Use of nucleoside phosphorylases for the preparation of 5-modified pyrimidine ribonucleosides

Journal Pre-proof Use of nucleoside phosphorylases for the preparation of 5-modified pyrimidine ribonucleosides

Cyril S. Alexeev, Mikhail S. Drenichev, Evgeniya O. Dorinova, Roman S. Esipov, Irina V. Kulikova, Sergey N. Mikhailov PII:

S1570-9639(19)30178-5

DOI:

https://doi.org/10.1016/j.bbapap.2019.140292

Reference:

BBAPAP 140292

To appear in:

BBA - Proteins and Proteomics

Received date:

24 July 2019

Revised date:

25 September 2019

Accepted date:

6 October 2019

Please cite this article as: C.S. Alexeev, M.S. Drenichev, E.O. Dorinova, et al., Use of nucleoside phosphorylases for the preparation of 5-modified pyrimidine ribonucleosides, BBA - Proteins and Proteomics(2019), https://doi.org/10.1016/j.bbapap.2019.140292

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© 2019 Published by Elsevier.

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Use of nucleoside phosphorylases for the preparation of 5modified pyrimidine ribonucleosides Cyril S.b Alexeeva, #1, Mikhail S. Drenicheva, ‡1, Evgeniya O. Dorinovaa, Roman S. a a Esipov , Irina V. Kulikova , Sergey N. Mikhailov * Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow, 119991 Russia. b Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia * Correspondence to [email protected], sergey.mikhailov1949@g mail.co m. 1 The authors contributed equally. # [email protected], [email protected] m ‡ [email protected]

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Abstract Enzymatic transglycosylation, a transfer of the carbohydrate moiety from one heterocyclic base to another, is catalyzed by nucleoside phosphorylases (NPs) and is being actively developed and applied for the synthesis of biologically important nucleosides. Here, we report an efficient one-step synthesis of 5substitited pyrimidine ribonucleosides starting from 7-methylguanosine hydroiodide in the presence of nucleoside phosphorylases (NPs).

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Keywords: Enzymes, nucleoside phosphorylases, nucleosides, phosphorolysis, transglycosylation, uridine.

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1. Introduction

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Nucleosides and their derivatives are the major components of natural products and are involved in various biochemical processes, particularly in the storage and transfer of genetic information. Investigation of nucleosides as natural compounds is among the most promising fields of research. Thus, about a hundred of 100 nucleoside-like drugs have been developed. One-half of antiviral drugs and a quarter of antitumor drugs are derived from nucleosides. Diverse therapeutic applications of nucleoside analogues and the need to develop new compounds of this series have facilitated efforts in optimizing their synthesis. Enzymatic transglycosylation, a transfer of the carbohydrate residue from one heterocyclic base to another, is being actively developed and applied for the synthesis of practically important nucleosides. These reactions are catalyzed by nucleoside phosphorylases (NPs) that perform reversible phosphorolysis of nucleosides to yield the corresponding heterocyclic base and monosaccharide 1-phosphate [1-3]. The equilibrium of these reactions is shifted towards the formation of nucleosides [4-5], more significantly in the case of purine. The overall transglycosylation process may be represented as two consecutive equilibrium reactions (Scheme 1). One or two different NPs may be utilized in this process; their choice depends on the starting nucleoside (Nuc-1), heterocyclic base (B2), and the resulting nucleosides (Nuc-2).

Scheme 1. Enzymatic phosphorolysis of ribonucleosides and transglycosylation; NP1 and NP2 are nucleoside phosphorylases, B1 and B2 are heterocyclic bases.

