and allopurinol (mesophilic cascade) were synthesized. For the first time, D-arabinosides of 2-chloro-6-methoxypurine and 2-fluoro-6-methoxypurine were synthesized using the mesophilic cascade. Despite the relatively small difference in temperatures when performing the cascade reactions (50 and 80 °C), the rate of product formation in the reactions with Escherichia coli enzymes was significantly higher. E. coli enzymes also provided a higher content of the target products in the reaction mixture. Therefore, they are more appropriate for use in the polyenzymatic synthesis of modified nucleosides.


Introduction
Nucleoside analogs can be synthesized by chemical or enzymatic methods or by a combination of these methods [1,2]. Chemical synthesis is a long multi-stage process involving the introduction and removal of various protective groups in the carbohydrate residue and heterocyclic base, which leads to a significant decrease in the efficiency of the process. Despite the use of selective glycosylation methods, racemic mixtures are formed in the synthesis of nucleosides, which complicates the isolation of target compounds. Enzymatic synthesis has a number of advantages over chemical synthesis: mild reaction conditions, high stereo-and regioselectivity, the minimal use of polluting chemicals and organic solvents, high efficiency, and the absence of undesirable impurities [1,3,4].
Biocatalytic reactions can involve either one enzyme that catalyzes one specific reaction or several enzymes that act sequentially in a cascade of reactions [3]. In recent years, the terms "cascade reactions" or "tandem reactions" have been used to denote poly-enzymatic reactions [5].
Bacterial glycosyltransferases that catalyze the transfer of the pentofuranosyl group to purine or pyrimidine bases are successfully used in the synthesis of various natural nucleoside analogs of biological and pharmaceutical significance. The donor of the pentofuranosyl residue can be a natural nucleoside or its derivative, while natural or modified heterocyclic bases serve as acceptors [3]. Nucleoside phosphorylases (NP) include purine nucleoside phosphorylases (PNP) and pyrimidine nucleoside phosphorylases (PyNP): uridine phosphorylases (UP) and thymidine phosphorylases (TP). One of the most studied polyenzymatic cascades in the synthesis of modified nucleosides is the sequential use of two NPs. The combination of PNP and PyNP (UP or TP) is often used in the enzymatic synthesis of nucleoside analogs. Such a combination allows obtaining purine nucleosides from pyrimidine ones and vice versa [6]. In addition, some enzymes are involved in the biosynthesis of uridyl peptide antibiotics, such as nikkomycin or tunicamycin [7]. One of the enzymes involved in the biosynthesis of the uridyl peptide antibiotic pacidamycin, dehydratase Pac13, can be used as a biocatalyst for the preparation of 3 -deoxynucleosides [8].
The use of nucleoside phosphorylase-producing microorganisms in the synthesis of modified nucleosides of biological and pharmaceutical significance has proven to be highly effective [9,10]. The transglycosylation reaction is usually carried out at 60 • C to inhibit other enzymes-for example, deaminases. At this temperature, some NPs retain most of their activity. However, reactions involving TP are usually carried out at 45 • C, since thymidine phosphorylase loses activity at temperatures above 50 • C.
Temperature restrictions can be overcome by using thermophilic microorganisms such as Geobacillus stearothermophilus [11] and Thermus thermophilus [4,12,13]. Several strains of T. thermophilus that can synthesize purine nucleosides from adenine or hypoxanthine have been selected [12]. An example of pyrimidine nucleoside synthesis is the enzymatic synthesis of 5-methyluridine from inosine and thymine with the participation of immobilized PNP and PyNP Bacillus stearothermophilus JTS 859 [14]. A similar approach has been used in the synthesis of several modified nucleosides: cladribine (2-chloro-2 -deoxyadenosine), fludarabine (2 -fluoradenine arabinoside), vidarabine (9-β-D-arabinosyladenine), and others. Partially purified preparations of PNP and PyNP G. stearothermophilus B-2194 immobilized on aminopropylated macroporous glass have shown high enzymatic activity and stability at 70 • C and reusability up to 20 times [15]. Immobilized thermophilic nucleoside phosphorylases have been used in the polyenzymatic synthesis of several halogenated nucleoside analogs: cladribine [4], 2-fluoro-2 -deoxyadenosine, and fludarabine [16]. The synthesis of halogenated nucleosides is of particular interest, since they exhibit a broad spectrum of biological activity [9,17].
Recently published data on the activity of the hyperthermophilic PyNP from Thermus thermophilus should be noted [13]. This enzyme works in a wide pH range (4)(5)(6)(7)(8)(9) at 100 • C and relatively high concentrations of organic solvents (up to 80% v/v DMSO and ethylene glycol).
Phosphopentomutases catalyze the reversible transfer of the phosphate group between the C1 and C5 atoms of ribose or deoxyribose [19]. The resulting α-D-ribose-1phosphate is a substrate for nucleoside phosphorylases in the synthesis of nucleosides. Tandem enzymatic cascades of nucleoside synthesis using two enzymes (E. coli PPM and NP combination) have been adapted for the synthesis of D-ribose, D-arabinose, and 2deoxy-D-ribose nucleosides [18,20]. The possibility of the polyenzymatic synthesis of 2 -deoxy-β-D-ribofuranosides-8-aza-purine and 8-aza-7-deazapurine from 2-deoxyribose using ribokinase, PPM, and NP has been shown [21].
Cascades of thermophilic microorganism enzymes can also be used for the synthesis of modified nucleotides. Therefore, in 2016, a cascade of thermophilic enzyme Thermus species 2.9 was presented for the synthesis of nucleotides from D-pentoses [22].
The synthesis of natural nucleosides using various nucleoside phosphorylases yields similar results. However, with modified heterocyclic bases or different carbohydrates, the results may vary significantly. It would be promising to obtain recombinant TthPPM and try the complete cascade of thermophilic RK → PPM → PNP to synthesize modified nucleosides. It is essential to carry out a comparative study of the features and efficiency of the cascade syntheses of nucleosides using mesophilic and thermophilic enzymes. In addition, some heterocyclic bases, such as 2-chloroadenine and 2-fluoroadenine, have low water solubility, so the use of thermophilic enzymes might be preferable. Carrying out the reaction with thermophilic enzymes at the operating temperatures of mesophilic enzymes reduces the benefits of this approach; therefore, the reactions with Thermus thermophilus enzymes were carried out at a higher temperature.
NMR spectra were recorded on Bruker Avance II 700 spectrometers (Bruker BioSpin, Rheinstetten, Germany) in DMSO-d6 at 303 K. Chemical shifts in ppm (δ) were measured relative to the residual solvent signals as internal standards (2.508 ppm). Coupling constants (J) were measured in Hz; s-singlet, br.s.-broad signal, d-doublet, m-multiplet, t-triplet. NMR spectra data were provided in the Supplementary Materials.
Liquid chromatography-mass spectrometry was performed on the Agilent 6210 TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA).
UV spectra were recorded on a Hitachi U-2900 spectrophotometer (Hitachi, Tokyo, Japan).

