ACS Chemical Biology
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(8 × 10 mL), and dried over Na2SO4. The crude product was
concentrated in vacuum and directly subjected to a flash silica gel
chromatography using CH2Cl2 containing 1% MeOH and 1% Et3N as
eluent. White solids with over 90% of isolated yield could be obtained
for each of compound 10.
contrast, the longer farnesyl group (C-15 lengths of terpene
chain) shows very similar effects to the overall duplex stability
and base-pairing specificity when compared to the geranyl
group. The molecular dynamic simulation studies suggested
that, for the (xT):A pair, the modified terpene chain interrupts
base pairing and widens the DNA duplex at the modified site, as
the terpene chain grows longer, resulting in a constant decline
in overall DNA duplex stability. In the case of xT:G-pair-
containing duplexes, the long-chain terpenes can fit into the
minor groove of DNA duplex and increase the duplex stability.
This phenomenon could not be observed with the shorter
carbon chains like the methyl and dimethylallyl groups. In our
data set, the geranyl group (C-10) was the first carbon chain
length, wherein base-pairing discrimination was observed.
Although the farnesyl group shows comparable base-pairing
specificity to the geranyl group, the ges2T provides relatively
higher stability to overall DNA duplexes than fas2T does, and it
is hypothesized to be a more economical way for cells to use
geranyl group with less carbon atoms than farnesyl or other
longer terpene groups in modifying the 2-thiouridine, which
represents an evolutionary advantage of the geranyl group. Of
course, the availability of building blocks in cells and a working
enzyme to recognize these substrates are other two important
factors to be considered to fully understand nature’s unique
selection of geranyl group in tRNA modification. Most likely,
the farnesyl or other terpene pyrophosphates are not efficient
substrates of SelU, the natural enzyme that installs geranyl
group at 2-thiouridine residues in tRNAs.
1
10a. H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.52−7.14 (m,
9H), 6.86−6.75 (m, 4H), 6.21 (t, J = 6.0 Hz, 1H), 4.64 (m, 1H), 4.13
(m, 1H), 3.78 (s, 6H), 3.54 (dd, J1 = 3.2 Hz, J2 = 10.8 Hz, 1H,), 3.41
(dd, J1 = 2.8, J2 = 10.8 Hz, 1H), 3.2 (s, 1H), 2.58 (s, 3H), 2.51−2.45
(m, 1H), 2.34−2.25 (m, 1H), 1.55 (s, 3H); 13C NMR (100 HMz,
CDCl3) δ 169.94, 161.08, 159.04, 144.478, 135.58, 135.52, 134.76,
130.32, 128.36, 128.27, 127.44, 119.28, 113.57, 88.30, 87.35, 87.02,
72.31, 63.58, 55.50, 41.96, 14.95, 13.57;39 ESI-MS [M]: 574.0396
(calcd MS: 574.2138)
1
10b. H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.40−7.23 (m,
9H), 6.84−6.82 (m, 4H), 6.23 (t, J = 4.0 Hz, 1H), 5.32−5.22 (m, 1H),
4.61 (m, 1H), 4.35−4.25 (m, 1H), 4.21−4.13 (m, 1H), 4.09−4.11 (m,
1H), 3.85−3.95 (m, 2H), 3.78 (s, 6H), 3.50 (dd, J1 = 3.2 Hz, J2 = 10.8
Hz, 1H,), 3.39 (dd, J1 = 2.8, J2 = 10.8 Hz, 1H), 2.80 (s, 1H), 2.51−2.40
(m, 1H), 2.35−2.28 (m, 1H), 1.72 (s, 3H), 1.71 (s, 3H), 1.55 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 169.89, 160.84, 158.58, 144.25,
138.82, 135.13, 135.04, 130.00, 128.05, 126.99, 118.72, 116.73, 113.16,
88.34, 86.83, 74.35, 72.28, 69.04, 62.18, 55.11, 45.92, 41.81, 34.01,
29.37, 25.61, 17.89, 13.97,13.13; ESI-MS: [M + H]+: 629.2643 (calcd
MS: 629.2607).
