Synthesis and Properties of Oligonucleotide–Cyanine Conjugates
FULL PAPER
1
1
7
33.20, 128.29, 127.48, 125.91, 124.83, 124.76, 124.57, 122.88, 117.17,
144.98, 139.84, 139.26, 138.42, 133.50, 132.98, 129.89, 127.04, 125.63,
125.54, 125.48, 125.44, 122.23, 117.60, 115.91, 110.39, 92.72, 49.80, 43.08,
12.20, 109.29, 88.06, 47.36, 43.10, 33.44, 30.43, 29.35, 28.41, 27.56, 27.07,
+
.46 ppm; ESI-MS: m/z calcd for C26
H
30IN
2
S: 529.5, found: 529.1 [M+H
33.68, 30.68, 29.52, 28.80, 27.48, 27.22, 7.61 ppm; ESI-MS: m/z calcd for
À1
À1
+
]
; UV/Vis (MeOH): lmax (e)=503nm (58900 mol cm ).
C
28
H
32IN
2
: 523.5, found: 523.1 [M+H ]; UV/Vis (MeOH): lmax(e)=
À1
À1
5
57 nm (97000 mol cm ).
Synthesis of N-(8-iodooctyl)quinaldinium iodide 7b: A solution of diio-
dooctane (2.33 mL, 11.7 mmol) in dry dioxane (6 mL) was heated to
Synthesis of 1-(8-iodooctyl)-4-[(2,3-dihydro-3-methylbenzothiazol-2-ylide-
8
08C, and quinaldine (315 mL, 2.33 mmol) was added slowly over 45 min
ne)methyl]quinolinium iodide (15): The synthesis was performed as pre-
[
70] 13
producing a brown solution. After 5 h, the solution was concentrated,
and pentane (20 mL) added to give a black solid, which was collected by
filtration, washed with pentane several times, and purified on a silica gel
viously reported for 8a and 7c.
3
C NMR (500 MHz,CDCl ): d=
160.02, 149.07, 144.83, 140.28, 137.05, 132.99, 128.27, 127.27, 126.95,
124.80, 124.69, 124.54, 122.64, 116.78, 112.14, 109.25, 88.70, 55.10, 35.61,
column with a MeOH gradient (0 to 4%) in CH
2
Cl
, TMS): d=9.01 (d, J=
.5 Hz, 1H; HAr), 8.39 (d, J=9.0 Hz, 1H; HAr), 8.31 (d, J=8 Hz, 1H;
2
to give a brown
33.51, 30.43, 29.50, 29.11, 28.42, 26.62, 7.54; ESI-MS: m/z calcd for
1
+
solid (120 mg, 10%). H NMR (500 MHz, CDCl
3
C
26
H
30IN
2
S: 529.5, found: 529 [M+H ]; UV/Vis (MeOH):
l
max(e)=
À1
À1
8
503nm (86400 mol cm ).
H
Ar), 8.19 (t, J=8 Hz, 1H; HAr), 8.13(d, J=8.25 Hz, 1H; HAr), 7.90 (d,
Synthesis of 1-(8-iodooctyl)-4-[(1,2-dihydro-1-methylquinolin-2-ylidene)-
methyl]quinolinium iodide (16): A mixture of 8b (71 mg, 180 mmol), 7c
(90 mg, 172 mmol CH Cl (1 mL), MeOH (1 mL), and TEA (140 mL,
+
J=7.5 Hz, 1H; HAr), 5.08 (t, J=8.4 Hz, 2H; CH
CH ), 3.18 (t, J=7.0 Hz, 2H; CH I), 2.0 (q, J=8.0 Hz, 2H; CH
J=7.0 Hz, 2H; CH I), 1.65 (q, 2H; J=7.5 Hz, CH ), 1.47–1.33 ppm (m,
H; 3CH ); ESI-MS: m/z calcd for C18 25IN: 382.3, found: 382.1 [M+H
2
N), 3.34 (s, 3H; N
3
2
2
), 1.80 (q,
2
2
2
2
1 mol) was agitated to produce a dark purple solution. After 24 h, the
mixture was concentrated and purified on a silica column with a MeOH
gradient (0 to 2%) in CH Cl to give a purple paste (43mg, 38%).
