SCHEME 2. Synthesis of dTHU from dCyt and Competing
Undesired Reactions (dR ) Deoxyribose)
SCHEME 5. Methanolysis of dTHU (dR ) Deoxyribose)
be the most reliable method of purification, reproducibly
resulting in >95% purity dTHU samples in consistent yields
around 40% (based on 2′-deoxyuridine). Although certain
factors, such as greater exposure to ambient oxygen, often make
preparatory TLC an inferior method to column chromatography
for purifying sensitive compounds,14 column chromatography
on silica gel using a number of solvent combinations, including
basic methanol, dichloromethane, and chloroform, was ineffec-
tive in this case. We speculated that silica surface area, acidity,
or metal content are potential factors contributing to differences
between these chromatographic techniques.15 Once purified, to
prevent loss of dTHU to C4-hydrolysis or isomerization, samples
SCHEME 3. Synthesis of dTHU from dUrd (R )
Deoxyribose)
1
were dried to a colorless film. H NMR and MS analysis of
dTHU samples stored at -20 °C in microcentrifuge tubes for 4
months showed no change in spectra.
In conclusion, we report a simple and reliable procedure for
the synthesis of dTHU on an approximately 150-mg scale
reaction in 40% overall yield from dUrd. Although dTHU has
been used in various studies over several decades, and is a
fundamental pyrimidine modification, a reliable preparation and
characterization data, to our knowledge, were not previously
available. We anticipate that the approach described here, and
the high purity product which can be obtained following this
procedure, will improve the quality of data obtained using dTHU
as an experimental tool and will expand research in related areas.
Furthermore, we expect that the spectroscopic data reported here
will facilitate the identification of dTHU in complex mixtures.
SCHEME 4. Over-reduction of dTHU Is Observed if the
Reaction Is Carried out with 1 Equiv NaBH4
result from incomplete hydrogenation interrupted by hydrolysis
(5) or over-reduction (6).
Hanze explored an alternative approach to synthesize THU
by catalytic hydrogenation of uridine, followed by sodium
borohydride reduction.1 However, this approach was abandoned
due to even more severe complications such as impurities and
the instability of the product to reaction conditions. Revisiting
this strategy, we describe its success for the synthesis of dTHU
on an approximately 150-mg scale, as illustrated in Scheme 3.
The starting material dUrd is quantitatively converted to
deoxydihydrouridine 6. The efficiency of the subsequent boro-
hydride reduction of 6 to dTHU is extremely sensitive to reagent
stoichiometry. With 1 equiv of NaBH4, a significant amount of
inseparable over-reduction side product 7 is produced, which
results from a reaction pathway common in borohydride
reduction of pyrimidines (Scheme 4).13 To circumvent this
problem, NaBH4 is used with precise hydride equivalence by
combining it with 4 equiv of 6. By carrying out the reaction
with excess nucleoside, the only observed products are dTHU
and remaining 6, but no 7. Acidic quench of the crude
borohydride reduction mixture, as described by Hanze, was
eliminated to prevent acid-catalyzed hydrolysis of the dTHU
4-OH.
Experimental Section
For general methods and considerations, see the Supporting
Information.
2′-Deoxy-5,6-dihydrouridine (6). A suspension of 2′-deoxyuri-
dine (0.338 g, 1.48 mmol) and 5% Rh/alumina catalyst (0.125 g,
0.06 mmol Rh) in 15 mL of water was placed on a Parr shaker
under hydrogen gas (45 psi) for 18 h. The reaction solution was
filtered through Celite to remove the Rh/alumina catalyst and
concentrated under vacuum to afford a pale yellow oil. TLC analysis
indicated the formation of one product (Rf 0.57). The oil was
redissolved in 10 mL of MeOH. Solvent was removed under
vacuum to yield a white crystalline solid (341 mg, 1.48 mmol,
1
quantitative): H NMR (300 MHz, D2O) δ 2.05-2.13 (m, 1H),
2.23-2.32 (m, 1H), 2.70 (t, J ) 6.9 Hz, 2H), 3.47-3.54 (m, 2H),
3.62-3.76 (m, 2H), 3.85-3.89 (m, 1H), 4.31-4.36 (m, 1H), 6.23
(t, J ) 7.2 Hz, 1H); 13C NMR (D2O) δ 174.2, 154.7, 86.4, 84.7,
71.3, 61.7, 35.4, 35.6, 30.8; MS (ESI+) m/z 231 (M + H+), 253
(M + Na+).
2′-Deoxytetrahydrouridine (dTHU). A solution of 6 (341 mg,
1.48 mmol) in water (10 mL) was cooled to 0 °C. Sodium
borohydride (14 mg, 0.4 mmol) was added to the cold solution.
The reaction mixture stirred at 0 °C for 2 h. TLC analysis (30%
MeOH-NH3/CHCl3) of the crude reaction mixture indicated the
formation of two products (Rf 0.68 and 0.44). The reaction was
A further difficulty in the synthesis of pure dTHU is its
instability. For example, storage of pure dTHU in basic methanol
at 25 °C for 12 h results in a mixture of dTHU and a new
1
(14) Fried, B.; Sherma, J. In Thin-Layer Chromatography: Chromatographic
Science; Marcel Dekker, Inc.: New York, 1999; Vol. 81.
(15) Nyiredy, S.; Dallenbach-Toelke, K.; Zogg, G. C.; Sticher, O. J. Chro-
matogr. 1990, 499, 453–462.
product with an m/z of 269 and a H NMR similar to dTHU
containing a singlet at 3.33 ppm, consistent with the metha-
nolysis product 8 (Scheme 5). Preparatory TLC was found to
2222 J. Org. Chem. Vol. 74, No. 5, 2009