Cloning, Expression, and Purification of Recombinant TthPPM
The TT_RS08405 gene encoding the phosphopentomutase from Thermus thermophilus HB27 was amplified from genomic DNA by PCR with primer PPM-forward (5 -GGTGGTC ATATGAAGGCGGTGGCCATCGTTTTG-3 ) and PPM-reverse (5 -GGTGGTGCGGCCGCG ACGAGGCTCGTTCCGGGG-3 ) and cloned into the pET-23a+ expression vector at the NdeI and NotI restriction sites. The resulting plasmid pER-PPM-Tth contained the gene encoding the TthPPM with the C-terminal His-tag. The producing strain E. coli NiCo21 (DE3)/pER-PPM-Tth was obtained. Cultivation of the strain was carried out in a lysogeny broth (LB) medium containing 100 µg/mL ampicillin. After reaching an absorbance of A 595 = 0.8, the cultures were added with IPTG to a final concentration of 0.4 mM, and cultivation was continued for 4 h at 37 • C. After culturing, the cell biomass was separated by centrifugation (2.8 g of wet biomass per liter).
The cell biomass (5.6 g) was disrupted in 50 mM KH 2 PO 4 , pH 6.8, 10 mM EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF) in a ratio of 1:10 (w/v) using an ultrasonicator. The clarified cell lysate was heat-treated at 80 • C for 10 min to precipitate the contaminated proteins and DNA. To purify TthPPM, a two-stage technique was developed. In the first step, the protein was purified by anion exchange chromatography using an XK 16/20 column packed with 10 mL of DEAE Sepharose Fast Flow resin and equilibrated with 10 mM KH 2 PO 4 and 1 mM PMSF, pH 6.8. The target protein was eluted with a linear gradient from 0-to 0.4-M NaCl (100 mL, 2 mL/min). The chromatography eluate was loaded onto an XK 16/20 column packed with 20 mL of Chelating Sepharose Fast Flow and equilibrated with 50 mM Tris·HCl, pH 8.5. The second chromatography was performed on an XK 16/20 column packed with 20 mL of Chelating Sepharose Fast Flow and equilibrated with 50 mM Tris·HCl, pH 8.5, and 5 mM EDTA in a linear gradient of 50-200 mM imidazole (100 mL, 2 mL/min). The eluate was concentrated using a 30-kDa cut-off polyethersulfone membrane and then loaded on a HiLoad 16/60 Superdex 75-g size-exclusion column equilibrated with 20 mM Tris·HCl, pH 8.0, supplemented with 50 mM NaCl, 5% glycerol, and 0.04% (w/v) NaN 3 . Fractions containing a target enzyme with purity higher than 90% were combined and concentrated to the final concentration of 10.0 ± 0.5 mg/mL.

