“Click” Conjugation of 8-Aza-7-deazaadenine-DNA
Bioconjugate Chem., Vol. 21, No. 9, 2010 1631
residue was subjected to FC (silica gel, column 10 × 4 cm,
eluted with CH2Cl2/MeOH 97:3 f 90:10). Evaporation of the
main zone afforded compound 9 (0.33 g, 79%) as a colorless
foam. TLC (CH2Cl2/MeOH 9:1). Rf 0.6. UV λmax (MeOH)/nm
322 (ε/dm3 mol-1 cm-1 24 400). Anal. (C21H26N6O3) C, H, N.
1H NMR [DMSO (d6), 300 MHz]: δ 1.62-1.67 (m, 4H, 2 ×
CH2), 2.21-2.24 (m, 3H, CH2, C2′-HR), 2.48-2.55 (m, 2H,
CH2), 2.77-2.81 (m, 2H, C2′-Hꢀ, CtCH), 3.19, 3.22 (2s, 6H,
2 × CH3), 3.47-3.52 (m, 1H, C5′-H), 3.79-3.81 (d, J ) 3.6
Hz, 1H, C4′-H), 4.41 (s, 1H, C3′-H), 4.77-4.81 (m, 1H,
C5′-OH), 5.25-5.26 (d, J ) 4.2 Hz, 1H, C3′-OH), 6.55-6.59
(t, J ) 6.3 Hz, 1H, C1′-H), 8.43 (s, 1H, C2-H), 8.90 (s, 1H,
NdCH).
column 10 × 3 cm, CH2Cl2/MeOH 88:12). From the main zone,
compound 12 (0.21 g, 85%) was isolated as a yellowish foam.
TLC (silica gel, CH2Cl2/MeOH 8:2). Rf 0.5. UV λmax (MeOH)/
nm 348 (ε/dm3 mol-1 cm-1 5600), 365 (7600), 386 (6900). Anal.
1
(C33H32N8O3) C, H, N. H NMR [DMSO (d6), 300 MHz]: δ
1.13-1.19 (m, 2H, CH2), 1.55-1.65 (m, 4H, 2 × CH2),
2.19-2.27 (m, 1H, C2′-HR), 2.48-2.54 (m, 2H, CH2), 2.73-2.78
(m, 1H, C2′-Hꢀ), 3.79-3.84 (m, 1H, C4′-H), 4.40 (br s, 1H,
C3′-H), 4.81 (br s, 1H, C5′-OH), 5.29-5.34 (m, 1H, C3′-OH),
6.50-6.54 (m, 1H, C1′-H), 6.58 (s, 2H, CH2), 7.53-7.66 (m,
4H, Ar-H), 7.71 (s, 1H, triazole-H), 8.13 (s, 1H, Ar-H), 8.16
(s, 1H, Ar-H), 8.22 (s, 1H, C2-H), 8.56 (s, 1H, Ar-H), 8.60
(s, 1H, Ar-H), 8.72 (s, 1H, Ar-H). ESI-TOF: [M + Na]+ calcd
611.66. Found 611.20.
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-ꢀ-D-erythropento-
furanosyl]-4-{[(N,N-dimethylamino)methylidene]amino}-3-
(octa-1,7-diynyl)-1H-pyrazolo[3,4-d]pyrimidine (10). Com-
pound 9 (0.59 g, 1.44 mmol) was dried by repeated coevaporation
with dry pyridine (3 × 5 mL). The residue was dissolved in
dry pyridine and stirred with 4,4′-dimethoxytrityl chloride (0.58
g, 1.72 mmol) at rt for 6 h. After completion of the reaction
(monitored by TLC), methanol (6 mL) was added to the reaction
mixture, and stirring was continued for another half an hour.
The reaction mixture was evaporated to dryness under reduced
pressure and the remaining residue was dissolved in dichlo-
romethane (50 mL) and washed with 5% aq NaHCO3 solution
(2 × 250 mL) and water (80 mL). The organic layer was dried
over Na2SO4, the solvent was evaporated under reduced
pressure, and the residue was subjected to FC (silica gel, column
10 × 4 cm, eluted with CH2Cl2/acetone 95:5 f 90:10).