We showed that the initial concentrations of the starting compounds and the phosphorolysis equilibrium constants of the starting and final glycosides determine the concentrations of all the components at the equilibrium state [5]. Therefore, the main requirement for substrates of phosphorolytic transglycosylation is as follows: the higher the Keq 2/Keq 1 ratio, the higher the outcome of the products (Nuc-2). The constants for phosphorolysis of natural pyrimidine nucleosides are approximately 20 times higher than the constants for purine nucleosides [5]. Hence, the starting pyrimidine nucleoside (Nuc-1) is preferable over purine nucleoside. The analysis of these reactions shows that the highest yield of Nuc-2 may be expected when the equilibrium of step 1 is shifted towards the formation of α-D-ribofuranose-1-phosphate (Rib-1-P) and the equilibrium 2 is shifted towards the target nucleoside (Nuc-2). Practically irreversible phosphorolysis of 7-methylguanosine (7-MeGuo) may further increase the concentration of Rib-1-P and lead to a significant shift of the transglycosylation equilibrium towards the target Nuc-2. The phosphorolysis of 7methylguanosine in the presence of PNP was shown to produce Rib-1-P and 7-methylguanine (7-MeGua) in practically quantitative yields [12]. Recently, we have demonstrated that 7-methyl-2ʹ-deoxyguanosine is an excellent source of 2′-deoxyribose-1-phosphate in transglycosylation reactions [14]. In this paper, we report our recent results in the efficient synthesis of 5-substituted pyrimidine ribonucleosides. 7-

Journal Pre-proof Methylguanosine (7-MeGuo) is often used for the preparation of purine ribonucleosides [6-13]; however, its application for the synthesis of pyrimidine ribonucleosides is not well-documented. Nucleoside phosphorylases are involved in salvage pathways of nucleoside biosynthesis. This class of enzymes includes thymidine phosphorylase (TP; EC 2.4.2.4), uridine phosphorylase (UP; EC 2.4.2.3), and purine nucleoside phosphorylase (PNP; EC 2.4.2.1). Thymidine phosphorylase is the most specific enzyme, utilizing thymidine as the substrate, UP catalyzes phosphorolysis of uridine, while PNP may cleave the N-glycosidic bond both in purine ribo- and 2'-deoxyribonucleosides [15-16]. These enzymes are found in virtually all organisms. They are widely used as catalysts in the synthesis of biologically important nucleosides via the transglycosylation reaction [17-19]. In the methodology proposed for the synthesis of 5-substituted uridine derivatives, we utilized a small excess of 7-MeGuo (1.5 equiv.) as a donor of Rib-1-P and 0.5 equiv. of phosphate compared to the starting 5-substituted uracil. The use of reagents in a small excess reduces the cost of the synthesis and greatly simplifies the separation of the target compounds by chromatography. We synthesized pyrimidine ribonucleosides using E.coli PNP, which catalyzed the phosphorolysis of 7-MeGuo, and E.coli UP, which catalyzed the ribosylation of 5-substituted uracils.

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2. Material and methods

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The solvents and materials were of reagent grade and were used as received. Column chromatography was performed on silica gel (Kieselgel 60 Merck, 0.0630.200 mm). TLC was carried out on Alugram SIL G/UV254 (Macherey-Nagel) with visualization by UV light ( = 254 nm). UV spectra were recorded on a Cary 300 UV/VIS spectrometer (Varian, Australia). HPLC analysis was performed using an Akvilon HPLC gradient system (Russia; 2×Stayer pumps (2nd series), Stayer MS16 dynamic mixer, and Stayer 104M UV-Vis detector). HPLC-analysis of transglycosylation reactions was performed on chromatographic column 4×150mm Dr. Maisch HPLC column (5µm, Reprosil-Pur C18-AQ 120 Å, Part No r15.aq.s1504, Dr. Maisch HPLC GmbH (Germany), in linear gradient of acetonitrile in deionized water (unmodified mobile phase) from 2 to 12% for 10 min (flushing with 12-80% acetonitrile/deionized water for 10-10.1 min, then 80-2% for 10.1-10.8 min) at flow rate 1 mL/min with UV detection at optimal (260 nm for uracil (Ura), 5-methyluracil (5-MeUra), 5-ethyluracil (5-EtUra), 5-fluorouracil (5FUra), 276 nm 5-chlorouracil (5-ClUra), 275 nm for 5-bromouracil (5-BrUra), 285 nm for 5-iodouracil (5-IUra)) wavelength, injection volume 20 μL. HPLC analysis of transglycosylation products was performed under the same conditions with UV detection at optimal wavelength. The phosphorolysis constants (Kp ) were determined as described in [5]. Column chromatography was performed on Kieselgel (0.040–0.063 mm, Merck). 1 H NMR spectra were measured on a Bruker AMX 300 NMR spectrometer (Germany) at 300 MHz and 303 K and on a Bruker AMX 400 NMR at 400 MHz and 303 K. Chemical shifts were measured in ppm relative to the residual signals of the solvent as internal standards (in DMSO-d 6 , 1 H, 2.50 ppm; in D2 O, 1 H, 4.79 ppm). Melting points were determined on a Electrothermal apparatus (Great Britain) and are uncorrected. Spin-spin coupling constants (J) are given in Hz. Guanosine dihydrate, uracil, thymine (5-methyluracil), 5-fluorouracil, 5-ethyluracil, 5-chlorouracil, 5-bromouracil, and 5-iodouracil and the corresponding ribonucleosides were purchased from Sigma Aldrich (sigmaaldrich.com).

