Chou et al.
(54)
US 6,858,742 B2
(51)
(52)
(58)
546/303; 436/6
(75)
(56)
References Clted
U.S. PATENT DOCUMENTS
4,312,883 A *
(1)
.
(*)
Notice:
* Cited by examiner
(57)
ABSTRACT
(22) Filed?
(65)
2003.
US 6,858,742 B2
1
nitroXyl (HNO).
ACRIDINIUM-9-CARBOXAMIDE
DERIVATIVES AND PROCESS OF
PREPARING DNA LABELING COMPOUNDS
Maciej
AdamcZyk
et
al.,
Neopentyl
2003.
FIELD OF THE INVENTION
10
as chemiluminescent labels.
biotin.
Furocoumarins
40
40454049 (1983)).
25
dimethylangelicin.
dimethylangelicin)acridinium carboXamide.
50
(1977).
spacer.
US 6,858,742 B2
3
Especially, the hydrochloride of 4-(Aminomethyl)-4,5
dimethylangelicin is excellent reagent to bind the DNA
through the irradiation by UV 365 nm and showed good
20 g crude product
Binding Enhancers
Recovery yield
Extracted method
HCl gas method
30%
90%
180 mins
30 mins
CH3
N+
15
o
20
R
R2
labels. 9-(4-(Aminomethyl)-4,5-Dimethyl-angelicin)
of detectable groups are biotin, acridinium ester or 25 acridinium carboxamide is the preferred chemiluminescent
label.
acridinium-9-carboxamide.
ments.
30
of
mixture;
(b) dissolving the mixture in organic extracting solvent;
35
40
TABLE 1
Chemiluminescence in Water
Chemiluminescence in pH 8
Buffer at RT 5 mins (RLU/
pmol)
embodiment.
It is difficult to purify the hydrochloride of 4
pmol)
0.5-1 x 10
2x107
5x107
1.8 X 107
s X 106
1.6 X 107
5 X 105
2 X 107
s X 106
s X 106
3 X 105
4x106
8x104
Derivatives
pmol)
50
1-3 x 107
(RLU/pmol)
Chemiluminescence in pH 8
Buffer at RT" 0 min (RLU/
Acridinium ester
45
Acridinium-9-carbo
xamide Derivatives
Chemiluminescence in pH 8
Buffer at RT 10 mins (RLU/
Chemiluminescence in pH 8
55 Buffer at 60 C. 0 min
(RLU/pmol)
Chemiluminescence in pH 8
Buffer at 60 C. 5 mins
(RLU/pmol)
Chemiluminscence in pH 8
60 Buffer at 60 C. 10 mins
(RLU/pmol)
*RT is 25 C.
65
US 6,858,742 B2
5
EXAMPLES
Example 5
dimethylangelicin
invention.
dimethylangelicin
Example 1
10
Example 6
dimethylangelicin
20
(300 HZ, CD3OD): delta 7.58(d, 1H, J=8.8 HZ), 6.82(dd, 1H,
J=8.8&2.4 HZ), 6.69(1H, d, J=2.4 HZ), 6.08(s, 1H), 2.49(s,
3H).
Example 7
(Aminomethyl)-4.5-dimethylangelicin
35
to half, the HCl gas Was purged into the organic layer until
the precipitation Was emerged. The precipitation Was col
lected and Washed With chloroform (50 mL) to afford the
methylcoumarin
Example 8
50
Example 4
Example 3
2.48(s, 3H).
Example 2
methylcoumarin
60
ture for 24 hours. Filtered the solid and Washed With acetone
1H, J=8.8 HZ), 7.32(d, 1H, J=8.8 HZ), 6.72(s, 1H), 6.24(s,
(15 ml) to afford the desired compound (950 mg). The yield
is 75%, m.p.210 C., Hl-NMR (300 HZ, DMSO-d6): delta.
65
US 6,85 8,742 B2
8
7
1H), 4.28(m, 1H), 4.11(m, 1H), 3.01(m, 1H), 2.98(m, 1H),
2.80(m, 1H), 2.54(m, 1H), 2.1m, 2H, J=7.2 HZ), 2.020, 2H,
Example 9
angelicin
6-(biotinyl)amino)hexanoic acid (500 mg) and
N-hydroxysuccimide (175 mg) Were taken into the single
10
into the single neck reactor. After all solid Were dissolved in
25 mL dimethylsul?de, Stirred at room temperature for 36
1.491.20(m, 12H).
(m, 1H), 2.54(m, 1H), 2.48(s, 3H), 2.45(s, 3H), 2.05(m, 4H),
1.491.17(m, 12H).
Derivatives
Example 10
solution
55
9-(4 -(Aminomethyl)-4,5 -
HZ), 6.43(s, 1H), 5.02(d, 2H, J=7.2 HZ), 4.81(s, 3H), 2.65(s,
3H), 2.63(s, 3H).
rated all the solvent, the residue Washed With methanol (10
6.11(s, 1H), 5.02(d, 2H, J=7.5 HZ), 2.80(s, 3H), 2.47(s, 3H),
1.41(br, 1H).
60
product compound;
(b) dissolving the product compound in an organic
65
US 6,85 8,742 B2
9
10
re?ux.
3. The process according to claim 2 Wherein the solvent
and6. step
The process according to claim 1 Wherein the organic
is ethanol.
5 solvent in step (d) is chloroform.
4. The process according to claim 1 Wherein the organic
extracting solvent in step (b) is chloroform.