+
6
2
H
]
.
2
2
1
Synthesis of 1-(8-iodooctyl)-2-[(2,3-dihydro-3-methylbenzothiazol-2-ylide-
3
H NMR (500 MHz, CDCl , TMS): d=8.65 (d, J=7.5 Hz, 1H; HAr), 8.34
ne)methyl]quinolinium iodide (12): A mixture of 8a (29 mg, 90 mmol),
(d, J=8.5 Hz, 1H; HAr), 8.02 (d, J=10.0 Hz, 1H; HAr), 7.87(d, J=9.0 Hz,
1H; HAr), 7.79 (t, J=7.7 Hz, 1H; HAr), 7.67 (m, 4H; HAr), 7.59 (m, 2H;
7
2 2
b (40 mg, 79 mmol), CH Cl (1 mL), MeOH (1 mL), and TEA (24 mL,
H
Ar), 7.40 (t, J=7.5 Hz, 1H; HAr), 6.24 (s, 1H; =CH
À), 4.51(t, J=7.5 Hz,
1
70 mmol) was agitated to produce a dark orange solution. After 24 h,
+
the mixture was concentrated and purified on a silica column with a
MeOH gradient (0 to 3%) in CH Cl and then on preparative TLC
plates (CH Cl /acetone/MeOH 75/22/3) to give an orange powder
18.5 mg, 36%). H NMR (500 MHz, CDCl
2H; CH
2
N), 4.02 (s, 3H; N CH
3
), 3.17 (t, J=7.0 Hz, 2H; CH
2
I), 1.94 (q,
CH I), 1.20–
): d=154.55,
150.32, 143.77, 140.53, 137.87, 137.77, 133.06, 132.67, 129.21, 126.71,
J=7.5 Hz, 2H; NCH
2
CH
2
), 1.79 (q, J=7.0 Hz, 2H; CH
2
2
2
2
1
3
1
.60 ppm (m, 8H; 4CH ); C NMR (500 MHz, CDCl
2
3
2
2
1
(
9
3
, TMS): d=8.11 (d, J=
.5 Hz, 1H; HAr), 8.00 (d, J=9.0 Hz, 1H; HAr), 7.78 (m, 3H; HAr), 7.69
1
3
C
26.66, 125.32, 125.25, 125.00, 122.12, 116.89, 116.34, 110.14, 94.77, 54.82,
9.14, 33.52, 30.45, 29.46, 29.15, 28.43, 26.68, 7.60 ppm; ESI-MS calcd for
(
1
d, J=8.0 Hz, 1H; HAr), 7. 52 (t, J=8 Hz, 1H; HAr), 7.50 (t, J=7.5 Hz,
H; HAr), 7.45 (d, J=8.5 Hz, 1H; HAr), 7.34 (t, J=8.0 Hz, 1H; HAr), 6.34
+
28
H
32IN
2
: 523.5, found: 523.1 [M+H ]; UV/Vis (MeOH): lmax(e)=
À1
À1
+
556 nm (98000 mol cm ).
