Communication
dried using a vacuum pump and then in a vacuum desiccator over
Enzymatic Synthesis of Modified Nucleosides
9-(ꢀ- -Ribofuranosyl)-allopurinol: Allopurinol
phosphorus pentoxide to yield 248 mg (82 %) of crystals. Rf 0.21 (2-
D
(100
mg,
1
propanol/NH4OH/H2O, 70:10:20, v/v). H NMR (400 MHz, D2O): 5.70
0.735 mmol) and α- -ribofuranose-1-O-phosphate barium salt
D
dd (1H, J1,2 = 3.9 Hz, JH-P = 6.2 Hz, H1), 4.28 m (1H, J4,3 = 4.1 Hz,
H4), 4.16 dd (1H, J2,3 = 6.1 Hz, H2), 4.11 dd (1H, H3), 3.85 dd (1H,
J5a,4 = 3.3 Hz, J5a,5b = –12.4 Hz, H5a), 3.75 dd (1H, J5b,4 = 4.7 Hz,
H5′b), 3.23 m (2H, C6H11), 2.05 m (4H, C6H11), 1.87 m (4H, C6H11),
1.72 m (2H, C6H11), 1.41 m (10H, C6H11). 13C NMR (100 MHz, D2O):
100.10 (C1), 86.90 (C4), 74.36 (C2), 72.62 (C3), 64.27 (C5), 53.10
(C6H11), 33.09 (C6H11), 27.04 (C6H11), 26.54 (C6H11). 31P NMR
(121.5 MHz, D2O): 1.92. HMBC spectrum is characterized by the
presence of cross-peaks corresponding to interaction of HMBC
spectrum is characterized by the presence of cross-peaks corre-
sponding to interaction of 31P nucleus with 1H1 (higher intensity)
and 1H2 (lower intensity) nuclei. HRMS of C17H37O8N2P: calculated
for [M + H]+ 429.2360, found 429.2362.
(179 mg, 0.490 mmol) were dissolved in 184 mL of 50 m Tris-
M
HCl buffer (pH 7.5). The reaction was initiated by adding purine
nucleoside phosphorylase (PNP) E. coli (800 μL of PNP E. coli solu-
tion with 10.6 U/mL activity, 8.5 IU). The reaction mixture was stirred
(orbital shaker) for 4 days at 37 °C. The conversion of the initial
Allopurinol was controlled via HPLC (66 % conversion after 4 days).
