Synthesis of (S)-5-(4,5-dihydroxypentyl)uracil from pseudouridine and clarification of stereochemistry

Full text of DOI:10.1021/jo980975q

J . Org. Chem. 1998, 63, 7539-7541
7539
Sch em e 1a
Syn th esis of
(S)-5-(4,5-Dih yd r oxyp en tyl)u r a cil fr om
P seu d ou r id in e a n d Cla r ifica tion of
Ster eoch em istr y1
Isamu Maeba,† Noriaki Morishita,† Paula Francom,‡ and
Morris J . Robins*,‡
Department of Chemistry and Biochemistry, Brigham
Young University, Provo, Utah 84602-5700 and Faculty
of Pharmacy, Meijo University,
Tempaku-ku, Nagoya 468, J apan
Received May 21, 1998
Approximately half of the thymine bases in the large
transducing bacteriophage SP-15 of Bacillus subtilis were
replaced by (S)-5-(4,5-dihydroxypentyl)uracil (DHPU,
7).2,3 Incorporation of DHPU with its chiral dihydroxy-
pentyl side chain in place of thymine confers novel
physical properties on SP-15 DNA including a lower
melting temperature (Tm ) 61.5 °C), increased buoyant
density (CsCl), and alkaline lability.2 Phosphoglucu-
ronidation of one hydroxyl on the pentyl side chain and
glucosidation of the other occurs, and this hypermodifi-
cation is responsible for the observed increase in buoyant
density.2,3 Presence of the hypermodified DHPU has also
been associated with high levels of resistance to endo-
nucleases4 and DNA T4 ligase,5 although potential roles
of DHPU in the control of bacteriophage transcription
and translation are not well understood.6-8
DHPU was isolated from SP-15 DNA after formic acid
hydrolysis.2 Neither elucidation of its biosynthetic path-
way nor identification of the sequence of steps leading
to its incorporation into SP-15 DNA have been estab-
lished.6-10 Potential biosynthetic precursors including
uracil,6 ribose,9 and glutamic acid10 have been suggested.
An obvious candidate, pseudouridine (1), was discounted
as a precursor since its Cahn-Ingold-Prelog configura-
tion descriptor at C4′ (R) was noted to be opposite to that
of DHPU at C4′ (S).10 However, C-I-P configuration
descriptors are based on rules with C3′ of higher priority
than C5′ in pseudouridine (1), but C5′ of higher priority
than C3′ in DHPU (no hydroxyl group on C3′). Thus,
a
(a) TBDPSCl/pyridine. (b) (Imidazole)2CS/DMF/∆. (c) P(OEt)3/
∆. (d) NH4F/MeOH/∆. (e) H2/Pd-C. (f) BzCl/pyridine.
the absolute stereochemistry at C4′ is the same in both
1 and DHPU.
(S)-DHPU (7) was synthesized in seven steps from (S)-
malic acid.10 Two syntheses of the racemic product have
involved an analogous route10 or generation of an initial
intermediate by photochemical addition of vinylene car-
bonate to uracil.11 We now describe a synthesis of DHPU
from pseudouridine [5-(â-D-ribofuranosyl)uracil, 1] that
employs hydrogenolysis of the benzylic C1′-O4′ bond
with retention of configuration at C4′ (Scheme 1).
Treatment of pseudouridine (1) with tert-butyldiphen-
ylsilyl chloride in pyridine gave 5-(5-O-TBDPS-â-D-ribo-
furanosyl)uracil (2, 76%). Thiocarbonyldiimidazole/DMF/
90 °C converted
2 into its cyclic 2′,3′-O-thiono-
carbonate 3 (94%) which was heated with triethyl phos-
phite12 (110 °C) to give 5-[5(S)-(TBDPS)oxymethyl-2,5-
dihydrofuran-2(R)-yl]uracil (4, 94%). Deprotection of 4
(NH4F/MeOH)13 gave 5-[2,5-dihydro-5(S)-hydroxymeth-
ylfuran-2(R)-yl]uracil (5, 71%). Concomitant hydrogenol-
ysis of the C1′-O4′ bond and hydrogenation of the
furanyl double bond occurred with Pd-C to give 5-[5-
(TBDPS)oxy-4(S)-hydroxypentyl]uracil (6, 54%). Depro-
tection of 6 (NH4F/MeOH) gave the desired (S)-5-(4,5-
dihydroxypentyl)uracil (7, 99%). Treatment of 5 with H2/
Pd-C also gave 7. The mp, UV, NMR, and HRMS data
for 7 were consistent with those reported previously for
(S)-DHPU.10
* To whom inquiries should be addressed at Brigham Young
University [FAX: (801) 378-5474].
† Meijo University.
‡ Brigham Young University.
(1) Nucleic Acid Related Compounds. 104. Paper 103: Robins, M.