(
(
s, 1H; =CH), 4.18 (s, 3H; N CH
t, J=7.0 Hz, 2H; CH I), 1.98 (m, 2H; CH
), 1.45–1.65 ppm (m, 8H; 4CH
3
), 3.77 (t, J=7.5 Hz, 2H; CH
), 1.80 (q, J=7.0 Hz, 2H;
); C NMR (500 MHz,CDCl ): d=
2
N), 3.18
2
2
Synthesis of 1-(8-iodooctyl)-4-[(1,4-dihydro-1-methylquinolin-4-ylidene)-
methyl]quinolinium iodide (17): A mixture of 7c (100 mg, 196 mmol), 8c
1
3
CH
2
2
3
1
1
3
62.37, 152.87, 140.86, 140.00, 139.45, 133.76, 129.94, 128.47, 125.96,
25.24, 124.69, 123.88, 122.58, 119.15, 116.57, 112.86, 86.90, 50.16, 36.58,
(67 mg, 200 mmol), CH
1.1 mmol) was agitated to produce a dark blue solution. After 24 h, the
mixture was concentrated and purified on a silica column (CH Cl
MeOH 98/2) to give an intensely blue paste (35 mg, 39%). H NMR
(500 MHz, CDCl , TMS): d=7.51~8.34 (m, 12H; HAr), 7.03(s, 1H; CH),
.36 (t, J=7,5 Hz, 2H; CH N), 4.07 (s, 3H; NCH ), 3.16 (t, J=7.0 Hz,
H; CH I), 1.92 (q, J=7.5 Hz, 2H; NCH CH ), 1.78 (q, J=7.0 Hz, 2H;
CH I), 1.20–1.40 ppm (m, 8H 4CH ); C NMR (500 MHz,CDCl
d=149.43, 149.35, 143.31, 142.62, 138.69, 137.76, 132.68, 132.58, 126.30,
2 2
Cl (1 mL), MeOH (1 mL), and TEA (25.3 mL,
3.56, 30.49, 29.51, 28.49, 27.51, 27.00, 7.77 ppm; ESI-MS calcd for
2
2
/
+
1
C
26
H
30IN
2
S: 529.5, found: 529.1 [M+H ]; UV/Vis (MeOH): lmax(e)=
À1
À1
4
84 nm (69400 mol cm ).
3
4
2
CH
2
3
Synthesis of 1-(8-iodooctyl)-2-[(1,2-dihydro-1-methylquinolin-2-ylidene)-
methyl]quinolinium iodide (13): A mixture of 8b (71 mg, 180 mmol), 7b
2
2
2
1
3
(
1
91 mg, 147 mmol), CH
mmol) was agitated to produce a dark red solution. After 24 h, the mix-
ture was concentrated and purified on a silica column with a MeOH gra-
dient (0 to 2%) in CH Cl to give an oil, which was further purified on a
preparative TLC plate with CH Cl /MeOH (99/1, 3), CH Cl /MeOH
/MeOH (96/42) to give a purple-red paste
18 mg, 19%). H NMR (500 MHz, CDCl , TMS): d=7.40~7.95 (m,
2H; HAr), 5.85 (s, 1H; CH), 4.58 (m, 2H; CH N), 4.21 (s, 3H; NCH ),
.18 (t, J=7.0 Hz, 2H; CH I), 1.96 (q, J=7.5 Hz, 2H; NCH CH ), 1.81
CH I), 1.20–1.40 ppm (m, 8H; 4CH ); C NMR
): d=154.91, 153.11, 140.26, 139.24, 138.70, 138.55,
32.94, 129.64, 129.20, 125.44, 125.28, 125.02, 124.88, 121.83, 121.64,
16.76, 115.93, 91.28, 49.79, 39.96, 33.37, 30.28, 29.19, 28.35, 27.22, 26.80,
2 2
Cl (1 mL), MeOH (1 mL), and TEA (140 mL,
2
2
2
3
)
1
4
26.14, 125.77, 125.48, 125.41, 116.83, 116.76, 109.87, 109.73, 97.22, 54.61,
2.58, 33.34, 30.26, 29.18, 28.96, 28.25, 26.53, 7.43 ppm. ESI-MS: m/z
2
2
+
calcd for C28
l
H
32IN
2
: 523.5, found: 523.2 [M+H ]; UV/Vis (MeOH):
2
2
2
2
À1
À1
max(e)=589 nm (70470mol cm ).