Analytical Methods
The protein concentration was determined by the Lowry method, using bovine serum albumin (BSA) as the standard [30]. Protein purity was determined using protein electrophoresis in polyacrylamide gel under denaturing conditions [31].
The reaction mixtures were incubated at 37 • C. Substrate and product quantities were determined using HPLC system 2.

Nucleosides Synthesis
To a solution of MeONa in MeOH (376 mg, 16.2 mmol Na in 8 mL of MeOH), 1.0 g (2.2 mmol) of dichloride (1) was added at room temperature under stirring. In 2 h, the reaction mixture was cooled up to 0 • C, and conc. HCl was added up to pH 7.0. NaCl was filtered off, and the precipitate was washed by hot MeOH (3 × 3 mL). The filtrate was evaporated to a quarter of the volume, 1 mL of H 2 O was added, and the solution was heated to boiling. The clear solution was left at room temperature. The crystals were filtered off and washed with iPrOH and Et 2 O. Yield 560 mg, purity 74%. The desired product was purified by chromatography on silica gel (3 x 30 cm, elution by gradient of MeOH in CHCl 3 (0-20%), 1 L, with a flow rate of 6 mL/min. Yield 310 mg (0.97 mmol, 44%), purity 92.9% ( (5) A 70% solution of HF in pyridine (7.5 mL) was cooled to −18 • C, and 3.20 g (7.4 mmol) of monochloride (4) was added. Some tert-butyl (2.15 mL) (28.5 mmol) was added dropwise within 8 min, and the reaction mixture was stirred at −18 • C for 30 min and at room temperature for 20 min. The mixture was poured into a glass with ice (150 g) and CaCO 3 (15 g), filtered off, and the precipitate was washed by CHCl 3 (150 mL). The organic layer was washed by water (100 mL), 5% NaHCO 3 (100 mL), water (150 mL), dried by MgSO 4, and concentrated. The desired product was isolated by column chromatography on silica gel (2×9 cm; 7 cm of silica gel covered by 2 cm of activated carbon, elution by 200 mL of CHCl 3 , then by 5% MeOH in CHCl 3 , with a flow rate 0.9 mL/min   (6) To a solution of MeONa in MeOH (376 mg, 16.2 mmol Na in 8 mL of MeOH), 1.0 g (2.3 mmol) of nucleoside (5) was added at room temperature under stirring. In 2 h, the reaction mixture was cooled up to 0 • C, and conc. HCl was added up to pH 7.0. NaCl was filtered off, and the precipitate was washed by hot MeOH (3 x 3 mL) and concentrated. The desired product was isolated by column chromatography on silica gel (4.6 x 40 cm, elution by a gradient of MeOH in CHCl 3 (0-30%, 1 L, flow rate 6 mL/min      2.8.7. 9-(β-D-Arabinofuranosyl)-2-fluoro-6-methoxypurine (11) Thirty-six milligrams (0.12 mmol) of riboside (6) were dissolved in 10 mL of 10 mM KH 2 PO 4 (pH 7.0). Fifty microliters of 20 mM Na 2 HAsO 4 and EcPNP (0.068 mg, 15 units, concentration in the reaction mixture 6.8 µg/mL) were added. The solution was heated up to 50 • C and kept for 3 h until the complete conversion of riboside (6) to base (7) (monitored by HPLC System 3, RT(6) = 7.9 min, RT(7) = 6.2 min). The reaction mixture was concentrated twice, cooled down to 4 • C, and kept for 16 h. The supernatant was removed, and the precipitate was washed with cold (4 • C) 10 mM KH 2 PO 4 (pH 7.0) (2 × 5 mL) and

