residue chromatographed on silica gel (eluent: CH3CN–H2O–sat.
KNO3, 40 : 1 : 1, v/v/v). Fractions containing the product were
combined and the solvent removed under vacuum. Excess KNO3
was filtered off from a CH3CN solution to obtain the red colored
final complex 7 (75 mg, 30%). 1H-NMR (CD3CN, 300 MHz) d 1.45
(s, 9H), 4.4 (s, 2H), 4.6 (s, 2H), 7.1 (ddd, J = 7.5, 6.6, 1.2 Hz, 2H),
7.43 (d, J = 5.2 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.94 (ddd, J =
8.7, 7.7, 1.5 Hz, 2H), 8.18 (d, J = 8.3 Hz, 2H), 8.67 (d, J = 7.8 Hz,
2H), 9.03 (s, 2H); 13C-NMR (CD3CN, 75 MHz) d 28.0 (CH3), 42.6
(CH2), 76.0 (CH2), 82.7 (quat), 122.2 (CH), 125.2 (CH), 128.1
(CH), 128.6 (CH), 129.3 (CH), 136.3 (quat), 138.7 (CH), 142.2
(quat), 148.7 (quat), 153.1 (CH), 156.1 (quat), 158.9 (quat), 170.1
(quat); m/z (ESI) 562.1 (M2+/2); UV (l nm, CH3CN) 490, 310,
285.
Oligonucleotide synthesis
The oligonucleotides 10a–d functionalized at the 3¢-end by an
aldehyde group were prepared following a previously reported
protocol by using a post-synthetic oxidation of a diol moiety.10
General procedure for the coupling reaction between
oligonucleotides and metal (Ru2+ and Fe2+) complexes
(A) Coupling of mono ODN to [Ru(ttpy)(ttpy¢¢)]2+ complex 3:
conjugates 11a–d. To a solution (300 ml) of aldehyde-containing
oligonucleotide 10a (1.0 mg, 0.19 mmol) in 0.4 M ammonium
acetate buffer (pH = 4.5), an aqueous solution of mono-oxyamino
containing Ru2+ metal complex 3 (0.4 mg, 0.47 mmol) was added
so that the final concentration of the metal complex remained
at ~0.02 M. The resultant reaction mixture was stirred at room
temperature. The reaction was followed by RP-HPLC, which
indicates almost 100% consumption of 10a into conjugate 11a
within a 2 h time period. The crude product was purified either
by reverse phase HPLC or by 30% denaturing gel electrophoresis
to afford the final conjugate 11a in ~40% (460 mg) isolated yield.
m/z (MALDI) calcd 6093.5, found 6085.3. Conjugates 11b, 11c
and 11d were obtained using a similar protocol.
Protected [Ru(ttpy¢¢)(ttpy)]2+ complex 8.
A mixture of
[Ru(ttpy)Cl3] (100 mg, 0.14 mmol), compound 5 (70 mg,
0.14 mmol) and AgNO3 (57 mg, 0.33 mmol) were added into
a degassed CHCl3–EtOH solution (1 : 2, v/v, 5 ml) and the
resultant solution was heated at reflux under nitrogen for 8 h. The
red solution was then filtered, evaporated under vacuum and the
residue chromatographed on a silica gel column (eluent CH3CN–
H2O–sat. KNO3, 40 : 0.5 : 0.5, v/v/v). Fractions containing the
product were combined and the solvent was removed. Excess
KNO3 was filtered off from a CH3CN solution to finally obtain 8
(B) Coupling of bis-ODNs to [Ru(ttpy¢¢)2]2+ complex 2: conjugates
12a–d. Aldehyde functionalized ODN 10b (1.0 mg, 0.19 mmol)
was dissolved in 0.4 M ammonium acetate buffer solution (300 ml,
pH = 4.5) and an aqueous solution of bis-oxyamino containing
metal complex 2 (85 mg, 0.09 mmol) was added dropwise in
3 steps. The progress of the reaction was followed by reverse
phase HPLC. After complete addition of the metal complex the
reaction mixture was left overnight. This process actually converts
all aldehyde functionalized ODN into the bis-ODN tethered
metal complex, 12b. The crude product was purified by reverse
phase HPLC to afford final bis-ODN–metal complex conjugate
12b in 35% isolated yield. m/z (MALDI) calcd 11566.3, found
11545.8. Conjugates 12a, 12c and 12d were obtainedusinga similar
protocol.
