column chromatography eluting with CH2Cl2 : MeOH : Et3N
(94 : 4 : 2). The resulting solid was precipitated from hexane at
room temperature and dried over P2O5 in a desiccator to afford
an amorphous solid (1.00 g, 97%); mp: 130 ЊC; Rf 0.29 (CH2Cl2
eluting with a gradient of hexane to EtOAc. This afforded an
orange solid that was dried in vacuo over P2O5 (0.71 g, 76%). Rf
0.50 (EtOAc); 1H NMR (CDCl3): 9.99 (br s, 1H, CONH), 8.52
(s, 1H, CH2), 8.30 (s, 1H, CH6), 8.32–8.28 (m, 1H, CH Ar), 7.80
(s, 0.5 H, CHN, Z–E pair), 7.75 (d, J = 8.0 Hz, 2H, CH Ar), 7.74
(s, 0.5 H, CHN, Z–E pair), 7.50–7.30 (m, 3H, CH Ar), 7.35–
7.10 (m, 9H, CH Ar), 6.72–6.64 (m, 4H, CH Ar), 6.70–6.60 (m,
1H, H1Ј), 6.07 (d, J = 8.0 Hz, 2H, CH Ar), 5.60 (br s, 1H, OH),
4.60–4.40 (m, 3H, H3Ј, CH2NH), 4.03–4.00 (m, 1H, H4Ј), 3.62 (s,
6H, OCH3), 3.56–3.07 (m, 4H, CH2), 3.33–3.12 (m, 2H, H5Ј),
2.50 (s, 6H, N(CH3)2), 2.40–2.32 (m, 2H, H2Ј,2Љ), 1.49–1.28 (m,
4H, CH2), 1.17–1.00 (m, 4H, CH2), 0.76 (t, J = 9.0 Hz, 3H,
CH3), 0.57 (t, J = 9.0 Hz, 3H, CH3); MS ESϩ: 996.0 (M ϩ H)ϩ,
1018.0 (M ϩ Na)ϩ HRMS requires: (M ϩ H) 996.5131 found:
996.5131.
1
: MeOH, 95 : 5 ϩ 2% Et3N); H NMR (CDCl3): 8.15 (s, 1H,
CH2), 7.38 (s, 1H, CH6), 7.35–7.10 (m, 9H, CH Ar), 6.80–6.69
(m, 4H, CH Ar), 6.70–6.50 (m, 1H, H1Ј), 5.65 (br s, 2H, NH2),
4.60–4.48 (m, 1H, H3Ј), 3.98–3.10 (m, 1H, H4Ј), 3.27–3.23 (m,
2H, CH2NH), 3.70 (s, 6H, OCH3), 3.43–3.39 (m, 2H, H5Ј), 2.50–
2.30 (m, 2H, H2Ј,2Љ); MS ESϩ: 702.0 (M ϩ H)ϩ, 724.0 (M ϩ
Na)ϩ, 740.0 (M ϩ K)ϩ; HRMS M ϩ Hϩ requires: 702.2534
found: 702.2682.
4-Amino-5-(prop-2-ynylamino)-7-(5Ј-(4,4Ј-dimethoxytrityl)-
2Ј-deoxy-1Ј-ꢀ-D-ribofuranosyl)pyrrolo(2,3-d)pyrimidine
(11).
Compound 10 (1.21 g, 1.76 mmol) was dissolved in MeOH
(10 ml) to which a solution of NaOH (0.21 g, 5.28 mmol) in
H2O (1 ml) was added dropwise, with stirring. The reaction was
stirred overnight at room temperature, and then diluted with
CH2Cl2 (80 ml) and washed with saturated brine (100 ml). The
organic phase was dried (anhydrous Na2SO4), evaporated to
dryness, and purified by flash silica gel column chromatography
eluting with CH2Cl2 : MeOH : Et3N (91.8 : 8 : 0.2) to give
the product as a white foam which was dried in vacuo over
P2O5 (0.92 g, 87%). Rf 0.29 (CH2Cl2 : MeOH, 9 : 1 ϩ 4%
4-(N,N-di-n-butylformamidinyl)-5-(2-(4-N,NЈ-dimethylamino-
phenylazo)phenyl)amidoprop-2-ynyl)-7-(5Ј-(4,4Ј-dimethoxy-
trityl))-3Ј-cyanoethoxy-N,N-(diisopropylamino)phosphinyl-2Ј-
deoxy
-1Ј-ꢀ-D-ribofuranosyl)pyrrolo(2,3-d)pyrimidine
(14).