2.1. Enzymes

In the present work, we used recombinant enzymes. E. coli purine nucleoside phosphorylase (PNP, 295 U/mL, 32 mg/mL solution) was purchased from Sigma–Aldrich (United States)) and E. coli uridine phosphorylase (8.4 mg/mL solution, 81.9 U/mg) was kindly provided by Dr. Roman Esipov (Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences , 16/10 ul. Miklukho-Maklaya, 117997 Moscow, Russia) [20].

2.2. General transglycosylation protocol (analytic reactions, Table 1) To a sample solution (1 mL), 2 µL of 2.50 mg/mL solution of E. coli UP (0.182 U) and 3 µL of 3.99 mg/mL solution of E. coli PNP (0.24 U) were added. The reaction mixture was incubated at 20o C. The reaction was monitored by HPLC. The equilibrium was considered to be established when the concentrations of the components in the direct and the reverse reactions were similar.

2.3. Nucleoside synthesis 2.3.1. 7-Methylguanosine hydroiodi de (1) To a suspension of guanosine dihydrate (5 g, 15.6 mmol) in dry DMF (100 mL), iodomethane (4.4 mL, 70.5 mmol) was added dropwise with vigorous stirring. The reaction mixture was refluxed in a 250 mL flask at 30°C for 24 h until the formation of the transparent yellow solution and then allowed to stand for 1 h on air in a fume hood. The

Journal Pre-proof reaction mixture was filtered through celite (15 mL). The transparent liquid filtrate was added dropwise to dichloromethane (1.1 L) at 0°С, and the mixture was allowed to stand at 0°С for 16 h. The precipitate was filtered off, washed with diethyl ether (100 mL) and dichloromethane (100 mL), and dried using a vacuum pump at 45°C for 1 h to yield 5.6 g (84%) as white powder. M.p.159-160°C dec. Rf 0.095 (dichloromethane–ethanol, 1 : 1, v/v). 1 H NMR (300 MHz, DMSO-d 6 , δ, J/Hz): 11.66 br s (1H, NH 7-MeGua), 9.34 s (1H, H8 7-MeGua), 7.19 br s (2H, NH2 7-MeGua), 5.83 d (1H, J1′ 2′ = 3.9, H1′ ), 5.60 d (1H, 3 J = 5.1, OH), 5.30 d (1H, 3 J = 4.0, OH), 5.14-5.02 m (1H, 5ʹOH), 4.37 ddd (1H, J2′ 3′ = 5.3, J2′ 1′ = 3.9, J2′ OH = 4.0, H2′ ), 4.15 ddd (1H, J3′ 2′ = 5.3, J3′ 4′ = 3.6, J3′ OH = 5.1, H2′ ), 4.033.96 m (4H, CH3 + H4′ ), 3.72 br d (1H, J5′ a5′ b = -12.3, H5′ a), 3.60 br d (1H, J5′ b5′ a = -12.3, H5′ b). UV (H2 O): pH 6: λ max (ε) = 257 nm (11600); λ max (ε) = 279 nm (8000) (Lit. pH 7: λ max (ε) = 258 nm (8500); λ max (ε) = 281 nm (7400) [21]); UV (50 mM Tris-HCl buffer, pH 7.5): λ max (ε) = 257 nm (7800); λ max (ε) = 281 nm (8600). 2.3.2. Ribothymi dine (2b)

