´
E. Trevisiol, E. Defrancq, J. Lhomme, A. Laayoun, P. Cros
FULL PAPER
Protected Nucleoside 12: To a solution of acid 3 (0.40 g, 2 mmol)
in dry THF (10 mL), cooled at 0°C and under argon, were added,
successively, N-methylmorpholine (0.23 mL, 2 mmol), isobutyl
chloroformate (0.26 mL, 2 mmol), and then, after 15 min, com-
pound 10 (1 g, 2 mmol); the reaction mixture was stirred at room
temp. for 2 h. The solvent was evaporated and the oily residue ob-
tained was dissolved in CH2Cl2. The organic layer was washed suc-
cessively with 0.1 aq. NaOH, 1 aq. HCl, and brine, then it was
H, CH2ϪCH2), 3.30 (m, 2 H, CH2ϪN), 3.59 (m, 2 H, CH2ϪN),
4.30Ϫ4.20 (overlapping m, 2 H, 5Ј,5Љ-H), 4.27 (s, 2 H, CH2ϪO),
4.73 (m, 1 H, 4Ј-H), 5.32 (m, 1 H, 3Ј-H), 5.42 (t, 1 H, 2Ј-H), 6.29
(d, 1 H, 1Ј-H), 8.27 (br. s, 1 H, 2-H), 8.47 (s, 1 H, 8-H). Compound
2: tR ϭ 30 min. Ϫ 31P NMR (160 MHz, D2O): δ ϭ Ϫ21.83 (t, Pβ),
Ϫ10.90 (d, J ϭ 19 Hz, Pα), Ϫ5.95 (d, J ϭ 18 Hz, Pγ), Ϫ H NMR
(400 MHz, D2O): δ ϭ 1.74 (m, 4 H, CH2ϪCH2), 3.34 (m, 2 H,
CH2ϪN), 3.62 (m, 2 H, CH2ϪN), 4.17 (s, 2 H, CH2ONH2),
1
dried (Na2SO4). Evaporation of the solvent afforded product 11 4.23Ϫ4.34 (m, 2 H, 5Ј,5ЈЈ-H), 4.43 (m, 1 H, 4Ј-H), 4.69 (m, 1 H,
(1.1 g, 82%) as a white powder which was used without further 3Ј-H), 4.86 (m, 1 H, 2Ј-H), 6.16 (d, 1 H, 1Ј-H), 8.27 (br. s, 1 H, 2-
1
purification for the next reaction. Ϫ H NMR (200 MHz, CDCl3): H), 8.55 (s, 1 H, 8-H). Ϫ MS (electrospray, negative mode); m/z:
δ ϭ Ϫ0.01 (s, 6 H, SiϪCH3), 0.82 [s, 9 H, SiϪC(CH3)3], 1.37 (s, 3 650 [M Ϫ H]Ϫ, 672 [M Ϫ 2H ϩ Na]Ϫ, 694 [M Ϫ 3H ϩ 2Na]Ϫ.
H, CH3), 1.43 [s, 9 H, C(CH3)3], 1.60 (s, 3 H, CH3), 1.70 (m, 4 H,
Protected Aldehydic Fluorophore 14: To a solution of fluorescein
isothiocyanate (0.91 g, 2.35 mmol) in dry DMF (10 mL), 4-amino-
butyraldehyde diethylacetal (0.4 mL, 2.35 mmol) was added, and
the reaction mixture was stirred at room temp. for 1 h under argon.
The solvent was then evaporated under reduced pressure and the
crude product was purified by silica gel chromatography (CH2Cl2/
CH2ϪCH2), 3.37 (q, 2 H, CH2ϪN), 3.62 (m, 2 H, CH2ϪN), 3.70
(m, 2 H, 5Ј,5Љ-H), 4.29 (s, 2 H, CH2ϪONH), 4.35 (m, 1 H, 4Ј-H),
4.95 (dd, 1 H, 3Ј-H), 5.25 (dd, 1 H, 2Ј-H), 6.12 (d, 1 H, 1Ј-H), 7.60
(br. s, 1 H, NH), 7.95 (s, 1 H, 8-H), 8.15 (br. s, 1 H, NH), 8.36 (s,
1 H, 2-H). Ϫ MS (DCI); m/z: 666 [M ϩ H]ϩ. Ϫ To the crude
product 11 (1 g, 1.5 mmol), dissolved in THF (5 mL), was added a
1 TBAF (3 mL, 3 mmol) solution in THF and the reaction mix-
ture was stirred for 1 h. The solvent was evaporated and the residue
obtained was dissolved in CH2Cl2. The organic layer was washed
MeOH, 90:10) to give compound 14 (1.21 g, 94%) as an orange
powder, m.p. 200°C (dec.). Ϫ IR (KBr): ν˜ ϭ 3263 cmϪ1, 2929,
1744, 1610, 1443. Ϫ UV (MeOH): λmax (ε) ϭ 480 nm (20800), 454
(20000), 273 (28000), 226 (77400).