The reaction mixture was cooled using an ice bath to 0 °C, and
then filtered through nitrocellulose membrane Whatman (0.2 μm,
25 mm) to remove the white precipitate of barium phosphate. The
clear supernatant was evaporated in vacuo. The residue was chro-
matographed with silica gel column (10 mL), the column was
washed with dichloromethane; dichloromethane/ethanol, 95:5,
v/v; dichloromethane/ethanol, 90:10, v/v; and the product was
eluted with dichloromethane/ethanol, 80:20, v/v. Yield: 130 mg
(0.48 mmol) (98 %) as a foam. Rf 0.26 (dichloromethane/ethanol,
80:20, v/v). 1H NMR Spectrum (400 MHz, D2O): 8.32 s (1H, H2), 8.29 s
(1H, H7), 6.35 d (1H, J1′,2′ = 4.9 Hz, H1′), 4.92 dd (1H, J2′,3 ′ = 5.1 Hz,
H2′), 4.58 dd (1H, J3′,4′ = 4.9 Hz, H3′), 4.29 ddd (1H, H4′), 3.92 dd
α- -2′-Deoxyribofuranose-1-O-phosphate Bis(cyclohexylammo-
D
nium) Salt: Procedure equivalent to above, but starting from 7-
methyl-2′-deoxyguanosine hydroiodide (300 mg, 0.733 mmol) and
dicyclohexylamine hydrophosphate (239 mg, 0.806 mmol) yielded
224 mg (74 %) of the product as an amorphous powder. Rf 0.26 (2-
(1H, J5′a,4 ′ = 3.52 Hz, J5′a,5′b = –12.51 Hz, H5′a), 3.81 dd (1H, J5′b, 4′
5.3 Hz, H5′b).
=
1
propanol/NH4OH/H2O, 70:10:20, v/v). H NMR (400 MHz, D2O): 5.83
ddd (1H, J1,2a = 6.4 Hz, J1,2b = 1.3 Hz, JH-P = 5.9 Hz, H1), 4.29 m (2H,
H3, H4), 3.79 dd (1H, J5a,4 = 3.4 Hz, J5a,5b = –12.2 Hz, H5a), 3.53 dd
9-(2-Deoxy-ꢀ-
Chloro-adenine (45 mg, 0.267 mmol) and α-
1-O-phosphate bis(cyclohexylammonium)
0.373 mmol, 1.4 equiv.) were suspended in 170 mL of 2.2 m
D
-ribofuranosyl)-2-chloroadenine (Cladribine): 2-
-2′-deoxyribofuranose-
salt (154 mg,
(1H, J5b,4 = 5.2 Hz, H5′b), 3.22 m (2H, C6H11), 2.43 ddd (1H, J2a,3
=
D
6.4 Hz, J2a,2b = -14.1 Hz, H2a), 2.15 ddd (1H, J2b,3 = 2.6 Hz, H2b),
2.04 m (4H, C6H11), 1.87 m (4H, C6H11), 1.73 m (2H, C6H11), 1.69 m
(2H, C6H11), 1.40 m (10H, C6H11). 13C NMR (100 MHz, D2O): 99.63
(C1), 86.07 (C4), 71.21 (C3), 61.69 (C5), 50.39 (C6H11), 41.72 (C2),
30.39 (C6H11), 24.33 (C6H11), 23.83 (C6H11). 31P NMR (121.5 MHz,
D2O): 1,67. HRMS of C17H37O7N2P: calculated for [M + H]+ 413.2411,
found 413.2412.
M
Ca(OH)2 solution (pH Ӎ 9.3). The reaction was initiated by adding
purine nucleoside phosphorylase (PNP) E. coli (60 μL of PNP E. coli
solution with 10.6 U/mL activity, 0.65 IU). The reaction mixture was
stirred (orbital shaker) for 2 days at 45 °C. The conversion of the
initial 2-chloro-adenine was controlled via HPLC (approximately
80 % conversion after 48 h). The reaction mixture was cooled using
an ice bath to 0 °C, and then filtered through nitrocellulose mem-
brane Whatman (0.2 μm, 25 mm) to remove the white precipitate
of bis(cyclohexylammonium) phosphate and excess of 2-chloro-
adenine. The clear supernatant was evaporated in vacuo. The resi-
due was chromatographed with a silica gel column (40 mL), the
column was washed with dichloromethane; dichloromethane/eth-
anol, 95:5, v/v; and the product was eluted with dichloromethane/
ethanol, 92.5:7.5, v/v. Yield: 55 mg (72 %) as a foam. Rf 0.49 (di-
chloromethane/ethanol, 90:10, v/v). 1H NMR Spectrum (400 MHz,
D2O): 8.30 s (1H, H8), 7.06 brs (2H, NH2), 6.42 dd (1H, J1′,2a′ = 7.1 Hz,
J1′,2b′ = 6.3 Hz, H1′), 5.00–4.55 m (1H, overlapping with HOD signal,
H3′), 4.23 ddd (1H, J4′,3 ′ = 7.3 Hz, J4′,5a′ = 3.3 Hz, J4′,5b′ = 4.3 Hz,
H4′), 3.86 dd (1H, J5a′,5b′ = –12.6 Hz, H5′a), 3.75 dd (1H, H5′b), 2.85
ddd (1H, J2a′,1 ′ = 7.1 Hz, J2a′,3 ′ = 6.1 Hz, J2a′,2b′ = –14.1 Hz, H2′a),
2.62 ddd (1H, J2b′,1 ′ = 6.3 Hz, J2b′,3 ′ = 3.4 Hz, H2′b).