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To a solution of thymine (Thy, 37 mg, 0.3 mmol), 7-methylguanosine hydroiodide (7-MeGuo, 191 mg, 0.45 mmol) and potassium dihydrophosphate (KH2 PO4 , 20 mg, 0.15 mmol) in 50 mM Tris -HCl buffer, pH 7.5 80 mL) at ambient temperature, 5 µL of 32 mg/mL E. coli purine nucleoside phosphorylase (PNP) solution (1.48 U) and 12.5 µL E. coli uridine phosphorylase (UP) (8.60 U) were added in one portion. The reaction mixture was allowed to stand for 5 h at ambient temperature under neat stirring and then allowed to stand for 15 h at ambient temperature without stirring. The conversion of the initial 7-methylguanosine was controlled by HPLC. The reaction mixture was cooled to 0°C and then filtered through nitrocellulose membrane Whatman (0.2 µm, 25 mm) to remove the white precipitate of 7-methylguanine (7-MeGua). The precipitate was washed with milli-Q water (15 mL). The combined transparent filtrate was concentrated under reduced pressure using a rotary evaporator to ca. 2 mL (bath temperature ˂ 35°C) and diluted with ethanol (25 mL). Then silica gel (5 mL) was then added to the suspension. The resulting mixture was concentrated to near dryness and co-evaporated with ethanol (2×20 mL). The dry residue was applied on a chromatographic column (diameter 20 mm) with silica gel (20 mL) for purification. For the protection, the silica gel layer was topped with ca 0.5-cm layer of sand. The column was washed with dichloromethane (25 mL), a mixture of dichloromethane and ethanol (95:5, v/v, 50 mL), and a mixture of dichloromethane and ethanol (90:10, v/v, 100 mL). The product was eluted with dichloromethane : ethanol (80:20, v/v, 100 mL) and 10 mL fractions were collected and evaporated in vacuo to dryness. The residue was co-evaporated 5 times with dichloromethane and dried using a vacuum pump at r.t. for 1 h. Yield 58 mg (75%) as a white powder. M.p. 166-171°C dec. (Lit. 183-187°C [22]). Rf 0.74 (dichloromethane : ethanol, 9:1, v/v). 1 H NMR (300 MHz, D2 O, δ, J/Hz): 7.76 q (1H, 4 JH-Me=1.2, H6 Thy), 5.99 d (1H, J1′ 2′ = 4.8, H1′ ), 4.42 dd (1H, J2′ 1′ = 4.8, J2′ 3′ = 5.4, H2′ ), 4.32 t (1H, J3′ 2′ = J3′ 4′ = 5.4, H3′ ), 4.20 ddd (1H, J4′ 3′ = 5.4, J4′ 5′ a= 3.1, J4′ 5′ b = 4.3, H4′ ), 3.99 dd (1H, J5′ a4′ = 3.1, J5′ a5′ b = -12.7, H5′ a), 3.88 dd (1H, J5′ b4′ = 4.3, J5′ b5′ a = -12.7, H5′ b), 1.97 d (1H, 4 JMe-H =1.2, CH3 Thy). UV (H2 O): pH 2-7: λ max (ε) = 266 nm (9600); pH 13: λ max (ε) = 266 nm (7400). 2.3.3. 5-Ethyluridine (2c)