Evaporation of the main zone afforded 10 (0.72 g, 70%) as a
colorless foam. TLC (silica gel, CH2Cl2/MeOH 9:1). Rf 0.7. UV
4-Amino-1-[2-deoxy-ꢀ-D-erythropentofuranosyl]-3-[(7-hy-
droxycoumarin-1′,2′,3′-triazol-4′-yl)hexylidyne]-1H-pyrazo-
lo[3,4-d]pyrimidine (13). As described above for 12. A solution
of 1 (0.100 g, 0.28 mmol) and 3-azido-7-hydroxy-coumarin 3
(0.068 g, 0.43 mmol) in THF/H2O/t-BuOH (3:1:1, 4 mL),
sodium ascorbate (280 µL, 0.28 mmol), copper(II) sulfate
pentahydrate 7.5% in water (242 µL, 0.07 mmol). FC (silica
gel, column 10 × 3 cm, CH2Cl2/MeOH 88:12) afforded 13
(0.135 g, 86%) as a yellowish solid. TLC (silica gel, CH2Cl2/
MeOH 8:2). Rf 0.6. UV λmax (MeOH)/nm 287 (ε/dm3 mol-1
cm-1 15 200), 346 (19 500). 1H NMR [DMSO (d6), 300 MHz]:
δ 1.65-1.80 (m, 4H, 2 × CH2), 2.17-2.25 (m, 1H, C2′-HR),
2.58-2.63 (m, 2H, CH2), 2.73-2.79 (m, 3H, C2′-Hꢀ, CH2),
3.47-3.52 (m, 2H, C5′-H), 3.76-3.79 (m, 1H, C4′-H), 4.39
(br s, 1H, C3′-H), 4.78 (br s, 1H, C5′-OH), 5.27 (br s, 1H,
C3′-OH), 6.48-6.53 (t, J ) 6.3 Hz, 1H, C1′-H), 6.83 (s, 1H,
C7′′-H), 6.87-6.91 (d, J ) 3.0 Hz, 1H, C9′′-H), 7.70-7.73 (d,
J ) 8.7 Hz, 1H, triazole-H), 8.21 (s, 1H, C6′′-H), 8.33 (s, 1H,
C2-H), 8.55 (s, 1H, C4′′-H). ESI-TOF: [M + Na]+ calcd
581.55. Found 581.18.
λ
max (MeOH)/nm 225 (ε/dm3 mol-1 cm-1 41 800), 320 (24 900).
Anal. (C42H44N6O5) C, H, N. 1H NMR [DMSO (d6), 300 MHz]:
δ 1.58-1.69 (m, 4H, 2 × CH2), 2.17-2.22 (m, 2H, CH2),
2.28-2.32 (m, 1H, CH), 2.49-2.54 (m, 2H, CH2), 2.77-2.88
(m, 2H, CtCH, C2′-Hꢀ), 3.00-3.09 (m, 2H, C5′-H), 3.19-3.22
(s, 6H, 2 × CH3), 3.69 (s, 6H, 2 × OCH3), 3.90-3.93 (m, 1H,
C4′-H), 4.51-4.52 (m, 1H, C3′-H), 5.30-5.32 (d, J ) 4.8 Hz,
1H, C3′-OH), 6.58-6.62 (m, 1H, C1′-H), 6.70-6.77 (m, 4H,
Ar-H), 7.14-7.21 (m, 7H, Ar-H), 7.29-7.31 (m, 2H, Ar-H),
8.46 (s, 1H, C2-H), 8.90 (s, 1H, NdCH).
4-Amino-1-[2-deoxy-ꢀ-D-erythropentofuranosyl]-3-{[(1,2,3,4-
tetrahydro-5-methyl-2,4-dioxopyrimidin-1-yl)furan-3-yl]-
(1,2,3-triazol-4′-yl)hexylidyne}-1H-pyrazolo[3,4-d]pyrimi-
dine (14). As described above for 12. A solution of 1 (0.05 g,
0.14 mmol) and AZT 5 (0.045 g, 0.16 mmol) in THF/H2O/t-
BuOH, (3:1:1, 3 mL), sodium ascorbate (150 µL, 0.15 mmol),
copper(II) sulfate pentahydrate 7.5% in water (130 µL, 0.03
mmol). FC (silica gel, column 10 × 3 cm, CH2Cl2/MeOH 80:
20) afforded 14 (0.071 g, 81%) as a colorless solid. TLC (silica
gel, CH2Cl2/MeOH 7:3). Rf 0.2. UV λmax (MeOH)/nm 269 (ε/
dm3 mol-1 cm-1 17 900). Anal. (C28H34N10O7) C, H, N. 1H NMR
[DMSO (d6), 300 MHz]: δ 1.66-1.76 (m, 4H, CH2), 1.80 (s,
3H, CH3), 2.08 (s, 3H, CH3), 2.13-2.44 (m, 1H, C2′-HR),
2.57-2.62 (m, 3H, CH2, C2′-Hꢀ), 2.66-2.80 (m, 4H, CH2),
3.47-3.52 (m, 2H, C5′-H), 3.57-3.67 (m, 2H, C5′-H), 3.70-3.82
(m, 2H, C4′-H), 4.18-4.19 (m, 1H, C3′-H), 4.39-4.42 (m, 1H,
C3′-H), 4.72-4.77 (m, 1H, 3′-OH), 5.27-5.31 (m, 2H,
5′-OH), 6.38-6.42 (t, J ) 6.6 Hz, 1H, C1′-H), 6.49-6.54 (t,
J ) 6.3 Hz, 1H, C1′-H), 7.81 (s, 1H, triazole-H), 8.06 (s, 1H,
C2-H), 8.22 (s, 1H, C2-H), 11.36 (s, 1H, NH). ESI-TOF: [M
+ Na]+ calcd 645.63. Found 645.25.