Results
To create a polyenzymatic cascade for the synthesis of modified nucleosides, it was necessary to obtain a sufficient amount of recombinant ribokinase, phosphopentomutase, and nucleoside phosphorylases. We believe that the enzymes from Escherichia coli and thermophilic bacteria Thermus species 2.9 and Thermus thermophilus HB27 have the best prospects for research and subsequent use. The purifications of all the enzymes (except TthPPM) were obtained earlier, the methods of their isolation were optimized, and the substrate characteristics were studied [21][22][23][24]28,29].
To obtain TthPPM, an expression vector was constructed. The TT_RS08405 gene obtained by the amplification of genomic DNA from Thermus thermophilus HB27 was cloned into the pET-23a+ vector. A strain E. coli NiCo21(DE3)/pER-PPM-Tth producing a soluble-form enzyme was created, and a purification protocol was developed. The technique includes the stages of heat precipitation of contaminating proteins and DNA, anion exchange, metal chelate affinity, and size-exclusion chromatography.
Preparations of purified enzymes were characterized by the purity, the content of oligomeric forms, and enzymatic activity (Table 1).

Study of the Influence of Various Factors on TthPPM Activity
The effect of pH, reaction mixture temperature, divalent cations, and cofactor (glucose-1,6-bisphosphate) on TthPPM activity was studied. The enzymatic activity of TthPPM was calculated, allowing for the content of impurity proteins.
The studied phosphopentomutase showed the maximal activity at pH 8.0 (Figure 1a). The enzyme was active in a wide temperature range; the highest activity was observed in the temperature range from 80 to 90 • C (Figure 1b). The measurement of activity in the presence of chlorides of various metals showed that phosphopentomutase can bind various divalent cations, while the maximal activity was observed in the presence of a manganese cation. In the absence of divalent metal cations, the activity of the enzyme significantly decreased (Figure 2a). The studied phosphopentomutase exhibited maximal activity at a concentration of manganese ions of 0.5 mM (Figure 2b). Glucose-1,6-bisphosphate increased the activity of TthPPM (Figure 3), although its presence in the reaction was not necessary.
The data obtained indicate that the affinity of D-ribose-5-phosphate and 2-deoxy-Dribose-5-phosphate for the active site of TthPPM differs insignificantly. Thus, the presence of an OH group in the second position does not significantly affect the binding to the active site. At the same time, the reaction rate in the active site for D-ribose-5-phosphate is 7.1 times higher.
The activity of all the enzymes used in this study was determined (Table 3). For thermophilic enzymes, the activity was determined at 80 • C and, for mesophilic enzymes, at 37 • C. As the activity of TspRK was 16 times lower compared to EcRK, more proteins were added to the reaction mixture. In this case, the activity of phosphopentomutase and purine nucleoside phosphorylase II (with respect to adenosine) of Thermus thermophilus was higher than that of E. coli enzymes, while the activity of TthPNPI (with respect to inosine) and EcPNP was the same. Table 2. Kinetics of α-D-ribose 1-phosphate and 2-deoxy-α-D-ribose 1-phosphate synthesis by phosphopentomutase Thermus thermophilus HB27. Each reaction mixture (100 µL, 20 mM Tris·HCl, pH 8.0) contained 1-mM adenine, 0.5 mM MnCl 2 , and 0.25 µM α-D-glucose 1,6-bisphosphate, from 0.011 to 1.8 mM D-ribose 5-phosphate or 2-deoxy-D-ribose 5-phosphate, 0.02 or 0.05 µg TthPPM, and 0.5 µg TthPNPII. The reaction mixtures were incubated 2 min at 80 • C.