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Compound 2c was synthesized in a similar way from 5-ethyluracil (80 mg, 0.57 mmol) in a yield of 104 mg (67%) as white powder. M.p. 187-190°C dec. Rf 0.55 (dichloromethane : ethanol, 8:2, v/v). 1 H NMR (300 MHz, D2 O, δ, J/Hz): 7.75 t (1H, 4 J = 1.0, H6 5-Et-Ura), 5.98 d (1H, J1′ 2′ = 4.4, H1′ ), 4.42 dd (1H, J2′ 1′ = 4.4, J2′ 3′ = 5.4, H2′ ), 4.32 dd (1H, J3′ 2′ = 5.4, J3′ 4′ = 5.5, H3′ ), 4.19 ddd (1H, J4′ 3′ = 5.5, J4′ 5′ a= 3.0, J4′ 5′ b = 3.9, H4′ ), 4.00 dd (1H, J5′ a4′ = 3.0, J5′ a5′ b = 12.8, H5′ a), 3.87 dd (1H, J5′ b4′ = 3.9, J5′ b5′ a = -12.8, H5′ b), 2.38 qd (2H, 3 J = 7.5, 4 J = 1.0, CH2 ), 1.15 t (3H, 3 J = 7.5, CH3 ). UV (H2 O): pH 2-7: λ max (ε) = 266 nm (9400); pH 13: λ max (ε) = 266 nm (7200). 2.3.4. 5-Fluorouridine (2d) Compound 2d was synthesized in a similar way from 5-fluorouracil (80 mg, 0.61 mmol) in a yield of 122 mg (76%) as white powder. M.p. 174-176°C dec. (Lit. 182°C [22]). Rf 0.23 (dichloromethane : ethanol, 9:1, v/v). 1 H NMR (300 MHz, D2 O, δ, J/Hz): 8.15 d (1H, 3 JH-F=6.5, H6 5-F-Ura), 5.97 dd (1H, J1′ 2′ = 4.6, 5 JH-F=1.5, H1′ ), 4.39 dd (1H, J2′ 1′ = 4.6, J2′ 3′ = 5.2, H2′ ), 4.29 dd (1H, J3′ 2′ = 5.2, J3′ 4′ = 5.4, H3′ ), 4.20 ddd (1H, J4′ 3′ = 5.4, J4′ 5′ a= 2.9, J4′ 5′ b = 4.1, H4′ ), 4.00 dd (1H, J5′ a4′ = 2.9, J5′ a5′ b = -12.8, H5′ a), 3.87 dd (1H, J5′ b4′ = 4.1, J5′ b5′ a = -12.8, H5′ b). UV (H2 O): pH 2-7: λ max (ε) = 269 nm (8900); pH 13: λ max (ε) = 269 nm (6800). 2.3.5. 5-Chlorouridine (2e) Compound 2e was synthesized in a similar way from 5-clorouracil (50 mg, 0.34 mmol) in a yield of 61 mg (65%) as slightly gray powder. M.p. 196.5-197°C dec. (Lit. 212-214°C dec, 245°C, 220-223°C [23]). Rf 0.25 (dichloromethane : ethanol, 9:1, v/v). 1 H NMR (400 MHz, D2 O, δ, J/Hz): 8.28 s (1H, H6 5-ClUra), 5.95 d (1H, J1′ 2′ = 4.0, H1′ ), 4.39 dd (1H, J2′ 1′ = 4.0, J2′ 3′ = 5.2, H2′ ), 4.29 t (1H, J3′ 2′ = 5.2, J3′ 4′ = 5.2, H3′ ), 4.19 ddd (1H, J4′ 3′ = 5.2, J4′ 5′ a=

Journal Pre-proof 2.8, J4′ 5′ b = 3.9, H4′ ), 4.01 dd (1H, J5′ a4′ = 2.8, J5′ a5′ b = -12.9, H5′ a), 3.88 dd (1H, J5′ b4′ = 3.9, J5′ b5′ a = -12.9, H5′ b). UV (H2 O): pH 2-7: λ max (ε) = 277 nm (10400) (Lit. λ max (ε) = 277 nm (10600) [23]); pH 13: λ max (ε) = 275 nm (7500). 2.3.6. 5-Bromouridine (2f) Compound 2f was synthesized in a similar way from 5-bromouracil (50 mg, 0.34 mmol) in a yield of 112 mg as white powder. M.p. 192.5-193°C dec. (Lit. 203-204°C dec, 181-184°C [23]). Rf 0.30 (dichloromethane : ethanol, 9:1, v/v). 1 H NMR (400 MHz, D2 O, δ, J/Hz): 8.40 s (1H, H6 5-BrUra), 5.95 d (1H, J1′ 2′ = 3.8, H1′ ), 4.38 dd (1H, J2′ 1′ = 3.8, J2′ 3′ = 5.2, H2′ ), 4.29 dd (1H, J3′ 2′ = 5.2, J3′ 4′ = 6.6, H3′ ), 4.19 ddd (1H, J4′ 3′ = 6.6, J4′ 5′ a= 2.8, J4′ 5′ b = 3.8, H4′ ), 4.01 dd (1H, J5′ a4′ = 2.8, J5′ a5′ b = -12.9, H5′ a), 3.88 dd (1H, J5′ b4′ = 3.8, J5′ b5′ a = -12.9, H5′ b). UV (H2 O): pH 2-7: λ max (ε) = 279 nm (10500) (Lit. λ max (ε) = 279 nm (10600) [23]); pH 13: λ max (ε) = 277 nm (7800). 2.3.7. 5-Iodouri dine (2g)