1
as a red colored solid in 40% yield (50 mg). H-NMR (CD3CN,
300 MHz) d 1.44 (s, 9H), 2.5 (s, 3H), 4.35 (s, 2H), 4.6 (s, 2H),
7.14–7.19 (m, 4H), 7.4–7.44 (m, 4H), 7.54 (d, J = 8.0 Hz,
2H), 7.65 (d, J = 8.2 Hz, 2H), 7.92 (ddd, J = 7.8, 1.4 Hz,
4H), 8.1 (d, J = 8.2 Hz, 2H), 8.17 (d, J = 8.3 Hz, 2H),
8.68 (d, J = 8.2 Hz, 4H), 9.0 (s, 4H); 13C-NMR (CD3CN,
75 MHz) d 21.1 (CH3), 28.1 (CH3), 42.8 (CH2), 76.2 (CH2), 82.9
(quat), 122.1 (CH), 122.3 (CH), 125.4 (CH), 128.0 (CH), 128.4
(CH), 128.7 (CH), 129.4 (CH), 131.1 (CH), 134.8 (quat), 136.6
(quat), 138.8 (CH), 141.9 (quat), 142.1 (quat), 148.9 (quat), 149.4
(quat), 153.1 (CH), 156.3 (quat), 156.4 (quat), 159.1 (quat), 170.7
(quat); m/z (ESI) 468.0 (M2+/2); UV (l nm, CH3CN) 490, 310,
284.
(C) Coupling of bis-ODNs to [Fe(ttpy¢¢)2]2+ complex 1: conjugates
13a–d. Conjugates 13a–d were obtained using a similar protocol
as for 12a–d. 13b m/z (MALDI) calcd 11521.2, found 11499.1.
Complex 1. Boc protected [Fe(ttpy¢¢)2]2+ complex 6 (10 mg,
9.3 mmol) was dissolved in CH3CN–1 N HCl (1 : 1, v/v, 2 mL) and
the reaction mixture was stirred for 2 h at room temperature. The
resultant solution was evaporated and dried in vacuum to obtain
the violet colored [Fe(ttpy¢¢)2]2+ complex 1 in 80% yield (7.0 mg).
1H-NMR (D2O, 300 MHz) d 4.4 (s, 2H), 4.6 (s, 2H), 7.1 (t, J =
6.8 Hz, 2H), 7.25 (d, J = 5.6 Hz, 2H), 7.76 (d, J = 7.9 Hz, 2H),
7.9 (t, J = 7.8 Hz, 2H), 8.3 (d, J = 7.8 Hz, 2H), 8.6 (d, J = 7.9 Hz,
2H), 9.24 (s, 2H); m/z (ESI) 439.0 (M2+/2); UV (l nm, CH3CN)
580, 291.
UV-melting experiment
The modified single stranded metal–ODN conjugate (1.3 mM) was
mixed with the appropriate concentrations of the complementary
ODN stretches in 10 mM tris-HCl buffer at pH = 7.4 by varying
the salt concentrations (e.g. 40, 100 and 500 mM NaCl). In
one case the hybridization was carried out by heating the ODN
mixtures at 60 ◦C for 3 min followed by incubation at 4 ◦C
for 24 h. Modified duplexes with ODNs containing the Fe(II)
complex were incubated at 17 ◦C for 24 h and heated at 50 ◦C
for 3 min followed by incubation at 4 ◦C for 12 h. The stability of
such modified hybrid duplexes was compared with the respective
unmodified duplexes by thermal denaturation experiments. The
melting experiments were acquired on a CARY 400 Scan UV-Vis
spectrophotometer equipped with a temperature programmable
cell-block, by following the changes in absorbance at 260 nm as a
function of temperature ranging from 10–80 ◦C. The samples were
heated at 1.0 ◦C per min and the absorbance values were recorded
Complex 2. The cleavage of the Boc protecting group on
complex 7 was carried out under the same conditions as for 6
to afford the complex 2 in 80% yield. ESI-MS: m/z (ESI) 461.9
(M2+/2); UV (l nm, CH3CN) 490, 314, 285.
Complex 3. The cleavage of the Boc protecting group on
complex 8 was carried out under the same conditions as for 6
to afford complex 3 in 80% yield. m/z (ESI) 417.0 (M2+/2); UV
(l nm, CH3CN) 490, 310, 284.
2736 | Org. Biomol. Chem., 2009, 7, 2729–2737
This journal is
The Royal Society of Chemistry 2009
©