J .; Wnuk, S. F.; Yang, X.; Yuan, C.-S.; Borchardt, R. T.; Balzarini, J .;
De Clercq, E. J . Med. Chem., in press.
(2) (a) Marmur, J .; Brandon, C.; Neubort, S.; Ehrlich, M.; Mandel,
M.; Konvicka, J . Nature (London), New Biol. 1972, 239, 68-70. (b)
Brandon, C.; Gallop, P. M.; Marmur, J .; Hayashi, H.; Nakanishi, K.
Nature (London), New Biol. 1972, 239, 70-71.
(3) Ehrlich, M.; Ehrlich, K. C. J . Biol. Chem. 1981, 256, 9966-9972.
(4) Huang, L.-H.; Farnet, C. M.; Ehrlich, K. C.; Ehrlich, M. Nucl.
Acids Res. 1982, 10, 1579-1591.
(5) Cao, X.-M.; Huang, L.-H.; Farnet, C. M.; Ehrlich, M. Biochim.
Biophys. Acta 1983, 741, 237-243.
(6) Neubort, S.; Marmur, J . J . Virol. 1973, 12, 1078-1084.
(7) Walker, M. S.; Mandel, M. J . Virol. 1978, 25, 500-509.
(8) Dosmar, M.; Markewych, O.; Witmer, H. J . Virol. 1977, 21, 924-
931.
(9) Walker, M. W.; Mandel, M. J . Virol. 1978, 25, 695-697.
(10) (a) Hayashi, H.; Nakanishi, K. J . Am. Chem. Soc. 1973, 95,
4081-4083. (b) Hayashi, H.; Nakanishi, K.; Brandon, C.; Marmur, J .
J . Am. Chem. Soc. 1973, 95, 8749-8757.
Treatment of 7 with benzoyl chloride/pyridine gave an
amorphous tribenzoyl derivative 8 (80%). The bisignate
CD spectrum of 8 (Figure 1) has a positive first Cotton
(11) Bergstrom, D. E.; Agosta, W. C. Tetrahedron Lett. 1974, 13,
1087-1090.
(12) (a) Corey, E. J .; Winter, R. A. E. J . Am. Chem. Soc. 1963, 85,
2677-2678. (b) Chu, C. K.; Bhadti, V. S.; Doboszewski, B.; Gu, Z. P.;
Kosugi, Y.; Pullaiah, K. C.; Van Roey, P. J . Org. Chem. 1989, 54, 2217-
2225.
(13) Zhang, W.; Robins, M. J . Tetrahedron Lett. 1992, 33, 1177-
1180.
S0022-3263(98)00975-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/24/1998

7540 J . Org. Chem., Vol. 63, No. 21, 1998
Notes
5-[5-O-(ter t-Bu tyld ip h en ylsilyl)-2,3-O-th ioca r bon yl-â-D-
r ibofu r a n osyl]u r a cil (3). A solution of 2 (5.49 g, 11 mmol)
and thiocarbonyldiimidazole (3.0 g, 17 mmol) in DMF (150 mL)
was stirred for 6 h at 90 °C under N2. Volatiles were evaporated,
and the residue was chromatographed (MeOH/CHCl3, 1:99).
Crystallization of the solid residue (CHCl3) gave 3 (5.4 g, 94%;
fine colorless needles): mp 183-185 °C; 1H NMR δ 1.05 (s, 9H),
3.82 (m, 2H), 4.27 (q, J ) 5.0 Hz, 1H), 5.00 (d, J ) 3.0 Hz, 1H),
5.62 (dd, J ) 4.4, 7.4 Hz, 1H), 5.72 (dd, J ) 3.0, 7.4 Hz, 1H),
7.47-7.71 (m, 11H), 11.34 (br s, 2H); 13C NMR δ 18.72, 26.50,
63.24, 80.56, 84.19, 86.53, 88.81, 108.41, 127.83-134.97 (arom),
141.40, 151.00, 163.11, 190.60. Anal. Calcd for C26H28N2O6SSi
(524.7): C, 59.52; H, 5.38; N, 5.34. Found: C, 59.51; H, 5.42;
N, 5.33.
5-[5(S)-[[(ter t-Bu tyld ip h en ylsilyl)oxy]m eth yl]-2,5-d ih y-
d r ofu r a n -2(R)-yl]u r a cil (4). A solution of 3 (2.0 g, 3.8 mmol)
in triethyl phosphite (80 mL) was stirred for 17 h at 110 °C.