[D6]DMSO): δ ϭ 1.09 (t, 6 H, 2 CH2ϪCH3), 1.57 (m, 4 H,
CH2ϪCH2), 3.50 (overlapping m, H, CH2ϪN and
Ϫ
1H NMR (200 MHz,
with brine, dried (Na2SO4) and concentrated. The crude product
was purified by silica gel chromatography (CH2Cl2/MeOH, 95:5)
to give compound 12 (0.75 g, 91%) as a white powder, m.p.
89Ϫ91°C. Ϫ IR (KBr): ν˜ ϭ 3306 cmϪ1, 2981, 2944, 1737, 1628. Ϫ
6
2
CH3ϪCH2ϪO), 4.48 (t, 1 H, CH(OEt)2), 6.61 (m, 6 H, H-Ar), 7.15
(d, 1 H, H-Ar), 7.70 (d, 1 H, H-Ar), 8.10 (br. s, 1 H, NH or OH),
8.20 (s, 1 H, H-Ar), 9.85 (br. s, 1 H, NH or OH), 10.11 (br. s, 2 H,
NH or OH). Ϫ 13C NMR (50 MHz, [D6]DMSO): δ ϭ 15.2 (CH3),
23.6 (CH2), 30.6 (CH2), 35.6 (CH), 43.4 (CH2), 60.4 (CH2), 82.8
(quat), 101.8 (CH), 102.0 (CH), 109.5 (quat), 112.3 (CH), 123.8
(CH), 126.3 (quat), 128.8 (CH), 141.2 (quat), 147.0 (quat), 151.7
(quat), 159.3 (quat), 162.1 (CH), 168.3 (quat), 180.2 (quat). Ϫ MS
(FAB, mNBA matrix); m/z: 551 [M ϩ H]ϩ.
1
UV (MeOH): λmax (ε) ϭ 267 nm (16600). Ϫ H NMR (200 MHz,
CDCl3): δ ϭ 1.35 (s, 3 H, CH3), 1.43 [s, 9 H, C(CH3)3], 1.62 (s, 3
H, CH3), 1.75 (m, 4 H, CH2ϪCH2), 3.32 (m, 2 H, CH2ϪN), 3.68
(m, 2 H, CH2ϪN), 3.92 (m, 2 H, 5Ј,5Љ-H), 4.28 (s, 2 H,
CH2ϪONH), 5.09 (m, 1 H, 3Ј-H), 5.16 (m, 1 H, 2Ј-H), 5.81 (d, 1
H, 1Ј-H), 6.00 (br. t, 1 H, 5Ј-OH), 6.75 (br., 1 H, NH), 7.65 (br., 1
H, NH), 7.74 (s, 1 H, 8-H), 8.15 (br., 1 H, NH), 8.28 (s, 1 H, 2-
H). Ϫ 13C NMR (50 MHz, CDCl3): δ ϭ 25.1 (CH3), 26.4 (CH2),
26.5 (CH2), 27.4 (CH3), 27.9 (CH3), 38.4 (CH2), 40.0 (CH2), 63.0
(CH2), 75.8 (CH2), 81.5 (CH), 82.6 (quat), 82.9 (CH), 85.9 (CH),
93.8 (CH), 113.8 (quat), 121.1 (quat), 139.3 (CH), 147.5 (quat),
152.6 (CH), 155.0 (quat), 157.9 (quat), 169.1 (quat). Ϫ MS (FAB,
mNBA matrix); m/z: 552 [M ϩ H]ϩ. Ϫ HRMS (FAB, mNBA ma-
trix): calcd. 552.2782, found 552.2780. Ϫ C24H37N7O8 (551.3):
calcd. C 52.26, H 6.76, N 17.78; found C 51.87, H 6.61, N 17.18.