Synthesis of Modified Heterocyclic Bases for Cascade Synthesis of Nucleosides
The use of a thermophilic cascade could be advantageous in the synthesis of 2,6disubstituted purine arabinosides, since the solubility of 2-chloro-and 2-fluoradenine (for example) is known to be under 0.4 mM at 50 • C. Model reactions to determine the effectiveness of the thermophilic and mesophilic cascades were performed using 2-chloroadenine as a pentose acceptor.
After identifying the polyenzymatic cascade conditions, it was useful to synthesize both already known nucleosides (8-azaguanosine and allopurinol riboside) and new modified nucleosides using the cascade approach.
We decided to synthesize two bases: 2-chloro-and 2-fluoro-6-methoxypurine, and to obtain the corresponding arabinosides by a cascade of enzymes. From our experience, the optimal approach to the synthesis of 2-chloro-and 2-fluoro-6-methoxypurines is chemicalenzymatic (Figures 5 and 6). This variant of the synthesis has not been previously described in the literature.
All chemical transformations of bases were carried out on ribonucleosides with acetyl protection of the ribose residue. In this case, the protected ribose acts as a protection for the highly reactive N9 in purine.
In addition, the solubility of the nucleosides (both protected and unprotected) in most organic solvents is significantly higher than the solubility of the corresponding bases.
E. coli PNP can perform arsenolysis of a nucleoside bond. 1-α-Ribose arsenate is formed in the active site instead of 1-α-phosphate. Ribose arsenate is rapidly hydrolyzed to ribose and inorganic arsenate, and the equilibrium of the enzymatic reaction shifts towards the formation of base (3). E. coli PNP and Na 2 HAsO 4 were added to the riboside (2) solution. The reaction mixture was incubated at 60 • C for several hours. Then, the mixture was concentrated to a minimal volume and kept at 4 • C for a while. The precipitate (2-chloro-6-methoxypurine (3)) was used in the synthesis of arabinoside without additional purification.
Product (5) was isolated by column chromatography on silica gel. 2-Fluoro-6-methoxyriboside (6) was prepared by a treatment with sodium methylate in methanol according to the procedure described above. The target riboside of 2-fluoro-6-methoxypurine was isolated by column chromatography on a silica gel. The yield of nucleoside (6) was 44%, and the byproduct, according to NMR data, was 2,6-dimethoxypurine riboside. The target product (7) was synthesized by the same method as base (3) and used in the subsequent transformation without purification.

Conditions for Poly-Enzymatic Cascades Using Mesophilic and Thermophilic Enzymes
A series of poly-enzymatic cascades using recombinant thermophilic enzymes and E. coli enzymes was performed. The cascade of enzymatic reactions includes the sequential conversion of D-pentoses to 5-monophosphates, catalyzed by ribokinase (RK), the conversion of 5-phosphates to α-D-pentose-1-phosphates, catalyzed by phosphopentomutase (PPM), and the condensation of them with the corresponding base, catalyzed by purine nucleoside phosphorylase (PNP). This leads to the formation of the desired β-D-nucleoside (Figure 7). The reactions were monitored using the liquid chromatography-mass spectrometry (LC-MS) method. The conditions for the polyenzymatic cascade with mesophilic E. coli enzymes were determined earlier [18,33]: temperature 50 • C, pH 8.0, and the presence of potassium chloride and manganese ions. In the case of thermophilic enzymes, the conditions differed only in temperature (80 • C).
When selecting conditions for cascade reactions, previous experience with these enzymes was taken into account [18,22,33]. High concentrations of arabinose and xylose were due to their poor affinity for the active site of ribokinase. In addition, at ATP concentrations above 3 mM, ribokinase inhibition is observed.
As we expected, at first glance, the conditions for conducting polyenzymatic cascades of nucleoside synthesis are very similar, except for the temperature of reaction mixtures. However, there is a significant difference in synthesis efficiency and the stability of products under the conditions of obtaining 2-chloroadenine nucleosides.
For 8-azaguanine, the efficiency of the thermophilic cascade differed slightly from the mesophilic one. Therefore, we decided to synthesize 8-azaguanosine (8) using thermophilic enzymes, since the increased temperature provided acceptable solubility of the starting heterocycle. E. coli enzymes were used to obtain the allopurinol riboside (9), as they provide higher conversion of the base to riboside (96% per day).
The products were isolated by column chromatography. The yields of nucleosides (10) and (11) were 54% and 42%, respectively.