Volatiles were evaporated, and the residue was chromato-
graphed (MeOH/CHCl3, 1:99) to give 4 (1.6 g, 94%; colorless solid
foam): 1H NMR δ 0.99 (s, 9H), 3.65 (dd, J ) 5.4, 10.1 Hz, 1H),
3.74 (dd, J ) 4.0 Hz, 1H), 4.87 (m, 1H), 5.57 (br s, 1H), 5.92 (dd
J ) 5.9 Hz, 1H), 6.04 (dd, 1H), 7.12 (s, 1H), 7.38-7.65 (m, 10H),
10.85 (br, 1H), 11.18 (br, 1H); 13C NMR δ 18.78, 26.62, 66.58,
80.38, 86.35, 112.91, 127.77-135.02 (arom and olefin), 138.18,
151.11, 163.28; MS m/z 449 (M + 1). Anal. Calcd for C25H28N2O4-
Si‚0.5H2O (457.6): C, 65.62; H, 6.39; N, 6.12. Found: C, 65.36;
H, 6.41; N, 5.92.
F igu r e 1. Circular dichroism spectrum of 8 in MeOH solution.
effect (236 nm, +5.65) and a negative second Cotton effect
(222 nm, -3.07) indicative of exciton splitting14 for the
vicinal (4′ and 5′) benzoyl chromophores. The signed
order of Cotton effects in the CD spectrum of 8 is
consistent with positive (+)-chirality between the primary
(5′) and secondary (4′) benzoyl chromophores with an (S)-
configuration at C4′ as found with (S)-4-O-acetyl-1,2-di-
O-benzoylbutane-1,2,4-triol (9) and analogous deriva-
tives15 (and assuming weak exciton splitting interactions
with the N-benzoyluracil chromophore, which is in a 1,4
relationship with the chiral center at C4′).
Synthesis of (S)-5-(4,5-dihydroxypentyl)uracil (DHPU,
7) from pseudouridine (1) was achieved in five steps with
retention of configuration at C4′. This establishes
pseudouridine as a prime precursor candidate for the
biosynthesis of DHPU by B. subtilis bacteriophage SP-
15.
5-[2,5-Dih yd r o-5(S)-(h yd r oxym eth yl)fu r a n -2(R)-yl]u r a -
cil (5). A solution of 4 (90 mg, 0.2 mmol) and NH4F (300 mg)
in MeOH (5 mL) was refluxed for 90 min. Volatiles were
evaporated, and the residue was chromatographed (MeOH/
CHCl3, 1:9). Crystallization of the solid residue (MeOH) gave 5
(30 mg, 71%; fine off-white needles): mp 215 °C; UVmax 263 nm
(ꢀ 8800); 1H NMR δ 3.47 (“s”, 2H), 4.75 (m, 2H), 5.52 (d, J ) 3.4
Hz, 1H), 5.92 (“t”, 2H), 7.35 (s, 1H), 10.85 (br, 1H), 11.12 (br,
1H); 13C NMR δ 63.89, 80.15, 86.94, 112.97, 128.18, 129.53,
138.94, 151.05, 163.40; MS m/z 211 (M + 1). Anal. Calcd for
C9H10N2O4 (210.2): C, 51.43; H, 4.80; N, 13.33. Found: C, 51.28;
H, 4.63; N, 13.49.
5-[5-[(ter t-Bu tyld ip h en ylsilyl)oxy]-4(S)-h yd r oxyp en tyl]-
u r a cil (6). A solution of 4 (950 mg, 2.1 mmol) was added to a
suspension of 10% Pd-C (300 mg) in MeOH (10 mL), the
mixture was stirred under H2 (1 atm) for 24 h at ambient
temperature, and the catalyst was removed by filtration. Vola-
tiles were evaporated, and the residual syrup was chromato-
graphed (MeOH/CHCl3, 1:49). Crystallization of the solid
residue (CHCl3) gave 6 (510 mg, 54%; fine colorless needles):
mp 168 °C; 1H NMR δ 0.99 (s, 9H), 1.23-1.59 (m, 4H), 2.18 (“t”,
2H), 3.42-3.46 (m, 2H), 3.56 (m, 1H), 4.56 (s, 1H), 7.18 (s, 1H),
7.40-7.65 (m, 10H), 10.64 (br, 1H), 11.00 (br, 1H); 13C NMR δ
18.78, 24.16, 26.03, 32.82, 26.62, 67.86, 70.32, 111.97, 127.77-
135.02 (arom), 137.66, 151.41, 164.51; MS m/z 453 (M + 1). Anal.
Calcd for C25H32N2O4Si‚0.33H2O (458.6): C, 65.47; H, 7.18; N,
6.11. Found: C, 65.46; H, 7.15; N, 6.03.
Exp er im en ta l Section
Uncorrected melting points were determined with a hot-stage
apparatus. UV spectra were recorded with solutions in H2O (pH
7.0) unless otherwise indicated. NMR spectra of solutions in
Me4Si/Me2SO-d6 were recorded at 270 or 400 MHz (1H) or 25
MHz (13C). Mass spectra were determined at 70 eV with
chemical ionization (CI, CH4) unless otherwise indicated. All
chemicals and solvents were of reagent quality, and pyridine
was dried by reflux over and distillation from CaH2. Column
chromatography was performed with silica gel (100-200 mesh).