Aldehydic Fluorophore 15: Compound 14 (0.34 g, 0.62 mmol) was
dissolved in 30% aq. acetic acid solution (20 mL) and the reaction
mixture was stirred at room temp. for 1 h. After the solvent was
evaporated under reduced pressure, the crude product was purified
by silica gel chromatography (CH2Cl2/MeOH, 90:10) to give alde-
hyde 15 (0.15 g, 50%) as an orange powder, m.p. 200°C (dec). Ϫ
IR (KBr): ν˜ ϭ 3254 cmϪ1, 2950, 1730, 1586, 1460. Ϫ UV (Tris
buffer, pH ϭ 9): λmax (ε) ϭ 492 nm (77000), 239 (54000) Ϫ 1H
NMR (200 MHz, [D6]DMSO): δ ϭ 1.90 (m, 2 H, CH2), 2.06 (m,
2 H, CH2ϪCO), 3.63Ϫ3.77 (m, 2 H, CH2ϪN), 5.72 [m, 1 H,
CH(OH)2], 6.62 (m, 6 H, H-Ar), 7.18 (d, 1 H, H-Ar), 7.84 (dd, 1
H, H-Ar), 8.12 (s, 1 H, H-Ar), 9.30 (br., 1 H, NH or OH), 10.14
(br., 2 H, NH or OH). Ϫ MS (FAB, mNBA matrix); m/z: 477 [M
ϩ H]ϩ, 390 [M Ϫ (NHϪ[CH2]3ϪCHO)]ϩ. Ϫ HRMS (FAB, mNBA
matrix): calcd. 477.1120, found 477.1121. Ϫ C25H20N2O6S·1H2O
(476.1): calcd. C 60.71, H 4.49, N 5.67; found C 60.98, H 4.44,
N 5.56.
Deprotection of Nucleoside 12 To Obtain 13: Deprotected nucleo-
side 13 was obtained from nucleoside 12 in the same manner as
described for compound 7 in 45% yield. Ϫ IR (KBr): ν˜ ϭ 3314
cmϪ1, 2930, 2865, 1624, 1094. Ϫ UV (H2O): λmax (ε) ϭ 267 nm
(16600). Ϫ 1H NMR (300 MHz, [D6]DMSO): δ ϭ 1.50 (m, 4 H,
CH2ϪCH2), 3.10 (q, 2 H, CH2ϪN), 3.40 (overlapping m, 4 H,
5Ј,5Љ-H and CH2ϪN), 3.87 (s, 2 H, CH2ϪONH2), 3.95 (m, 1 H,
4Ј-H), 4.12 (m, 1 H, 3Ј-H), 4.58 (m, 1 H, 2Ј-H), 5.16 (d, 1 H, 5Ј-
OH), 5.42 (m, 2 H, 3Ј-OH and 2Ј-OH), 5.86 (d, 1 H, 1Ј-H), 6.32
(br., 1 H, NH), 7.68 (br., 2 H, ONH2), 8.18 (br. s, 1 H, 2-H), 8.32
(s, 1 H, 8-H). Ϫ 13C NMR (75 MHz, [D6]DMSO) δ ϭ 27.0 (CH2).
38.1 (CH2), 60.5 (CH2), 63.2 (CH2), 70.9 (CH), 72.5 (CH2), 73.7
(CH), 74.7 (CH2), 86.2 (CH), 88.3 (CH), 140.0 (CH), 148.0 (quat),
152.7 (CH), 155.0 (quat), 170.0 (quat). Ϫ MS (DCI); m/z: 412 [M
ϩ H]ϩ.
Oxime Ether 16: To a solution of deprotected nucleoside 7 (5 mg,
0.01 mmol) in water (1 mL) was added fluorophore 15 (6.5 mg,
0.01 mmol) in DMSO (0.1 mL). The reaction mixture was stirred
for 1 h, then concentrated under reduced pressure. The crude prod-
uct was purified by reverse-phase chromatography to give com-
pound 16 as an orange powder (5.7 mg, 70%). Ϫ UV (Tris buffer,
pH ϭ 9): λmax (ε) ϭ 494 nm (70000), 279 (24000), 239 (50000). Ϫ
Nucleotide Triphosphate 2: Nucleotide 2 was obtained, as a white
powder in 20% overall yield, from protected nucleoside 12 by the
same procedure that was used to obtain 1. Protected nucleotide
intermediate: tR ϭ 36 min. Ϫ 31P NMR (160 MHz, D2O): δ ϭ
1H NMR (500 MHz, [D6]DMSO,
1.75Ϫ1.77 (m, 2 H, CH2ϪCH2ϪNH), 2.21 and 2.41 (2 q, 2 H, Nϭ
H, CH2ϪNHϪCϭS),
2 diastereoisomers): δ ϭ
Ϫ21.62 (t, Pβ), Ϫ11.05 (d, J ϭ 19 Hz, Pα), Ϫ5.66 (d, J ϭ 20 Hz, CHϪCH2), 3.54 (overlapping m,
2
Pγ). Ϫ 1H NMR (400 MHz, D2O): δ ϭ 1.28 [s, 9 H, C(CH3)3], 1.49 3.56Ϫ3.64 (m, 2 H, 5Ј,5Љ-H), 3.84 (q, 1 H, 4Ј-H), 3.95 (q, 1 H, 3Ј-
(s, 3 H, CH3), 1.72 (s, 3 H, CH3), 1.75Ϫ1.65 (overlapping m, 4 H), 4.03 (q, 1 H, 2Ј-H), 4.10 (d, 2 H, CH2ϪNHϪCO), 4.38 and
216
Eur. J. Org. Chem. 2000, 211Ϫ217