Transesterification of Ribonucleotide Esters
J. Am. Chem. Soc., Vol. 121, No. 49, 1999 11271
5.34 (d, J5,6 ) 8.2 Hz, 1H, H-5), 6.06 (d, J1′,2′ ) 2.7 Hz, 1H, H-1′),
7.26-7.41 (m, 15H, C6H5), 7.88 (d, J5,6 ) 8.2 Hz, 1H, H-6), 9.13 (s,
1H, H-N3); 31P NMR (CH2Cl2, 25 °C, 101 MHz) δ ) 1.11 ppm;
analytical RP-HPLC (40% CH3CN in 20 mM K2HPO4/KH2PO4 buffer,
pH 7.0; flow 0.8) tR ) 13 min.
nucleophile, metal ion binding sites and medium for the
ribozyme reaction, i.e., a catalysis of the catalysis, observed in
the non-ribozymic reaction.
Experimental Section
N3-Methyl-5′-O-trityluridine 2′-/3′-dimethyl phosphate 14: Yield
88%, 3′-isomer: 1H NMR (CDCl3, 25 °C, 250 MHz) δ ) 3.29 (s, 3H,
H3C-N3), 3.45 (dd, J4′,5′ ) 2.6 Hz, J5′,5′′ ) 10.0 Hz, 1H, H-5′), 3.48 (s,
1H, HO), 3.56 (dd, J4′,5′′ ) 2.6 Hz, J5′,5′′ ) 10.0 Hz, 1H, H-5′′), 3.72
1
General Procedures, Methods and Materials. H and 31P NMR
spectra were taken on a BRUKER Avance-DRX 250 spectrometer at
300 K with tetramethylsilan as internal and 85% H3PO4 as external
standard, respectively. Chemical shifts are reported in δ (ppm). Reverse
phase HPLC analyses were performed on Waters Liquid Chromatograph
equipped with absorbance detector model 441 set at 280 nm and column
Nucleosil 100-5C18 (12.5 cm × 4.6 mm) or aminocolumn Spherisorb
5m NH2 (25 cm × 5 mm) for analytical runs, or Nucleosil 100-5C18
(25 cm × 10 mm) for semi-preparative runs. Commercial solvents and
reagents were used as received unless otherwise noted. Pyridine was
dried over sodium hydroxide and distilled over calcium hydride before
use. Dichloromethane and methanol were dried using standard proce-
dures and distilled before use. A 2 M stock solution of sodium
methoxide in methanol was prepared prior to use by dissolving metallic
sodium in the dried methanol under anhydrous conditions. 5′-O-
Trityluridine,23 5′-O-methyluridine,24 5′-O-trityl-2′-deoxyuridine25 and
2,2′-anhydrouridine26 were prepared as described.
3
3
(d, JP,H ) 11.3 Hz, 3H, H3COP), 3.78 (d, JP,H ) 11.3 Hz, 3H, H3-
COP), 4.33 (dt, J4′,5′ ) 2.6 Hz, J3′,4′ ) 4.7 Hz, 1H, H-4′), 4.48 (dd, J2′,3′
) 4.6 Hz, J3′,4′ ) 4.7 Hz, 1H, H-3′), 4.88 (1H, H-2′), 5.34 (d, J5,6
)
8.2 Hz, 1H, H-5), 5.98 (d, J1′,2′ ) 4.3 Hz, 1H, H-1′), 7.26-7.41 (m,
15H, C6H5), 7.75 (d, J5,6 ) 8.2 Hz, 1H, H-6); 31P NMR (CH2Cl2, 25
°C, 101 MHz) δ ) 0.95 ppm; analytical RP-HPLC (40% CH3CN in
20 mM K2HPO4/KH2PO4 buffer, pH 7.0; flow 0.8) tR ) 16 min. 2′-
Isomer: 1H NMR (CDCl3, 25 °C, 250 MHz) δ ) 3.28 (s, 3H, H3C-
N3), 3.45 (dd, J4′,5′ ) 2.6 Hz, J5′,5′′ ) 10.0 Hz, 1H, H-5′), 3.54 (s, 1H,
HO), 3.56 (dd, J4′,5′′ ) 2.6 Hz, J5′,5′′ ) 10.0 Hz, 1H, H-5′′), 3.82 (d,
3
3JP,H ) 11.3 Hz, 3H, H3COP), 3.84 (d, JP,H ) 11.3 Hz, 3H, H3COP),
4.16 (dt, J4′,5′ ) 2.6 Hz, J3′,4′ ) 6.7 Hz, 1H, H-4′), 4.59 (dd, J2′,3′ ) 4.7
Hz, J3′,4′ ) 6.7 Hz, 1H, H-3′), 4.88 (1H, H-2′), 5.34 (d, J5,6 ) 8.2 Hz,
1H, H-5), 6.06 (d, J1′,2′ ) 2.7 Hz, 1H, H-1′), 7.26-7.41 (m, 15H, C6H5),
7.88 (d, J5,6 ) 8.2 Hz, 1H, H-6); 31P NMR (CH2Cl2, 25 °C, 101 MHz)
δ ) 1.2 ppm; analytical RP-HPLC (40% CH3CN in 20 mM K2HPO4/
KH2PO4 buffer, pH 7.0; flow 0.8) tR ) 16.9 min.