5-[5-O-(ter t-Bu t yld ip h en ylsilyl)-â-D-r ib ofu r a n osyl]u r a -
cil (2). TBDPSCl (6.15 g, 22 mmol) was added to a suspension
of 1 (3.66 g, 15 mmol) in dried pyridine (250 mL), and the
mixture was stirred for 48 h at ambient temperature. Volatiles
were evaporated, and the residue was chromatographed (MeOH/
CHCl3, 1:99). Crystallization of the solid residue (CHCl3) gave
(S)-5-(4,5-Dih yd r oxyp en tyl)u r a cil (7). A solution of 6 (300
mg, 0.65 mmol) and NH4F (300 mg) in MeOH (10 mL) was
refluxed for 90 min. Workup (as described for 5) gave 7 (138
mg, 99%; fine colorless needles): mp 225-226 °C (lit.10b mp 225-
1
226 °C); UVmax 265 nm (ꢀ 6600); H NMR δ 1.16-1.24 (m, 1H),
1.34-1.44 (m, 2H), 1.50-1.57 (m, 1H), 2.11-2.19 (m, 2H), 3.20-
3.29 (m, 2H), 3.37 (“s”, 1H), 4.38 (br s, 2H), 7.18 (s, 1H), 10.66
(br s, 1H), 10.94 (br s, 1H); 13C NMR δ 24.34, 25.98, 32.82, 65.82,
70.91, 111.97, 137.66, 151.29, 164.45; HRMS (EI) m/z 214.0953
(5%, M+ ) 214.0952). Anal. Calcd for C9H14N2O4 (214.2): C,
50.46; H, 6.59; N, 13.08. Found: C, 50.04; H, 6.60; N, 12.80.
1
2 (5.49 g, 76%; colorless needles): mp 114-115 °C; H NMR δ
1.05 (s, 9H), 3.73 (q, J ) 5.7 Hz, 1H), 3.88 (br d, 2H), 3.97-4.01
(m, 2H), 4.57 (d, J ) 4.7 Hz, 1H), 4.85 (br s, 1H), 5.03 (br s, 1H),
7.34 (s, 1H), 7.43-7.73 (m, 10H), 11.09 (br, 2H); 13C NMR δ
18.72, 26.62, 64.41, 70.73, 73.66, 78.69, 82.90, 110.92, 127.77-
135.02 (arom), 139.12, 151.17, 163.46; MS m/z 483 (M + 1). Anal.
Calcd for C25H30N2O6Si‚H2O (500.6): C, 59.98; H, 6.44; N, 5.60.
Found: C, 60.22; H, 6.26; N, 5.65.
Catalytic reduction of 5 (as described for 4 f 6) also gave 7.
(S)-5-(4,5-Di-O-ben zoyl-4,5-d ih yd r oxyp en tyl)-N-ben zoyl-
u r a cil (8). Benzoyl chloride (158 mg, 1.12 mmol) was added to
a solution of 7 (40 mg, 0.187 mmol) in dried pyridine (2 mL),
and the mixture was stirred for 1.5 h at 60 °C. Volatiles were
evaporated, and the residue was chromatographed (MeOH/
CHCl3, 1:99) to give 8 (79.2 mg, 80%; colorless solid foam): UV-
(MeOH)max 230, 252 nm (ꢀ 28 000, 20 000); CD (MeOH) 236 nm
(14) Nakanishi, K.; Berova, N. In Circular Dichroism: Principles
and Applications Nakanishi, K., Berova, N., Woody, R. W., Eds.;
VCH: New York, 1994; Chapter 13.
(15) Mori, Y.; Furukawa, H. Tetrahedron 1995, 51, 6725-6738.

Notes
J . Org. Chem., Vol. 63, No. 21, 1998 7541
(∆ꢀ ) +5.65), 222 nm (∆ꢀ ) -3.07); 1H NMR δ 1.59 (m, 2H),
1.83 (m, 2H), 2.33 (m, 2H), 4.45 (dd, J ) 6.7, 11.8 Hz, 1H), 4.62
(dd, J ) 2.7, 11.8 Hz, 1H), 5.44 (m, 1H), 7.47-7.96 (m, 16H),
11.48 (br s, 1H); 13C NMR δ 23.96, 26.31, 30.43, 65.44, 71.72,
114.15, 128.36-136.77 (arom), 151.33, 162.88, 166.09, 166.22,
168.80; MS (EI) m/z 526 (M+).
Ack n ow led gm en t. We thank Kyowa Hakko Kogyo
Co., Ltd. for a gift of pseudouridine, Dr. W. H. Muhs
for useful preliminary experiments, and Mrs. J eanny
Gordon for assistance with the manuscript.
J O980975Q