Discussion
To understand the peculiarities of the mesophilic and thermophilic cascades in the synthesis of 2-chloroadenine riboside, 2-deoxyriboside, arabinoside, 2-deoxy-2-fluoro-D-arabinoside, and xyloside, it is necessary to scrutinize the dynamics of nucleoside formation.

Possibilities of Using Various D-Pentoses in the Synthesis of 2-Chloroadenine Nucleosides
D-ribose, 2-deoxy-D-ribose, and their phosphates are natural substrates of nucleic acid and carbohydrate metabolism enzymes. Therefore, their conversion in a cascade with thermophilic enzymes proceeded quite intensively. The maximal content of 2-chloroadenosine (Ribo-2ClAde) was 78% after 30 min, and 2 -deoxy-2-chloroadenosine (dRibo-2ClAde) was 60% after 30 min (Figure 8). Interestingly, the equilibrium of the cascade synthesis in the synthesis of the 2-chloroadenine riboside by E. coli enzymes was shifted towards nucleoside formation. However, under the conditions of a thermophilic cascade, the maximal product concentration was observed after 30 min, and then, the equilibrium began to shift in the opposite direction, which led to a twofold decrease of product content in the reaction mixture. These results may be due to the increasing hydrolysis rate of α-D-ribose-1-phosphate with the temperature increasing [34]. The content of 2 -deoxy-2-chloroadenosine also sharply decreased over time due to the equilibrium shift towards the opposite reaction because of the hydrolysis of 2-deoxy-α-D-ribose-1-phosphate (Figure 8b).
In the cascade reaction with E. coli enzymes, the maximal content of 2-chloroadenosine (91%) was observed after one hour and 2 -deoxy-2-chloroadenosine (73%) after 30 min. The content of 2-chloroadenosine in the reaction mixture remained constant. The content of 2 -deoxy-2-chloroadenosine gradually decreased over time due to the shift of equilibrium towards the opposite reaction. However, the rate of the opposite reaction was noticeably lower than in the cascade with thermophilic enzymes.
The conversion of D-xylose in the thermophilic cascade was very slow, and the maximal conversion of 2-chloroadenine to 9-(β-D-xylofuranosyl)-2-chloroadenine was only 3.9% after 24 h (Figure 9a). The maximal content of 9-(β-D-xylofuranosyl)-2-chloroadenine was 26% after 24 h in the mesophilic cascade, which is much higher than in the cascade with thermophilic enzymes.
The conversion of 2-deoxy-2-fluoro-D-arabinose to 9-(2 -deoxy-2 -fluoro-β-D-Arabinofuranosyl)-2-chloroadenine (clofarabine) (Figure 9b) proceeded slower than the synthesis of arabinosides (the maximal content was 19% after 24 h, from a thermophilic cascade). The conversion of 2-deoxy-2-fluoro-D-arabinose to clofarabine proceeded much better in the mesophilic cascade than in the cascade with thermophilic enzymes (the maximal content of clofarabine was 46% versus 19% after 24 h). Kamel with coworkers [35] investigated the stability of some pentose-1-phosphates at various temperatures. The stability of α-D-arabinose-1-phosphate was higher than that of α-D-ribose-1-phosphate and 2-deoxy-α-D-ribose-1-phosphate, but all these compounds were hydrolyzed. The stability of 2-deoxy-2-fluoro-α-D-arabinose-1-phosphate was much higher than that of all other pentose-1-phosphates. A slow decreasing of the product content after 24h in the cascade with D-arabinose and the absence of decreasing in the cascade with 2-deoxy-2-fluoro-D-arabinose may be due to a low rate of 1-phosphate intermediate hydrolysis.
The lower rate of D-arabinosides synthesis in comparison with D-ribosides and 2deoxy-D-ribosides can be explained by the different conformations of carbohydrates in the active site of purine nucleoside phosphorylase. For this reason, the rate of D-arabinosides phosphorolysis is also much lower [36].