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Compound 2g was synthesized in a similar way from 5-iodouracil (51 mg, 0.213 mmol) in a yield of 45 mg (58%) as slightly yellow foam. M.p. 193.5-194°C dec (Lit. 205-208 dec, 208-209 dec, 208-210°C dec. [23]) Rf 0.39 (dichloromethane : ethanol, 9:1, v/v). 1 H NMR (300 MHz, D2 O, δ, J/Hz): 8.50 s (1H, H6), 5.94 d (1H, J1′ 2′ = 3.7, H1′ ), 4.39 dd (1H, J2′ 1′ = 3.7, J2′ 3′ = 5.2, H2′ ), 4.32 dd (1H, J3′ 2′ = 5.2, J3′ 4′ = 6.1, H3′ ), 4.20 ddd (1H, J4′ 3′ = 6.1, J4′ 5′ a= 2.8, J4′ 5′ b = 3.4, H4′ ), 4.05 dd (1H, J5′ a4′ = 2.8, J5′ a5′ b = -12.9, H5′ a), 3.95 dd (1H, J5′ b4′ = 3.4, J5′ b5′ a = -12.9, H5′ b). UV (H2 O): pH 2-7: λ max (ε) = 286 nm (7300) (Lit. λ max (ε) = 286 nm (7300) [23]); pH 13: λ max (ε) = 282 nm (5500).

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3. Results and Discussion

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Previously, it was shown that phosphorolysis of 7-MeGuo in the presence of PNP produces Rib-1-P and 7-Me-Gua in quantitative yields [12-13, 24]. When using 7-MeGuo as a donor of Rib-1-P in the nucleoside synthesis by enzymatic transglycosylation (see Scheme 1, step 1), the precipitation of 7MeGua from the reaction mixture shifts the equilibrium towards the formation of Rib-1-P. Therefore, we used 7-MeGuo as the hydroiodide salt for the preparation of pyrimidine ribonucleosides. We applied the original method, which was elaborated by Robins and co-workers [25], to synthesize 7methylguanosine in 65% yield as white fluffy crystals of high purity, which is in full agreement with the literature data. We also used an alternate reaction of guanosine monohydrate with MeI in dry N,Ndimethylformamide (DMF) at 30°C (Scheme 2, step i). The product was isolated in 84% yield as white powder after the precipitation from the DMF medium with dichloromethane. The purity of 7methylguanosine hydroiodide was estimated at > 98% by 1 H-NMR spectroscopy (see Supplementary Data). According to UV spectroscopy, 7-MeGuo is stable in Tris-HCl buffer at pH 7.5 for nearly one month at 20°C, without noticeable decomposition, which is consistent with our previous findings [14]. To optimize the reagent ratio, we performed a series of analytical transglycosylation reactions starting from 7-MeGuo as a donor of Rib-1-P and the appropriate pyrimidine base as an acceptor of Rib-1-P at room temperature (see Table 1). Pyrimidine nucleosides were synthesized in the presence of two enzymes, namely PNP, which catalyzed the phoshorolysis of 7-MeGuo, and UP, which catalyzed the transfer of the ribofuranosyl moiety from Rib-1-P to pyrimidine base. The formation of nucleosides in the transglycosylation reaction was studied using uracil (Ura) and 5-substituted uracil derivatives (Thy, 5EtUra, 5-FUra, 5-CF3 Ura, 5-iPrUra, 5-ClUra, 5-BrUra, 5-IUra) as substrates of UP. In the reactions with 5-substituted uracil derivatives at a concentration of about 0.2 mM, the equilibrium is usually established within 1 h. In the presence of a large excess of inorganic phosphate over heterocyclic base (≥5 equiv.), the target nucleosides were obtained in lower yields (see Supplementary Data). According to HPLC analysis, uridine (Urd) can be obtained in high yield (89%) using a small excess (1.5 equiv.) of 7-MeGuo hydroiodide over Ura and a slight deficiency of inorganic phosphate (0.5 equiv.) (Table 1, entry 1). The replacement of Ura with thymine (Thy) or 5-ethyluracil (5-Et-Ura) also resulted in the formation of the corresponding ribonucleosides (Thd and 5-EtUrd) in high yields (92–100%) according to HPLC. The reaction with 5-trifluoromethyluracil did not gave a noticeable amount of the product (Table 1, entry 4) probably because of steric contacts. The reaction with 5-fluorouracil (5-FUra) instead of Ura gave 5fluorouridine in comparable yield (90%). When using other 5-halogen-substituted uracil derivatives as the starting compounds (5-ClUra, 5-IUra), ribonucleosides were obtained in lower yields compared to uracil (73–78%). The lowest yield was observed with 5-iodouridine (73%). The use of a large excess of 7MeGuo (4 equiv.) increased the yields of 5-iodosubstituted uridine derivative to 92% (Table 1, entry 10). The replacement of 7-MeGuo with adenosine (Ado) led to a significant decrease in the yield of the transglycosylation product, which is associated with the equilibrium displacement of phosphorolysis to the formation of Ado (Table 1, entry 2). Nevertheless, the synthesis of purine nucleoside (Ado) from 7-