10c. 1 H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.39 (d, J = 7.16
Hz, 2H), 7.30−7.16 (m, 6H), 6.83 (s, 2H), 6.81 (s, 2H), 6.24 (t, J =
5.80 Hz, 1H), 5.31 (t, J = 7.88 Hz, 1H), 5.06 (t, J = 7.88 Hz, 1H), 4.64
(t, J = 3.08 Hz, 1H), 4.12 (d, J = 2.72 Hz, 1H), 3.96−3.86 (m, 2H),
3.77 (s, 6H), 3.50 (dd, J1 = 10.60 Hz, J2 = 3.08 Hz, 1H), 3.41−3.37
(m, 1H), 2.51−2.46 (m, 1H), 2.37−2.30 (m, 1H), 2.10−2.00 (m, 5H),
1.70 (s, 3H), 1.67 (s, 3H), 1.59 (s, 3H), 1.52 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 169.59, 160.71, 158.63, 144.12, 142.58, 135.22,
134.43, 131.68, 129.96, 129.02, 127.99, 127.90, 127.70 (127.65),
127.06, 126.65, 123.64, 118.93, 116.32, 113.19, 113.04 (113.00),
MATERIALS AND METHODS
■
Synthesis of 2-Thio-geranyluridine Derivative Phosphora-
midite. 5′-DMTr-2-Thiothymidine (Compound 9). The 2-thiothymi-
dine 8 (500 mg, 1.95 mmol) was coevaporated with dry pyridine (5
mL) for three times to remove the potential water in the starting
material 8, which was subsequently redissolved in the pyridine (5 mL),
followed by the slow addition of the pyridine solution of
dimethoxytrityl chloride (760 mg, 2.25 mmol) at room temperature.
The reaction mixture was kept stirring for 4 h before being quenched
with anhydrous methanol (5.0 mL) and evaporated in vacuum. The
residue was extracted by dichloromethane and water. The organic layer
was washed with saturated sodium bicarbonate solution, dried over
magnesium sulfate, filtered, and the solvent was removed in vacuum.
The residue was purified by silica gel flash chromatography (eluent 1%
methanol in CH2Cl2 containing 1% of Et3N) to afford 9 as a white
solid (922 mg, 85.2%). TLC Rf = 0.60 (1% MeOH in CH2Cl2 with 1%
triethylamine). 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 7.69 (t, J
= 7.84 Hz, 1H), 7.40 (t, J = 7.48 Hz, 2H), 7.30−7.26 (m, 4H), 7.23 (d,
J = 7.16 Hz, 1H), 6.97 (t, J = 6.16 Hz, 1H), 6.83 (dd, J1 = 8.88 Hz, J2 =
1.72 Hz, 4H), 4.62−4.59 (m, 1H), 4.13 (d, J = 3.08 Hz, 1H), 3.77 (s,
6H), 3.55 (dd, J1 = 10.92 Hz, J2 = 2.72 Hz, 1H), 3.48 (dd, J1 = 10.56
Hz, J2 = 2.72 Hz, 1H), 2.68 (dd, J1 = 14.36 Hz, J2 = 2.65 Hz, 1H), 2.63
(dd, J1 = 6.16 Hz, J2 = 4.12 Hz, 1H), 2.33−2.26 (m, 1H), 1.45 (s, 3H);
13C NMR (100 HMz, CDCl3) δ 174.26, 161.41, 158.61, 149.34,
105.43, 87.95, 86.93, 86.67, 71.90, 63.23, 55.13, 41.62, 39.46, 30.67,
31
26.26, 25.58, 17.61, 16.26, 13.27;
ESI-MS: [M + H]+: 697.3341
(calcd MS: 697.3311).