Synthesis of Nucleoside Dimethyl Phosphates. Uridine 2′-/3′-
dimethyl phosphates 9 and arabinouridine 3′-dimethyl phosphate 11
were prepared from the corresponding nucleoside phosphates as
described previously.9,10
5′-O-Methyluridine 2′-/3′-dimethyl phosphate 8: Yield 85%, 3′-
isomer: 1H NMR (CDCl3, 25 °C, 250 MHz) δ ) 1.77 (s, 3H,
H3COC5′), 3.45 (dd, J4′,5′ ) 2.6 Hz, J5′,5′′ ) 9.9 Hz, 1H, H-5′), 3.48 (s,
1H, HO), 3.56 (dd, J4′,5′′ ) 2.6 Hz, J5′,5′′ ) 9.9 Hz, 1H, H-5′′), 3.72 (d,
General Procedure for the Preparation of 5′-O-Protected Nu-
cleoside Dimethyl Phosphates. To a cooled (5 °C) solution of POCl3
(1 mmol) in dry CH2Cl2 (4 mL) pyridine (4 mmol) was added dropwise.
After little initial fuming the resulting clear solution was added to a
precooled mixture (5 °C) of 5′-O-protected nucleoside (0.2 mmol) and
pyridine (4 mmol) in 4 mL dry CH2Cl2 and stirred at room temperature
for 5-10 min. Methanol (4 mmol) was added, and the stirring continued
for another 20-40 min (1, 8, 14) or overnight (10), when the reactions
were completed as judged by RP-HPLC (isocratic elution with 40%
CH3CN in 20 mM K2HPO4/KH2PO4 buffer, pH 7.0 for 1, 14, and 10
or with 15% CH3CN in the same buffer for 8) and 31P NMR. Then the
reaction mixture was evaporated to dryness under reduced pressure,
dissolved in acetonitrile, and applied on a semi-preparative RP-HPLC
column (isocratic elution with 50% CH3CN in 20 mM K2HPO4/KH2-
PO4 buffer, pH 7.0 for 1, 14, and 10 or with 25% CH3CN in the same
buffer for 8). Appropriate fractions (2′- and 3′-isomers can be collected
separately) were evaporated immediately (reduced pressure, 40 °C),
dried several times by coevaporation with dry acetonitrile or dichlo-
romethane, and kept in a desiccator. The analytically pure samples of
the triesters were prepared by dry extraction in acetonitrile or
dichloromethane from the inorganic buffer salts. Total yields of the
isolated isomeric mixtures: 80-90%.
3
3JP,H ) 11.3 Hz, 3H, H3COP), 3.78 (d, JP,H ) 11.3 Hz, 3H, H3COP),
4.33 (dt, J4′,5′ ) 2.6 Hz, J3′,4′ ) 4.7 Hz, 1H, H-4′), 4.48 (dd, J2′,3′ ) 4.2
Hz, J3′,4′ ) 4.7 Hz, 1H, H-3′), 4.89 (1H, H-2′), 5.37 (d, J5,6 ) 8.2 Hz,
1H, H-5), 5.95 (d, J1′,2′ ) 4.2 Hz, 1H, H-1′), 7.75 (d, J5,6 ) 8.2 Hz,
1H, H-6); 31P NMR (CH2Cl2, 25 °C, 101 MHz) δ ) 0.9 ppm; ana-
lytical RP-HPLC (15% CH3CN in 20 mM K2HPO4/KH2PO4 buffer,
pH 7.0; flow 0.8) tR ) 8.5 min. 2′-Isomer: 1H NMR (CDCl3, 25 °C,
250 MHz) δ ) 1.77 (s, 3H, H3COC5′), 3.45 (dd, J4′,5′ ) 2.6 Hz, J5′,5′′
) 9.9 Hz, 1H, H-5′), 3.54 (s, 1H, HO), 3.56 (dd, J4′,5′′ ) 2.6 Hz, J5′,5′′
3
) 9.9 Hz, 1H, H-5′′), 3.82 (d, JP,H ) 11.3 Hz, 3H, H3COP), 3.84 (d,
3JP,H ) 11.3 Hz, 3H, H3COP), 4.18 (dt, J4′,5′ ) 2.6 Hz, J3′,4′ ) 6.5
Hz, 1H, H-4′), 4.58 (dd, J2′,3′ ) 4.2 Hz, J3′,4′ ) 6.5 Hz, 1H, H-3′),
4.89 (1H, H-2′), 5.31 (d, J5,6 ) 8.2 Hz, 1H, H-5), 6.04 (d, J1′,2′
)
2.5 Hz, 1H, H-1′), 7.88 (d, J5,6 ) 8.2 Hz, 1H, H-6); 31P NMR (CH2Cl2,
25 °C, 101 MHz) δ ) 1.02 ppm; analytical RP-HPLC (15%
CH3CN in 20 mM K2HPO4/KH2PO4 buffer, pH 7.0; flow 0.8) tR ) 9.2
min.