Journal Pre-proof MeGuo, Ade and PNP by the transglycosylation reaction is more efficient compared to the synthesis of pyrimidine nucleosides, which is in agreement with the phosphorolysis constants listed in Table 1 (entry 11). Table 1. Analytical reactions used for the synthesis of pyrimidine ribonucleosides by enzymatic transglycosylation starting from Ura and its derivatives (200 µM), 7-MeGuo (1), and potassium dihydrophosphate at 20°C at different reagent ratios for 1 h.a) Entry

1

Initial compound/Product

Equilibrium concentration μM

7-MeGuo:Base:Pi

Yield (HPLC)

Ura/Urd

21.29/178.71

1.5:1:0.5

89%

Ura/Urd

5.79/194.21

3:1:0.5

97%

Ura/Urd

108.38/91.62

1:1:5

46%

Keq for phosphorolysis by UP

0.15 [5] 3:1:0.5 27% (Ado:Ura:Pi) 3 5-MeUra/5-MeUrd 0/200 1.5:1:0.5 100%c) 0.11 b) 4 5-CF3 Ura/5- CF3 Urd 1.5:1:0.5 NR 5 5-EtUra/5-EtUrd 16.14/183.86 1.5:1:0.5 92% 0.09 6 5-FUra/5-FUrd 21.40/178,60 1.5:1:0.5 90% 0.13 7 5-ClUra/5-ClUrd 43.31/156.69 1.5:1:0.5 78% 0.14 8 5-BrUra/5-BrUrd 16.65/183.35 1.5:1:0.5 92% 0.16 9 5iPrUra/5-iPrUrd 1.5:1:0.5 NRb) 53.63/146.37 1.5:1:0.5 73% 10 5-IUra/5-IUrd 16.90/183.10 2.5:1:0.5 92% 0.13 6.22/193.78 4:1:0.5 93% 11 Ade/Ado 0/200 1.5:1:0.5 100%c) 0.0084* [5] a) The reactions were performed at 20°C for 1 h until the equilibrium of transglycos ylation was reached; HPLC was performed using the reversed-phase sorbent 5µm, Reprosil-Pur C18-AQ in aqueous acetonitrile as eluent with the addition of TFA, except of Ura, 5-MeUra, 5-EtUra, 5-FUra (without TFA). b)No reaction. c) No detectable peak of heterocyclic base. * - phosphorolysis by PNP Ura/Urd

146.62/53.38

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We also used a larger excess of 7-MeGuo and P i ; however, 1.5-fold excess of the former and 0.5-fold deficiency of the latter appeared to be optimal for the synthesis of pyrimidine nucleosides by transglycosylation. Thus, the reaction conditions were selected to ensure a high yield using a slight excess of reagents. The tested analytical conditions were extended to the semi-microscale synthesis (50-100 mg) of 5-modified uridines (Scheme 2).

Scheme 2. Synthesis of 5-modified uridine derivatives by the transglycosylation reaction. Reagents and conditions: (i) MeI/DMF, 30°C, 24 h followed by the precipitation from CH2 Cl2 , 84%; (ii) KH2 PO4 , Tris-HCl buffer (pH 7.5), pyrimidine base (B), E.coli PNP, E.coli TP, r.t., 20 h: 2a (B=Ura), 89% (HPLC); 2b (B=Thy), 75%; 2c (B=5EtUra), 67%; 2d (B=5-FUra), 76%; 2e (B=5-ClUra), 65%; 2f (B=5-BrUra), 84%; 2g (B=5-IUrd), 58%.

