5-[15-Trifluoroacetamido-3,7,12-tris-(N-trifluoroacetyl)-3,7,12-
triazapentadecyl]-5Ј-O-(4,4Ј-dimethoxytrityl)-uridine 16
quenched by addition of polymer-bound benzyl alcohol (0.27
g, 0.54 mmol), filtered and the filtrate then partitioned between
DCM (10 mL) and saturated aq. NaHCO3 solution (10 mL).
The aqueous layer was further extracted with DCM (2 × 10
mL) and the combined organic layers were dried (Na2SO4) and
evaporated to give a yellow oil. Purification by silica gel
chromatography [eluent (dried over Na2SO4 prior to use) 40%
EtOAc in DCM/1% TEA to EtOAc/1% TEA 1%] gave the
Compound 15 (0.67 g, 1.14 mmol) was treated with spermine
(1.15 g, 5.7 mmol) in dry pyridine and MeOH (3.5 mL each)
under argon at r.t. After stirring for 15 h, NaBH4 (91 mg, 2.4
mmol) was added and after 1 h the reaction was evaporated.
DMAP (6.9 mg, 0.06 mmol) in dry pyridine (14 mL) was then
added and the solution cooled in an ice bath. Trifluoroacetic
anhydride (3.7 mL, 26.2 mmol) was then added dropwise over
15 min and the reaction was allowed to warm to r.t. over 1 h.
Saturated aq. NaHCO3 solution (50 mL) was then carefully
added with cooling and the product extracted into DCM
(3 × 20 mL). The combined organic layers were then evaporated
to give a red/brown oil which was purified by silica gel chroma-
tography (eluent 2–50% MeOH in DCM containing 0.1% TEA)
to give the polyamine conjugate 16 as a pale brown foam (0.75
g, 57% yield). 1H NMR (250 MHz, CDCl3): δ 1.60–1.96 (9H, m,
CH2CH2N), 2.15–2.28 (1H, m, CH2CH2N), 3.06–3.62 (16H, m,
CH2CH2N and 5Ј-H), 3.77 (6H, s, ArOCH3), 4.20 (1H, bs,
4Ј-H), 4.35–4.38 (2H, m, 2Ј-H and 3Ј-H), 5.89–5.92 (1H, m,
1Ј-H), 6.80–6.84 (4H, m, ArH), 7.20–7.40 (10H, m, ArH and
H6), 7.55–7.84 (2H, m, NH); 13C NMR (250 MHz, CDCl3):
δ 23.4, 25.3, 26.0, 26.3, 29.3, 36.2, 36.6, 43.4, 44.1, 45.4, 45.6,
46.8, 54.8, 61.0, 70.0, 74.9, 83.7, 86.4, 89.9, 109.9, 110.5, 112.9,
114.2, 117.1, 120.2, 126.8, 127.6, 129.7, 134.8, 137.3, 143.9,
150.6, 156.4, 156.8, 157.1, 158.3, 163.4; 19F NMR (250 MHz,
CDCl3): (1H-decoupled) δ Ϫ76.5, Ϫ76.4, Ϫ76.4, Ϫ69.6, Ϫ69.5,
Ϫ69.5, Ϫ69.4, Ϫ69.4, Ϫ69.3, Ϫ69.3, Ϫ69.3; (1H-coupled)
δ Ϫ76.5, Ϫ76.4, Ϫ76.4, Ϫ69.6, Ϫ69.5, Ϫ69.5, Ϫ69.4, Ϫ69.4,
Ϫ69.4, Ϫ69.3, Ϫ69.3, Ϫ69.3, Ϫ69.3, Ϫ69.2; Electrospray-MS:
1181 (M ϩ Na)ϩ, Acc. Mass: 1181.3536, C50H54N6O12F12Na
requires 1181.3506 deviation 2.6 ppm.