Non-Natural Heterocyclic Bases in the Cascade Synthesis of Nucleosides
To determine the possibility of practical use of thermophilic or mesophilic cascades, we tried to synthesize several non-natural nucleosides. We selected 8-azaguanine, allopurinol, 2-chloro-6-methoxypurine (3), and 2-fluoro-6-methoxypurine (7) as the heterocyclic bases, taking into account the prospect of subsequent biological testing of the corresponding modified nucleosides.

Synthesis of 8-Azaguanine and Allopurinol Ribosides
We investigated the formation of 9-(β-D-ribofuranosyl)-8-azaguanine and 9-(β-Dribofuranosyl) allopurinol from 8-azaguanine and allopurinol, respectively. In both cases, the transformation proceeded at a high rate. The formation of 9-(β-D-ribofuranosyl) allopurinol (Figure 10a) in the reaction with E. coli enzymes proceeded at a high rate; its maximal content was 95% after eight hours and did not change over time. The maximal content of 9-(β-D-ribofuranosyl) allopurinol in the thermophilic cascade was 73% after eight hours; then, the content decreased. The rate of conversion of 8-azaguanine to 8azaguanosine  in the mesophilic cascade was higher than in the cascade with thermophilic enzymes (Figure 10b). 8-Azaguanosine gradually accumulated in the reaction mixture with E. coli enzymes, and its maximum content was 89% after 24 h.
All synthesized nucleosides were characterized by LC-MS data. The structure was confirmed by NMR spectra: 1   The data obtained indicated that, despite the relatively small difference in temperatures when performing the cascade reactions (50 and 80 • C), the rate of product formation in the reactions with E. coli enzymes was much higher. The E. coli enzymes also provided a higher content of target products in the reaction mixture. Therefore, they were more appropriate for use in the polyenzymatic synthesis of modified nucleosides.

Conclusions
To carry out a comparative study of the possibilities of using the RK → PPM → NP cascade in the synthesis of modified nucleosides, sufficient quantities of genetically engineered enzymes of both E. coli and thermophilic Thermus species 2.9 and Thermus thermophilus HB27 were obtained. Recombinant phosphopenthomutase PPM from Thermus thermophilus HB27 was obtained for the first time: a strain-producer of the soluble form of the enzyme was created, and a procedure for its isolation and chromatographic purification was developed. The influence of the pH, temperature, presence of divalent cations, and cofactor (glucose-1,6-bisphosphate) on the activity of TthPPM was studied. The maximum activity was observed at pH 8.0. The enzyme is active over a wide temperature range, with the maximum activity in the interval between 80 and 90 • C. Glucose-1,6-bisphosphate significantly increased the activity of TthPPM, although its presence in the reaction was not necessary.
The preparations of all purified enzymes were characterized by purity, the content of the oligomeric forms, and enzymatic activity.
Four D-ribose and D-arabinose nucleosides were synthesized on a large scale by a cascade of enzymes. 8-Azaguanine riboside was synthesized by the thermophilic cascade with a 69% yield. Allopurinol riboside was obtained by the mesophilic cascade with an 84% yield. For the first time, 2-chloro-6-methoxypurine and 2-fluoro-6-methoxypurine D-arabinosides were synthesized using the mesophilic cascade (yields 54% and 42%, respectively). The data obtained indicated that, despite the relatively small difference in temperatures when performing the cascade reactions (50 and 80 • C), the rate of the product formation in the reactions with E. coli enzymes was much higher. The E. coli enzymes also provided a higher content of products in the reaction mixtures. Therefore, they are more appropriate for use in the polyenzymatic synthesis of modified nucleosides. The use of thermophilic enzymes and a high reaction temperature might be preferable with low-soluble heterocyclic bases (2-chloroadenine and 2-fluoroadenine), as well as in the case of using carbohydrates that form 1-phosphates with high temperature stability (D-arabinose and 2-deoxy-2-fluoro-D-arabinose).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.