The enzymatic synthesis of Urd was chosen as the reference reaction without the isolation of the product (Table 2, entry 1). 5-Halogen substituted uridine derivatives (Table 2, entries 4–7) were also synthesized using 7-MeGuo, base, and Pi in a ratio of 1.5 : 1 : 0.5 (Table 2, entries 1–3).

Journal Pre-proof The chromatographic purification of nucleosides was significantly improved when using a small excess of 7-MeGuo. Table 2. Semi-microscale synthesis of nucleosides from 7-MeGuo by the transglycosylation reaction a) Yield (HPLC)

Isolated yield

1

2a. Uridine (Urd)

89%

-

2

2b. Ribothymidine (Thd)

100%

75%

3

2c. 5-Ethyluridine (5-EtUrd)

92 %

67%

4

2d. 5-Fluorouridine (5-FUrd)

5

2e. 5-Chlorouridine (5-ClUrd)

6

2f. 5-Bromouridine (5-BrUrd)

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Nucleoside

7

B

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Entry

2g. 5-Iodouridine (5-IUrd)

90%

76%

78%

65%

92%

84%

73%

58%

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The reactions were performed at 20°C for 20 h using the initial substrates 1, heterocyclic base, and:Pi in a ratio of 1.5 :1 : 0.5 (corresponding substrate concentrations are 5 mM : 3.33 mM : 1.67 mM) until the equilibrium of transglycosylation was established.

The yields, estimated by HPLC for the reactions, listed in table 2, were consistent with the yields of the corresponding analytic reactions. Compared to the analytical conditions we used lower concentrations of PNP and UP in the reaction media for the rational consumption of enzymes in semi-microscale methods. Therefore, it took a longer time (20 h) for the equilibrium to be established at r.t. The reactions were controlled using the reversed-phase Dr. Maisch HPLC column (5µm, Reprosil-Pur C18-AQ). Before the addition of the enzymes PNP and UP, the chromatogram showed two peaks corresponding to pyrimidine base and 7-MeGuo (Fig. 1, t0 ). The formation of 7-methylguanine (7-MeGua) and the corresponding nucleoside was accompanied by a significant decrease in the intensity of the peaks related to pyrimidine base and 7-MeGuo with time (Fig. 1).

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Figure 1. Semi-microscale synthesis of 5-BrUrd (2f) using transglycosylation reaction. A: t initial , 1 – 5-BrUra, 2 – 7MeGuo; B: 1.5 h, 1 – 7-MeGua, 2 – 5-BrUra, 3 – 7-MeGuo, 4 – 5-BrUrd; C: 4 h, 1 – 7-MeGua, 2 – 5-BrUra, 3 – 7MeGuo, 4 – 5-BrUrd; D: 20 h, 1 – 7-MeGua, 2 – 5-BrUrd.

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During our experiments a partial decomposition of 5-chloro- and 5-fluorouridines under transglycosylation conditions was observed after 24 h at r.t. that was confirmed by HPLC (see Supplementary Data). Other derivatives were stable at the estimated conditions at least for 5 days. After chromatographic purification on silica gel, products 2b-2g were isolated in good yields. The structures of nucleosides were confirmed by NMR and UV spectra and their comparison with the spectra of commercially available nucleosides.

4. Conclusions

In conclusion, the one-step synthesis of 5-modified uridines from 7-MeGuo and KH2 PO4 (Pi) in the presence of PNP and UP was developed. The use of 7-MeGuo : base : P i reagent ratio of 1.5 : 1 : 0.5 is optimal and allows the synthesis of uridine derivatives bearing various substituents at position 5 of pyrimidine base in high yields. The use of reagents in a small excess reduces the cost of the synthesis and greatly simplifies the separation of the target compounds by column chromatography.

Conflict of interests: The authors declare no conflicts of interests.

Funding This work was supported by the Russian Science Foundation [project No. 16-14-00178].

References

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Graphical abstract

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Highlights  The eco-friendly enzymatic synthesis of 5-modified uridines was developed.  The use of enzymes allows preparation 100% pure beta-anomers.  Uridine phosphorylase is tolerant to various substituents at pyrimidine 5 position.  Nucleosides with various substituents in pyrimidine base were obtained in high yield.  The method may be a base for low-cost big scale biotechnology of nucleosides.