1
phosphoramidite 18 as a white foam (0.3 g, 86% yield). H
NMR (250 MHz, CDCl3): δ 0.07 (6H, m, SiMe), 0.83–0.88 (9H,
m, t-Bu), 1.04–1.22 [12H, m, 2 × NCH(CH3)2], 1.55–2.20 (10H,
m, CH2CH2N), 2.23–2.39 [1H, m, NCH(CH3)2], 2.56–2.61
[1H, m, NCH(CH3)2], 3.01–3.89 (20H, m, OCH2CH2CN,
CH2CH2CN and 5Ј-H), 3.71 (6H, s, ArOCH3), 4.18–4.42 (3H,
m, 2Ј-H, 3Ј-H and 4Ј-H), 5.88–5.96 (1H, m, 1Ј-H), 6.73–6.78
(4H, m, ArH), 7.20–7.57 (11H, m, ArH, H6 and NH), 7.68–
7.70 (1H, m, NH); 13C NMR (250 MHz, CDCl3): δ Ϫ5.2, 17.6,
20.0, 21.1, 22.5, 23.4, 26.1, 29.3, 24.2, 25.3, 36.1, 36.6, 42.4,
42.8, 43.0, 43.3, 44.8, 45.5, 46.7, 54.8, 57.5, 62.3, 72.0, 75.5,
82.7, 86.4, 108.5, 109.6, 112.9, 114.5, 117.4, 120.0, 122.0, 124.5,
126.9, 127.6, 129.8, 134.7, 143.8, 149.6, 137.1, 156.6, 157.3,
158.4, 162.6; 31P NMR (250 MHz, CDCl3): δ 149.9, 150.3, 150.4
and 150.5; Electrospray-MS: 1473 (M + H)+, 1496 (M + Na)+;
1471 (M Ϫ H)Ϫ, Acc. Mass: 1473.5636, C65H86N8O13F12SiP
requires 1473.5630 deviation 0.4 ppm.
5-(15-Amino-3,7,12-triazapentadecyl)-uridine 19
Nucleoside 4 (0.1 g, 0.12 mmol) was treated with methanolic
ammonia (100 mL) at 55 ЊC for 20 h. The solvent was then
removed in vacuo and the residue was partitioned between
water (10 mL) and EtOAc (10 mL). The aqueous layer was
further extracted with EtOAc (2 × 10 mL) before it was evapor-
ated to dryness in vacuo to yield the deprotected polyamine
conjugate 19 as a brown oil (40 mg, 72 % yield). This was used
5-[15-Trifluoroacetamido-3,7,12-tris-(N-trifluoroacetyl)-3,7,12-
triazahexadecyl]-5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-O-tert-butyl-
dimethylsilyl-uridine 17
1
as an HPLC standard. H NMR (250 MHz, CD3OD): δ 1.80
(4H, m, CH2CH2N), 2.05–2.18 (4H, m, CH2CH2N), 2.69–2.75
(2H, t, J = 6.7, C5-CH2CH2N), 3.02–3.24 (14H, m, CH2N),
3.73–3.79 (1H, dd, J = 2.9, 12.4, 5Ј-H), 3.85–3.91 (1H, dd,
J = 2.6, 12.4, 5Ј-H), 4.00–4.02 (1H, m, 4Ј-H), 4.15–4.22 (2H, m,
2Ј-H and 3Ј-H), 5.89 (1H, d, J = 4.0, 1Ј-H), 8.04 (1H, s, H6);
Electrospray-MS: 473 (M ϩ H)ϩ, Acc. Mass: 473.3073,
C21H41N6O6 requires 473.3088 deviation 3.1 ppm.
Compound 16 (1.32 g, 1.14 mmol) and silver nitrate (0.58 g,
3.41 mmol) were dissolved in dry pyridine (6.5 mL) under argon
at r.t. with stirring. Dry THF (19.5 mL) and then tBDMSCl
(0.51 g, 3.41 mmol) were added and the reaction was stirred for
0.5 h. The reaction mixture was filtered and the filtrate was
dissolved in 5% aq. NaHCO3 solution (50 mL) and extracted
with DCM (3 × 20 mL). The organic layers were combined and
evaporated and the residue purified by silica gel chromato-
graphy (eluent 20% EtOAc in DCM/0.01% TEA to EtOAc/
0.01% TEA) to afford 17 as a cream-coloured foam (1 g, 69%
Oligoribonucleotide synthesis
RNA synthesis was carried out on an ABI-294 synthesiser on a
1 µmol scale using 2Ј-O-tert-butyldimethylsilyl-3Ј-O-(2-cyano-
ethyl diisopropylphosphoramidite) monomers and solid
supports with phenoxyacetyl protection for adenosine and
guanosine and acetyl protection for cytidine (Glen Research).