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-ꢀ-D-erythropento-
furanosyl]-4-{[(N,N-dimethylamino)methylidene]amino}-3-
(octa-1,7-diynyl)-1H-pyrazolo[3,4-d]pyrimidin-3′-(2-cyano-
ethyl)-N,N-diisopropylphosphoramidite (11). A solution of
10 (0.12 g, 0.16 mmol) in dry CH2Cl2 (10 mL) was stirred with
(i-Pr)2NEt (57 µL, 0.33 mmol) at rt. Then, 2-cyanoethyl
diisopropylphosphoramidochloridite (75 µL, 0.33 mmol) was
added, and the reaction mixture was stirred for 30 min. After
completion of the reaction (monitored by TLC), the reaction
mixture was diluted with CH2Cl2 (30 mL) and was poured into
5% NaHCO3 solution (30 mL) and extracted with CH2Cl2 (3 ×
30 mL). The combined organic phases were dried over Na2SO4,
and the solvent was evaporated. The residual foam was applied
to FC (silica gel, column 8 × 3 cm, eluted with CH2Cl2/acetone
100:0 f 95:5). Evaporation of the main zone afforded 11 (0.11
g, 74%) as a colorless foam. TLC (silica gel, CH2Cl2/acetone
9:1). Rf 0.7. 31P NMR (CDCl3, 121 MHz): 148.26, 148.39.
4-Amino-1-[2-deoxy-ꢀ-D-erythropentofuranosyl]-3-[(ben-
zyl-1′,2′,3′-triazol-4′-yl)hexylidyne]-1H-pyrazolo[3,4-d]pyri-
midine (15). As described above for 12. A solution of 1 (0.100
g, 0.28 mmol) and benzyl azide 4 (0.045 g, 0.33 mmol) in THF/
H2O/t-BuOH, (3:1:1, 5 mL), sodium ascorbate (280 µL, 0.28
mmol), copper(II) sulfate pentahydrate 7.5% in water (242 µL,
0.07 mmol). FC (silica gel, column 10 × 3 cm, CH2Cl2/MeOH
90:10) afforded 15 (0.124 g, 90%) as a colorless solid. TLC
(silica gel, CH2Cl2/MeOH 8:2). Rf 0.5. UV λmax (MeOH)/nm
249 (ε/dm3 mol-1 cm-1 10 500), 287 (10 100). Anal.
4-Amino-1-[2-deoxy-ꢀ-D-erythropentofuranosyl]-3-[(meth-
ylanthracene-1′,2′,3′-triazol-4′-yl)hexylidyne]-1H-pyrazolo[3,4-
d]pyrimidine (12). To a solution of 1 (0.15 g, 0.42 mmol) and
anthracene azide 2 (0.12 g, 0.50 mmol) in THF/H2O/t-BuOH,
(3:1:1; 5 mL) was added sodium ascorbate (419 µL, 0.41 mmol)
of a freshly prepared 1 M solution in water, followed by the
addition of copper(II) sulfate pentahydrate 7.5% in water (362
µL, 0.10 mmol). The emulsion was stirred for 12 h at rt, and
the solution was evaporated and applied to FC (silica gel,
1
(C25H28N8O3) C, H, N. H NMR [DMSO (d6), 300 MHz]: δ
1.60-1.73 (m, 4H, 2 × CH2), 2.22-2.25 (m, 1H, C2′-HR),