1
10d. H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.38−7.18 (m,
9H), 6.80−6.78 (m, 4H), 6.22 (t, J = 4.0 Hz, 1H), 5.34−5.06 (m, 3H),
4.64 (m, 1H), 4.15−4.11 (m, 1H), 3.92−3.86 (m, 2H), 3.74 (s, 6H),
3.48−3.45 (dd, J1 = 3.2 Hz, J2 = 10.8 Hz, 1H,), 3.38−3.36 (dd, J1 = 3.2,
J2 = 10.8 Hz, 1H), 2.50−2.30 (m, 2H), 2.06−2.01 (m, 8H), 1.68 (s,
3H), 1.65 (s, 3H), 1.57 (s, 6H), 1.47 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 170.93, 160.85, 158.65, 144.23, 142.10, 140.38, 135.33,
135.22, 131.20, 130.02, 128.07, 124.29, 123.56, 118.85, 113.20, 88.18,
86.91, 74.40, 69.06, 62.22, 57.62, 55.13, 49.90, 45.86, 41.75, 39.62,
33.58, 31.67, 29.34, 25.64, 22.51, 21.22, 17.63, 16.33, 14.05, 13.24;
ESI-MS: [M + H]+: 765.3902 (calcd MS: 765.3859).
S-Geranyl Analogues Modified 5′-DMTr-2-Thiothymidine Phos-
phoramidites (Compounds 11a−d). For the synthesis of compounds
11a−d, the solution of compound 10 (200 mg), N,N-diisopropylethyl-
amine (DIPEA) (2 equiv) in anhydrous CH2Cl2 (5.0 mL), and 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite (1.5 equiv) was
stirred at RT for 5 h under Ar protection. The resulting reaction
mixture with the two diastereomers was concentrated in vacuum and
directly subjected to silica gel flash chromatography using CH2Cl2
containing 0.5% MeOH and 1% Et3N as eluent to give 11 as a light
yellowish sticky liquid with yields ∼75−80%. The compounds were
characterized by 31P NMR and ESI-MS. 11a. 31P NMR (CDCl3) δ
149.84, 149.22; ESI-MS: [M + H]+: 776.5565 (calcd MS: 776.3216).
11b. 31P NMR (CDCl3) δ 149.16, 148.72; ESI-MS: [M + H]+:
829.3734 (calcd MS: 829.3686). 11c. 31P NMR (CDCl3) δ 149.16,
144.20, 136.43, 136.24, 135.27, 129.98, 128.02, 127.89, 127.05, 123.78,
116.22, 113.17, 89.79, 86.80, 86.56, 71.07, 62.92, 55.15, (45.56), 41.13,
12.04 (10.55); ESI-MS: [M + Na]+: 583.1892 (calcd: 583.1879).31
S-Geranyl Analogues Modified 5′-DMTr-2-Thiothymidines (Com-
pounds 10a−d). For the synthesis of compounds 10a−d, the
methanol solution containing 9 (200 mg, 0.36 mmol), N,N-
diisopropylethylamine (260 μL, 1.48 mmol) and the terpene
bromides, for example, iodomethane (66.48 μL, 1.08 mmol, for
10a), 3,3-dimethylallyl bromide (82.26 μL, 0.72 mmol, for 10b),
farnesyl bromide (170 uL, 0.74 mmol, for 10c), and farnesyl bromide
(202.4 uL, 0.68 mmol, for 10d), respectively, was stirred at 25 °C for
12 h when the starting material was completely consumed. The
resulting reaction mixture was quenched with water, washed with brine
31
148.77; ESI-MS: [M + H]+: 897.4419 (calcd MS: 897.4390). 11d.
31P NMR (CDCl3) δ 149.10, 148.62; ESI-MS: [M + H]+: 965.5061
(calcd MS: 965.4938).
Synthesis of DNA Oligonucleotides. The DNA oligonucleotides
were chemically synthesized at 1.0 μmol scales by solid-phase synthesis
using an Oligo-800 DNA synthesizer. The system was protected with
helium gas. All the reagents were purchased from ChemGenes. The
G
ACS Chem. Biol. XXXX, XXX, XXX−XXX