α-
D
-Ribofuranose-1-O-phosphate Disodium Salt: Dowex-50Na+
-ribofuran-
(1 mL) was added to an aqueous solution (10 mL) of α-
D
ose-1-O-phosphate bis(cyclohexylammonium) salt (248 mg,
0.58 mmol). The mixture was stirred for 30 min at ambient tempera-
ture then filtered from Dowex. Dowex was washed with water
(3 mL). The combined liquid filtrates were evaporated under re-
duced pressure using a rotary evaporator to dry residue (bath tem-
perature < 35 °C). The residue was dried in a vacuum desiccator
over phosphorus pentoxide to yield 154 mg (97 %) of a foam. Rf
0.17 (2-propanol/NH4OH/H2O, 70:10:20, v/v). 1H NMR (400 MHz,
D2O): 5.65 dd (1H, J1,2 = 3.9 Hz, JH-P = 6.4 Hz, H1), 4.24 ddd (1H,
J4,5a = 3.3 Hz, J4,5b = 4.7 Hz, J4,3 = 4.1 Hz, H4), 4.12 ddd (1H, J2,3
6.2 Hz, J2,P = 0.9 Hz, H2), 4.07 dd (1H, H3), 3.80 dd (1H, J5a,5b
=
=
–12.4 Hz, H5a), 3.67 dd (1H, H5′b). 13C NMR (100 MHz, D2O): 102.31
(C1), 88.14 (C4), 72.64 (C2), 70.26 (C3), 63.33 (C5). 31P NMR
(121.5 MHz, D2O): 1.93. HRMS of C5H9O8PNa2: calculated for [M –
Na + 2H]+ 253.0084, found 253.0084.
Acknowledgments
α-
D
-2′-Deoxyribofuranose-1-O-phosphate Disodium Salt: Proce-
-2′-Deoxyribofuran-
This work was supported by the Russian Science Foundation
(project No. 16-14-00178). Authors are grateful to Dr. Roman
S. Esipov, the director of the Group of Genetically Engineered
Biopharmaceutical Technologies of the Shemyakin-Ovchinnikov
Institute of bioorganic chemistry RAS, for providing PNP for this
research.
dure equivalent to above, but starting from α-
D
ose-1-O-phosphate bis(cyclohexylammonium) salt (224 mg,
0.54 mmol) yielded 135 mg (96 %) of the product as a foam. Rf 0.24
(2-propanol/NH4OH/H2O, 70:10:20, v/v). 1H NMR (400 MHz, D2O):
5.79 ddd (1H, J1,2a = 5.2 Hz, J1,2b = 1.4 Hz, JH-P = 6.0 Hz, H1), 4.25 m
(2H, H3, H4), 3.75 dd (1H, J5a,4 = 3.5 Hz, J5a,5b = –12.2 Hz, H5a), 3.65
dd (1H, J5b,4 = 5.1 Hz, H5′b), 2.39 ddd (1H, J2a,3 = 7.3 Hz, J2a,2b
=
-14.4 Hz, H2a), 2.11 ddd (1H, J2b,3 = 2.5 Hz, H2b). 13C NMR (100 MHz,
D2O): 98.77 (C1), 87.10 (C4), 70.98 (C3), 63.06 (C5), 40.78 (C2). 31P
NMR (121.5 MHz, D2O): 1.71. HRMS for C5H9O7PNa2: calculated for
[M – Na + 2H]+ 237.0135, found 237.0133.
Keywords: Enzyme catalysis · Nucleosides ·
Phosphorolysis · Synthetic methods
Eur. J. Org. Chem. 0000, 0–0
5
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