5′-O-Trityl-2′-deoxyuridine 3′-dimethyl phosphate 10: Yield 90%:
1H NMR (CDCl3, 25 °C, 250 MHz) δ ) 2.36 (ddd, J1′,2′ ) 7.5 Hz,
J2′,2′′ ) 14.2 Hz, J2′,3′ ) 6.6 Hz, 1H, H-2′), 2.66 (ddd, J1′,2′′ ) 5.9 Hz,
J2′,2′′ ) 14.2 Hz, J2′′,3′ ) 3.0 Hz, 1H, H-2′′), 3.48 (d, J4′,5′ ) 3.0 Hz,
2H, H-5′), 3.72 (d, 3JP,H ) 11.2 Hz, 3H, H3COP), 3.76 (d, 3JP,H ) 11.2
Hz, 3H, H3COP), 4.26 (dt, J4′,5′ ) 3.0 Hz, J3′,4′ ) 5.8 Hz, 1H, H-4′),
5.13 (ddd, J2′,3′ ) 6.6 Hz, J2′′,3′ ) 3.0 Hz, J3′,4′ ) 5.8 Hz, 1H, H-3′),
5.32 (d, J5,6 ) 8.2 Hz, 1H, H-5), 6.34 (dd, J1′,2′ ) 7.5 Hz, J1′,2′′ ) 5.9
Hz, 1H, H-1′), 7.18-7.41 (m, 15H, C6H5), 7.69 (d, J5,6 ) 8.2 Hz, 1H,
H-6), 7.97 (s, 1H, H-N3); 31P NMR (CH2Cl2, 25 °C, 101 MHz) δ )
0.78 ppm; analytical RP-HPLC (40% CH3CN in 20 mM K2HPO4/KH2-
PO4 buffer, pH 7.0; flow 0.8) tR ) 15 min.
Methanolysis Reactions. To a solution of 0.12 mmol of nucleoside
dimethyl phosphate in 7 mL of dry organic solvent (CH2Cl2 or MeOH)
3 mL of 0.13-2 M NaOMe in MeOH was added at 298.2 K. The
reaction mixture was divided into two samples, and the progress of
the reaction was followed simultaneously both by 31P NMR spectros-
copy and analytical HPLC.
5′-O-Trityluridine 2′-/3′-dimethyl phosphate 1: Yield 82%, 3′-
isomer: 1H NMR (CDCl3, 25 °C, 250 MHz) δ ) 3.45 (dd, J4′,5′ ) 2.6
Hz, J5′,5′′ ) 9.9 Hz, 1H, H-5′), 3.48 (s, 1H, HO), 3.56 (dd, J4′,5′ ) 2.6
Hz, J5′,5′′ ) 9.9 Hz, 1H, H-5′′), 3.72 (d, 3JP,H ) 11.3 Hz, 3H, H3COP),
3.78 (d, 3JP,H ) 11.3 Hz, 3H, H3COP), 4.33 (dt, J4′,5′ ) 2.6 Hz, J3′,4′
)
4.7 Hz, 1H, H-4′), 4.48 (dd, J2′,3′ ) 4.6 Hz, J3′,4′ ) 4.7 Hz, 1H, H-3′),
4.88 (1H, H-2′), 5.34 (d, J5,6 ) 8.2 Hz, 1H, H-5), 5.98 (d, J1′,2′ ) 4.3
Hz, 1H, H-1′), 7.26-7.41 (m, 15H, C6H5), 7.75 (d, J5,6 ) 8.2 Hz, 1H,
H-6), 9.35 (s, 1H, H-N3); 31P NMR (CH2Cl2, 25 °C, 101 MHz) δ )
0.92 ppm; analytical RP-HPLC (40% CH3CN in 20 mM K2HPO4/
KH2PO4 buffer, pH 7.0; flow 0.8) tR ) 12 min. 2′-Isomer: 1H NMR
(CDCl3, 25 °C, 250 MHz) δ ) 3.45 (dd, J4′,5′ ) 2.6 Hz, J5′,5′′ ) 9.9
Hz, 1H, H-5′), 3.54 (s, 1H, HO), 3.56 (dd, J4′,5′ ) 2.6 Hz, J5′,5′′ ) 9.9
Hz, 1H, H-5′′), 3.82 (d, 3JP,H ) 11.4 Hz, 3H, H3COP), 3.84 (d, 3JP,H
)
11.4 Hz, 3H, H3COP), 4.16 (dt, J4′,5′ ) 2.6 Hz, J3′,4′ ) 6.7 Hz, 1H,
H-4′), 4.59 (dd, J2′,3′ ) 4.7 Hz, J3′,4′ ) 6.7 Hz, 1H, H-3′), 4.88 (1H,
H-2′),
31P NMR Kinetic Studies. One of the samples was transferred to
the NMR tube, and the 31P NMR spectra were recorded every 5 min
during 1 h. The time-dependent phosphate product distribution was
determined from the signal intensities at different reaction times.
HPLC Kinetic Studies. Aliquots were withdrawn from the other
sample at appropriate time intervals, diluted with the mobile phase and
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