Expansion of the Genetic Alphabet
J. Am. Chem. Soc., Vol. 122, No. 14, 2000 3285
Compound 12. To a suspension of magnesium metal (52 mg, 2.167
mmol) in THF (2 mL) was added 5-bromo-m-xylene (293 µL, 2.164
mmol). The resulting suspension was heated to ∼50 °C. After 1 h,
400 µL of this solution was added to chloroglycoside 8 (126 mg, 0.324
mmol) in THF (1 mL). After 14 h, an additional 100 µL of the
aforementioned Grignard solution was added. After 15 h, the reaction
was partitioned between ethyl acetate (10 mL) and saturated aqueous
NH4Cl (10 mL). The layers were separated, and the aqueous layer was
extracted with ethyl acetate (2 × 15 mL). The combined organics were
dried over Na2SO4, filtered, and concentrated. Purification by column
chromatography on silica gel (5-15% ethyl acetate in hexane) afforded
nucleoside 12 (13 mg, 9%) and its R-anomer (48 mg, 32%). 1H NMR
(400 MHz, CDCl3) δ 7.99 (4H, m), 7.28 (2H, d, J ) 8.1 Hz), 7.24
(2H, d, J ) 8.1 Hz), 7.02 (2H, s), 6.92 (1H, s), 5.62 (1H, m), 5.20
(1H, dd, J ) 10.9, 5.0 Hz), 4.71 (1H, dd, J ) 11.8, 4.0 Hz), 4.66 (1H,
dd, J ) 11.8, 3.6 Hz), 4.53 (1H, m), 2.50 (1H, m), 2.44 (3H, s), 2.41
(3H, s), 2.26 (7H, m). HRMS calcd for C29H31O5 (MH+): 459.2171;
found: 459.2179.
Purification via column chromatography on silica gel (10-20% ethyl
acetate in hexane) afforded the desired S-diastereomer (712 mg, 48%;
the R-diastereomer was isolated in 42% yield), which was dissolved
in pyridine (9 mL) and cooled to 0 °C. Triethylamine (570 µL, 4.09
mmol) was added, followed by the slow dropwise addition of mesyl
chloride (90 µL, 1.17 mmol). The reaction mixture was warmed slowly
to room temperature over 1.5 h, at which time the reaction was
quenched by addition of saturated aqueous NaHCO3 (∼2 mL).
Concentration, followed by purification by column chromatography on
silica gel (5-15% ethyl acetate in hexane), afforded the protected
1
nucleoside 16 (176 mg, 17% over two steps). H NMR (400 MHz,
CDCl3) δ 8.17 (1H, m), 8.07 (1H, m), 7.83 (1H, s), 7.65 (2H, d, J )
7.9 Hz), 7.31-7.59 (11H, m), 6.92 (6H, m), 5.74 (1H, dd, J ) 10.6,
5.2 Hz), 4.58 (2H, s), 4.39 (1H, m), 4.32 (1H, m), 3.86 (3H, s), 3.84
(6H, s), 3.58 (1H, dd, J ) 9.9, 4.5 Hz), 3.47 (1H, dd, J ) 9.9, 3.7 Hz),
2.72 (3H, s), 2.67 (3H, s), 2.53 (1H, dd, J ) 13.1, 5.3 Hz), 2.06 (1H,
m). 13C NMR (100 MHz, CDCl3) δ 159.1, 158.4, 145.0, 136.2, 136.1,
132.7, 132.3, 132.0, 130.1, 130.1, 129.2, 128.2, 127.9, 127.7, 126.7,
125.4, 125.0, 124.5, 124.4, 124.1, 113.8, 113.0, 86.1, 84.1, 81.1, 77.5,
70.7, 64.3, 55.1, 55.0, 40.7, 19.4, 13.7. HRMS calcd for C46H46O6Cs
(MCs+): 827.2349; found: 827.2378.