Compound 13 (425 mg, 0.44 mmol) was dissolved in anhydrous
THF (3.0 ml) and DIPEA (0.31 ml, 1.75 mmol) and 2-cyano-
ethyl-N,N-diisopropylchlorophosphoramidite (0.114 ml, 0.48
mmol) were added. The reaction was stirred at room temper-
ature for 1 hour, after which a further portion of 2-cyanoethyl-
N,N-diisopropylchlorophosphoramidite (0.02 ml, 0.09 mmol)
was added with further stirring (15 minutes). The mixture was
diluted with CH2Cl2 (60 ml), sat. KCl (60 ml) was added with
vigorous stirring and the organic layer was removed by cannula,
dried (anhydrous Na2SO4) and evaporated to dryness in vacuo.
The residue was dissolved in anhydrous CH2Cl2 (2 ml) and pre-
cipitated by dropwise addition to stirred hexane at Ϫ78 ЊC. The
precipitate was collected by filtration under argon, and co-
evaporated with anhydrous CH2Cl2 to give a red foam which
was dissolved in anhydrous MeCN (3 ml), filtered and then
dried in vacuo over P2O5 for 5 hours. This afforded the title
compound as a red foam (0.52 g, 99%). Rf 0.70, 0.60 (EtOAc :
hexane, 1 : 2); 1H NMR (CDCl3): 9.99 (br s, 1H, CONH), 8.52
(s, 1H, CH2), 8.30 (s, 1H, CH6), 8.32–8.28 (m, 1H, CH Ar), 7.80
(s, 0.5 H, CHN, Z–E pair), 7.75 (d, J = 8.0 Hz, 2H, CH Ar), 7.74
(s, 0.5 H, CHN, Z–E pair), 7.50–7.30 (m, 3H, CH Ar), 7.35–
7.10 (m, 9H, CH Ar), 6.72–6.65 (m, 4H, CH Ar), 6.70–6.60 (m,
1H, H1Ј), 6.07 (d, J = 8.0 Hz, 2H, CH Ar), 5.60 (br s, 1H, OH),
4.60–4.40 (m, 3H, H3Ј, CH2NH), 4.03–4.00 (m, 1H, H4Ј), 3.62 (s,
6H, OCH3), 3.56–3.07 (m, 4H, CH2), 3.45–3.30 (m, 2H, CH2),
3.33–3.12 (m, 2H, H5Ј), 2.50 (s, 6H, N(CH3)2), 2.40–2.32 (m,
2H, H2Ј,2Љ), 1.60–1.50 (m, 2H, CH2), 1.49–1.28 (m, 4H, CH2),
1.25–1.15 (m, 2H, CH2), 1.17–1.10 (m, 16H, CH3CH, CH2),
0.76 (t, J = 9.0 Hz, 3H, CH3), 0.57 (t, J = 9.0 Hz, 3H, CH3); MS
ESϩ: 1197.0 (M ϩ H)ϩ; The phosphoramidite (14) was used
to prepare the oligonucleotide TTTTT(14)TTTTT as a test
sequence. Coupling efficiencies >98.5% were observed; ESϪ:
calc. 3734.8 found: 3735.0.
1
NH4OH(conc.)); H NMR (CDCl3): 8.15 (s, 1H, CH2), 7.38 (s,
1H, CH6), 7.35–6.69 (m, 13H, CH Ar), 6.70–6.50 (m, 1H, H1Ј),
5.60 (br s, 2H, NH2), 4.52–4.42 (m, 1H, H3Ј), 4.05–3.98 (m, 1H,
H4Ј), 3.70 (s, 6H, OCH3), 3.56 (s, 2H, CH2NH), 3.28–3.18 (m,
2H, H5Ј), 2.52–2.35 (m, 2H, H2Ј,2Љ).