5Ј-FAM substrates were prepared using 5Ј-fluorescein reagent
(Glen Research) (Fig. 1b). The syntheses were carried out using
standard RNA synthesis procedures46 except for the use of 5-
benzylmercaptotetrazole (EMP Biotech) instead of tetrazole.48
Deprotection of the oligoribonucleotides was carried out by
treatment with NH3 (aq.):EtOH (3:1) (1 mL) at 55 ЊC for 6 h
and then TBAF (1 mL) for 20 h followed by desalting using
Sephadex NAP10 columns.
1
yield). H NMR (250 MHz, CDCl3): 0.09 (6H, s, SiCH3), 0.85
(9H, s, t-Bu), 1.54–1.90 (9H, m, CH2CH2N), 2.05–2.2 (1H, m,
CH2CH2N), 2.57–2.62 (1H, m, 3Ј-OH), 3.15–3.55 (16H, m,
CH2CH2N), 3.72 (6H, s, ArOCH3), 4.07–4.09 (1H, m, 4Ј-H),
4.15–4.31 (1H, m, 3Ј-H), 4.35–4.39 (1H, m, 2Ј-H), 5.84–5.92
(1H, m, 1Ј-H), 6.73–6.79 (4H, m, ArH), 7.08–7.43 (10H, m,
ArH and H6), 7.56–7.71 (1H, m, NH), 8.97–9.18 (1H, m, NH);
13C NMR (250 MHz, CDCl3): δ Ϫ5.2, 18.0, 23.8, 24.7, 25.7,
26.4, 26.7, 28.3, 36.5, 37.0, 43.6, 44.2, 45.0, 45.9, 47.1, 55.2,
62.7, 70.9, 75.9, 83.8, 86.9, 88.4, 110.4, 111.2, 114.9, 117.8,
120.8, 113.2, 127.2, 127.7, 128.1, 129.1, 130.1, 135.0, 137.4,
144.3, 150.1, 157.1, 157.3, 157.7, 158.7, 163.2; Electrospray-
MS: 1295 (M ϩ Na)ϩ, Acc. Mass: 1295.4377, C56H68N6O12F12-
NaSi requires 1295.4371 deviation 0.5 ppm.
The following oligonucleotides were prepared, where U*
represents a polyamine-conjugated uridine synthesised using
phosphoramidite 18: 5Ј-FAM substrate, 14-mer, M = 4882.08,
E260 nm = 146.6 µMϪ1 cm2 (5Ј-FAM-UCG CAG UCC UAU UU-
3Ј); ribozyme strand A, 32-mer, M = 10369.48, E260 = 401.2
nm
5-[15-Trifluoroacetamido-3,7,12-tris-(N-trifluoroacetyl)-3,7,12-
triazapentadecyl]-5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-O-tert-butyl-
dimethylsilyl-3Ј-O-(2-cyanoethyl diisopropylphosphoramidite) 18
µMϪ1 cm2 (5Ј-AAA UAG AGA AGC GAA CCA GAG AAA
CAC ACG CC-3Ј); ribozyme strand B, 21-mer, M = 6729.09,
E260
= 238.2 µMϪ1 cm2 (5Ј-GGC GUG GUA CAU UAC
nm
To compound 17 (0.3 g, 0.24 mmol) in dry DCM (5 mL) were
added dry DIPEA (0.25 mL, 1.42 mmol) and chloro(2-cyano-
ethyl)(diisopropylamino)phosphine (0.16 mL, 0.71 mmol)
under argon at r.t. After stirring for 15 h the reaction was
CUG GUA-3Ј); modified ribozyme strand B synthesised using
phosphoramidite 18 in place of a single uridine residue, M =
6957.44, E260
= 238.2 µMϪ1 cm2, U*34 where U34 is replaced
nm
by the conjugate U*, U*37 where U37 is replaced by the conju-
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 1 0 3 – 2 1 1 2
2110