Compound 17. To a stirred solution of nucleoside 16 (190 mg, 0.274
mmol) in acetic acid (6 mL) and methanol (1 mL) was added
trifluoroacetic acid (10 drops). After stirring at room temperature for
20 min, the orange reaction mixture was concentrated. Purification via
column chromatography on silica gel (20-50% ethyl acetate in hexane)
afforded nucleoside 17 (93 mg, 86%). 1H NMR (400 MHz, CDCl3) δ
8.09 (1H, m), 8.01 (1H, m), 7.54 (2H, m), 7.32 (2H, d, J ) 8.5 Hz),
6.92 (2H, d, J ) 8.6 Hz), 5.61 (1H, dd, J ) 10.6, 5.4 Hz), 4.53 (2H,
m), 4.21 (1H, m), 4.16 (1H, m), 3.91 (1H, dd, J ) 11.7, 3.7 Hz), 3.82
(6H, s), 3.80 (1H, m), 2.43 (1H, dd, J ) 13.4, 5.4 Hz), 1.89 (1H, ddd,
J ) 13.4, 10.6, 6.7 Hz). 13C NMR (100 MHz, CDCl3) δ 159.3, 135.1,
132.7, 132.4, 132.0, 130.0, 129.3, 128.5, 125.6, 125.2, 124.6, 124.5,
123.4, 113.9, 113.8, 85.1, 80.4, 77.6, 63.6, 55.2, 40.4, 19.6, 13.8. HRMS
calcd for C25H28O4Na (MNa+): 415.1885; found: 415.1882.
Compound 2. To a solution of nucleoside 12 in CH3OH (10 mL)
was added 1 M NaOMe (2 mL). After 45 min, the excess NaOMe was
quenched with NH4Cl (∼100 mg). The resulting slurry was concen-
trated, and purified by column chromatography on silica gel (1-5%
1
CH3OH in CH2Cl2) to afford nucleoside 2 (105 mg, 82%). H NMR
(400 MHz, CD3OD) δ 6.98 (2H, s), 6.78 (1H, s), 5.03 (1H, dd, J )
10.6, 5.3 Hz), 4.29 (1H, m), 3.92 (1H, ddd, J ) 7.6, 5.2, 2.4 Hz), 3.66
(1H, m), 2.27 (6H, s), 2.14 (1H, ddd, J ) 13.1, 5.4, 1.6 Hz), 1.91 (1H,
ddd, J ) 13.1, 10.6, 5.9 Hz). 13C NMR (150 MHz, CDCl3) δ 140.8,
138.1, 129.5, 129.3, 123.8, 123.4, 87.2, 80.1, 63.4, 43.9, 21.3. HRMS
calcd for C13H19O3 (MH+): 223.1334; found: 223.1326.
Compound 13. To a solution of nucleoside 1 (90 mg, 0.405 mmol)
in pyridine (2 mL) and CH2Cl2 (2 mL) was added triethylamine (300
µL, 2.152 mmol), followed by DMTr-Cl (175 mg, 0.516 mmol) in two
portions over 30 min. After stirring at room temperature for 2 h, the
reaction mixture was concentrated and purified by column chroma-
tography on silica gel (50-80% ethyl acetate in hexane) to afford the
tritylated nucleoside (185 mg), which was dissolved in CH2Cl2 (3.5
mL). A catalytic amount of DMAP (∼2 mg) was added, followed by
triethylamine (350 µL, 2.512 mmol) and 2-cyanoethyl diisopropyl-
aminochloro phosphoramidite (160 µL, 0.718 mmol). After 30 min,
the reaction mixture was partitioned between ethyl acetate (20 mL)
and saturated aqueous NaHCO3 (20 mL). The layers were separated,
and the aqueous layer was extracted with 2 × 20 mL ethyl acetate.
The combined organics were dried over Na2SO4, filtered, and concen-
trated. Purification by column chromatography on silica gel (10-30%
ethyl acetate in 5% triethylamine/hexane) afforded phosphoramidite
13 (238 mg, 81% over two steps). 1H NMR (400 MHz, CDCl3) δ 7.20-
7.55 (9H, m), 7.09 (2H, s), 6.93 (1H, s), 6.83 (4H, m), 5.12 (1H, m),
4.25 (1H, m), 4.14 (1H, m), 3.60-3.90 (8H, m), 3.20-3.40 (2H, m),
2.40-2.80 (5H, m), 2.30 (6H, m), 2.07 (1H, m), 1.03-1.29 (12H, m).
31P NMR (140 MHz, CDCl3) δ 148.5, 148.3.
Compound 3. To stirred a solution of 17 (8 mg, 0.020 mmol) in
CH2Cl2 (1 mL) and H2O (1 drop) was added DDQ (7 mg, 0.031 mmol).