4-Amino-5-(2-(4-N,NЈ-dimethylaminophenylazo)phenyl)-
amidoprop-2-ynyl)-7-(5Ј-(4,4Ј-dimethoxytrityl))-2Ј-deoxy-1Ј-ꢀ-
D-ribofuranosyl)pyrrolo{2,3-d}pyrimidine (12). Compound 11
(0.96 g, 1.59 mmol) was placed in a flask with methyl red (0.51
g, 1.91 mmol), HOBT (0.32 g, 2.39 mmol) and DCC (0.49 g,
2.39 mmol). Anhydrous CH2Cl2 (12 ml) was added, followed by
anhydrous Et3N (0.44 ml, 3.18 mmol) and the reaction was
stirred overnight at room temperature. The reaction mixture
was then diluted with CH2Cl2 (80 ml), washed with saturated
brine (80 ml) and dried (anhydrous Na2SO4). Evaporation to
dryness in vacuo gave an orange foam that was purified by flash
silica gel column chromatography eluting with CH2Cl2 : MeOH
: NH3 (92 : 4 : 4). The material was further purified by silica gel
column chromatography, eluting with EtOAc. This afforded the
desired compound as orange solid that was dried over P2O5
in vacuo (1.02 g, 88%); mp 126–129; Rf 0.61 (CH2Cl2 : MeOH,
9 : 1 ϩ 4% NH4OH(conc.)); 1H NMR (CDCl3): 8.46–8.39 (m, 1H,
CH Ar), 8.15 (s, 1H, H2), 7.75 (d, J = 8.0 Hz, 2H, CH Ar), 7.50–
7.35 (m, 3H, CH Ar), 7.35 (s, 1H, CH6), 7.35–7.10 (m, 9H, CH
Ar), 6.80–6.69 (m, 4H, CH Ar), 6.79–6.59 (m, 1H, H1Ј), 6.30 (d,
J = 8.0 Hz, 2H, CH Ar), 5.65 (br s, 2H, NH2), 4.50–4.41 (m, 1H,
H3Ј), 4.46–4.40 (m, 2H, CH2NH), 4.02–3.95 (m, 1H, H4Ј), 3.66
(s, 6H, OCH3), 3.40–3.18 (m, 2H, H5Ј), 2.68 (s, 6H, Me2N),
2.50–2.33 (m, 2H, H2Ј, 2Љ); MS ESϩ: 857.0 (M ϩ H)ϩ, 879.0 (M
ϩ Na)ϩ, 895.0 (M ϩ K)ϩ; HRMS (M ϩ H)ϩrequires: 857.3770
found: 857.3762.
6-(Trifluoroacetamido)caproic acid (15). 6-Aminocaproic acid
(20 g, 0.15 mol) was suspended in anhydrous methanol (200 ml)
and Et3N (106 ml, 0.76 mol) was added. The mixture was
stirred with gentle heating to aid dissolution (nb. the acid did
not fully dissolve at this stage) and ethyl trifluoroacetate (32.5 g,
27.00 ml, 0.23 mol) was added dropwise to the stirring suspen-
sion. The reaction became clear after one hour and was stirred
under argon overnight at room temperature. Evaporation under
reduced pressure gave a yellow oil that was dissolved in EtOAc
(250 ml), washed with 1 M HCl (250 ml, ×3), sat. KCl (250 ml)
and then dried (anhydrous Na2SO4). Filtration and evaporation
of the solvent under reduced pressure gave a pale yellow solid
which was recrystallised from hot Et2O, washed with cold hex-
ane and dried in vacuo over P2O5 to give a white solid. (33 g,
95%); Rf = 0.37 (CH2Cl2 : MeOH, 9 : 1); 1H NMR (DMSO-d6):
9.40 (s, 1H, NH), 3.45 (br s, 1H, OH), 3.16 (q, J = 6.6 Hz, 2H,
4-(N,N-di-n-butylformamidinyl)-5-(2-(4-N,NЈ-dimethylamino-
phenylazo)phenyl)amidoprop-2-ynyl)-7-(5Ј-(4,4Ј-dimethoxy-
trityl))-2Ј-deoxy -1Ј-ꢀ-D-ribofuranosyl)-pyrrolo{2,3-d}pyrimidine
(13). Compound 12 (0.8 g, 0.93 mmol) was dissolved in
anhydrous DMF (60 ml), N,N-di-n-butylformamide di-
methylacetal (0.38 g, 0.44 ml 1.87 mmol) was added dropwise
and the reaction was stirred at room temperature for 48 hours.
The mixture was then diluted with Et2O (250 ml) and washed
with water (100 ml, ×3) and the organic layer dried (anhydrous
Na2SO4) and evaporated to dryness under reduced pressure.
The residue was purified by flash silica gel column chromato-
graphy using pre-equilibrated silica (Et3N : hexane, 99 : 1),
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 6 7 – 2 2 7 5
2273