(
(
1
3
(
(
1
1
7
2 2
98/2, 2), and CH Cl
1
Synthesis of the ODN–cyanines conjugates (Scheme 2)
3
2
3
Synthesis of oligonucleotides 4 and 5: The ODNs were assembled by
classical phosphoramidite chemistry on a controlled pore glass (CPG)
support on a micromole scale. After drying of the supports bearing the
ODNs with a free 5’-hydroxyl group (1 mmol), O-(6-dimethoxytrityloxy-
2
2
2
1
3
q, J=7.0 Hz, 2H; CH
500 MHz, CDCl
2
2
2
3
3,4-dithiahexyl-H-phosphonate (0.025 g, 0.04 mmol) in pyridine/CH CN
3
(50/50, 0.4 mL, dried overnight on 3and 4 molecular sieves) and a so-
+
.63ppm; ESI-MS: m/z calcd for C28
H
32IN
2
: 523.5, found: 523.1 [M+H
lution of pivaloyl chloride (0.020 g, 0.166 mmol) in CH CN (0.5 mL, pre-
3
À1
À1
]
; UV/Vis (MeOH): lmax(e)=522 nm (69900 mol cm ).
pared 1 h before use and dried over 3 molecular sieves) were added si-
Synthesis of 1-(8-iodooctyl)-2-[(1,4-dihydro-1-methylquinolin-4-ylidene)-
methyl]quinolinium iodide (14): A mixture of 8c (29 mg, 85 mmol), 7b
multaneously to the ODN chain bound to the support. After 2.5 min, the
solution was removed, and the support washed with anhydrous pyridine/
(
1
40 mg, 79 mmol), CH
70 mmol) was agitated to produce a dark purple solution. After 24 h, the
crude mixture was concentrated and purified on a silica column using a
MeOH gradient (0 to 3%) in CH Cl to give a purple paste (21 mg,
, TMS): d=8.68 (d, J=7.5 Hz, 1H;
Ar), 8.15 (d, J=8.5 Hz, 1H; HAr), 8.06 (d, J=9.5 Hz, 1H; HAr), 7.87 (d,
J=9.0 Hz, 1H; HAr), 7.84 (t, J=7.5 Hz, 1H; HAr), 7.70 (m, 4H; HAr),
.61 (m, 1H; HAr), 7.53(d, J=9.0 Hz, 1H; HAr), 7.40 (t, J=7.5 Hz, 1H;
Ar), 6.14 (s, 1H; =CHÀ), 4.34 (t, J=7.5 Hz, CH
N), 4.20 (s, 3H; NCH ),
.53 (t, J=7,0 Hz, 2H; CH ), 3.18 (t, J=7,0 Hz, 2H; CH I), 1.40~
); C NMR (500 MHz,CDCl ): d=153.76, 151.04,
2
Cl
2
(1 mL), MeOH (1 mL), and TEA (23 mL,
CH
dine (2.5 mL, 3/2)) was added. After 20 min, the solution was removed,
and the support washed with pyridine/CH CN (50/50, 31 mL) and then
3 8 2
CN (50/50, 31 mL). Then a sulfur solution (50 mg S in a CS /pyri-
3
with acetonitrile (31 mL). ODNs 4 and 5 were deprotected and cleaved
from the support by treatment with concentrated aqueous ammonia for
2
2
1
4
H
5%). H NMR (500 MHz, CDCl
3
6
h at 508C. After removal of the ammonia solution, the crude ODNs 4
and 5 were analyzed by reversed-phase chromatography with a linear
gradient of CH CN (0 to 24% in 30 min). Detection l=260 nm. R (4)=
9 min, 26 s. R (5)=14 min, 16 s.
7
H
3
1
3
t
1
t
2
3
2
2
Synthesis of conjugates of 4 and 5: Before conjugate synthesis, MeOH
was passed over Chelex resin 100 to remove divalent ions. Methanolic
1
3
.60 ppm (m, 10H; CH
2
3
Chem. Eur. J. 2006, 12, 2270 – 2281
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2279