After 1 h, the reaction mixture was partitioned between ethyl acetate
(15 mL) and saturated aqueous NaHCO3 (15 mL). The layers were
separated, and the aqueous layer was extracted with 2 × 15 mL ethyl
acetate. The combined organics were dried over Na2SO4, filtered, and
concentrated. Purification by column chromatography on silica gel (1-
1
5% CH3OH in CH2Cl2) afforded nucleoside 3 (3 mg, 55%). H NMR
(400 MHz, CDCl3) δ 8.07 (1H, m), 7.98 (1H, m), 7.50 (2H, m), 7.45
(1H, s), 5.24 (1H, dd, J ) 11.5, 1.7 Hz), 4.23 (1H, m), 3.96 (2H, m),
3.81 (1H, m), 2.67 (3H, s), 2.62 (3H, s), 2.10 (1H, m), 1.88 (1H, ddd,
J ) 14.2, 11.6, 2.3 Hz). 13C NMR (150 MHz, CDCl3) δ 135.5, 132.8,
132.7, 132.1, 127.6, 125.7, 125.3, 124.7, 124.5, 123.8, 76.0, 70.9, 69.2,
68.0, 36.5, 19.5, 13.8. HRMS calcd For C13H24NO3 (MNH4+): 290.1756;
found: 290.1764.
Compound 14. Proton sponge (23 mg, 0.107 mmol) and nucleoside
2 (16 mg, 0.072 mmol) were dissolved in trimethyl phosphate (0.36
mL) and cooled to 0 °C. POCl3 (8 µL, 0.090 mmol) was added
dropwise, and the lavender slurry was stirred at 0 °C for 2 h.
Tributylamine (105 µL, 0.441 mmol) was added, followed by a solution
of tributylammonium pyrophosphate (62 mg) in DMF (0.8 mL). After
1 min, the reaction was quenched via addition of 1 M triethylammonium
bicarbonate (7 mL). The resulting crude solution was lyophilized, and
purification via reverse phase (C18) HPLC (4-30% CH3CN in 0.1 M
TEA-bicarbonate, pH 7.5) afforded triphosphate 14 as a white solid.
Compound 16. To a stirred solution of 2-bromo-1,4-dimethylnaph-
thylene (640 mg, 2.72 mmol) in THF (15 mL) at -78 °C was added
nBuLi (2 mL, 2 M in cyclohexane) slowly dropwise. The lime-green
mixture was stirred for 15 min, at which time a solution of aldehyde
15 (1.214 g, 1.801 mmol) in THF (5 mL) was added dropwise down
the side of the flask. After 1 h, the bath was removed, and the reaction
mixture was warmed to room temperature. After a total of 2.5 h, the
reaction mixture was partitioned between ethyl acetate (100 mL) and
saturated aqueous NaHCO3 (100 mL). The layers were separated, and
the aqueous layer was extracted with 2 × 100 mL ethyl acetate. The
combined organics were dried over Na2SO4, filtered, and concentrated.
Compound 18. To a solution of nucleoside 3 (28 mg, 0.103 mmol)
in pyridine (1 mL) was added triethylamine (75 µL, 0.535 mmol),
followed by DMTr-Cl (70 mg, 0.206 mmol). After stirring at room
temperature for 30 min, the reaction mixture was concentrated;
purification via column chromatography on silica gel (50-80% ethyl
acetate in hexane) afforded the tritylated nucleoside (45 mg), which
was dissolved in CH2Cl2 (1 mL). A catalytic amount of DMAP (∼2
mg) was added, followed by triethylamine (65 µL, 0.470 mmol), and
2-cyanoethyl diisopropylaminochloro phosphoramidite (35 µL, 0.157
mmol). After 15 min, the reaction mixture was partitioned between
CH2Cl2 (20 mL) and saturated aqueous NaHCO3 (20 mL). The layers
were separated, and the aqueous layer was extracted with 2 × 20 mL
CH2Cl2. The combined organics were dried over Na2SO4, filtered, and
concentrated. Purification by column chromatography on silica gel (10-
30% ethyl acetate in 5% triethylamine/hexane) afforded phosphor-
amidite 18 (51 mg, 64% over two steps). 1H NMR (400 MHz, CDCl3)
δ 8.09 (1H, m), 7.98 (1H, m), 7.75 (1H, d, J ) 1.8 Hz), 7.53 (4H, m),
7.43 (4H, m), 7.20-7.30 (3H, m), 6.83 (4H, m), 5.66 (1H, m), 4.56
(1H, m), 4.26 (1H, m), 3.83 (1H, m), 3.78 (3H, s), 3.77 (3H, s), 3.45-
3.70 (4H, m), 3.32 (1H, m), 2.64 (3H, s), 2.62 (1H, m), 2.56 (3H, s),
2.46 (1H, dd, J ) 6.6, 6.5 Hz), 2.42 (1H, m), 2.08 (1H, m), 1.19 (9H,