derivative would be able to overcome these sensitivity issues.
In fact, the most sensitive method reported to date (0.24
abasic sites detected per 106 nucleotides), which has been
demonstrated to detect spontaneous abasic site formation
under physiological conditions, utilizes a chemiluminescent
substrate for the indirect detection of biotin-tagged abasic
sites coupled to streptaviden-conjugated horseradish peroxi-
dase in an ELISA-type slot-blot format.13 Chemiluminescent
detection of abasic sites in a direct manner would be less
cumbersome and would provide a sensitivity superior to that
obtained in an indirect format. We describe here the synthesis
and preliminary evaluation of the first chemiluminescent
acridinium hydroxylamine (AHA) for the direct detection
of abasic sites in damaged DNA at levels previously
unobtainable with nonradioactive labels.
boxamide using N-succinimidyl trifluoroacetate.17,18 Subse-
quent coupling of protected hydroxylamine 2 with acri-
dinium-NHS ester 3 and acidic hydrolysis provided 4 in
94% yield.16
The utility of AHA for the direct detection of abasic sites
in damaged DNA was demonstrated by labeling calf thymus
DNA containing varying concentrations of abasic sites
generated in a time-dependent manner. Thus, calf thymus
DNA (50 µg/mL) in 10 mM sodium citrate, 100 mM NaCl,
pH 5 was heated at 70 °C for 0-60 min.8 Aliquots were
withdrawn at 15 min intervals, and the DNA in each sample
was immediately ethanol-precipitated. The abasic site con-
taining DNA samples were redissolved at 25 µg/mL in 20
mM sodium phosphate, 317 µM AHA (∼0.1 mg AHA/
assay), pH 6.8 and heated at 37 °C for 2 h. The resulting
labeled DNA samples were again isolated by ethanol
precipitation and dialyzed against 20 mM sodium phosphate
buffer (pH 6.8). Total chemiluminescent emission from 10
ng of each labeled sample was subsequently determined in
a direct format using an EG&G Berthold MicroLumat Plus
luminometer. Chemiluminescence due to background was
eliminated by subtraction of total emission intensity obtained
from a DNA sample treated with AHA that had not been
subjected to the depurination/depyrimidination conditions.
The plot of total chemiluminescent emission intensity versus
time of the depurination/depyrimidination reaction is linear
with a correlation coefficient of 0.999 (Figure 1). The right-
The synthesis of AHA (4) is depicted in Scheme 1. Briefly,
O-alkylation of commercially available ethyl N-hydroxy-
acetimidate with N-(4-bromobutyl)phthalimide (1) followed
by hydrazinolysis of the phthalimide group provided the
Scheme 1
protected hydroxylamine 2 in 20% overall yield for the two
steps.14-16 N-Hydroxysuccinimidyl acridinium active ester
3 was prepared in 95% yield by activation of 10-(3-
sulfopropyl)-N-tosyl-N-(3-carboxypropyl)acridinium-9-car-
Figure 1. Chemiluminescent intensity of abasic site-labeled DNA
versus time of depurination/depyrimidination reaction.
(13) Nakamura, J.; Walker, V. E.; Upton, P. B.; Chiang, S.-Y.; Kow, Y.
W.; Swenberg, J. A. Cancer Res. 1998, 58, 222-225.
(14) Berninger, R. W.; Lodge, M. S.; Tarnowski, S. J., Jr.; Colvin, F.
W.; Brinkley, J. M. PCT Int. Appl. WO9640662, 1996; Chem. Abstr. 1996,
126, 129004.
hand axis corresponds to literature values for the ratio of
expected abasic sites per 100 000 nucleotides under the
depurination/depyrimidination conditions utilized.8 The ab-
solute sensitivity of a direct chemiluminescent detection
format is dependent upon both the quantity of DNA available
for analysis and the number of abasic sites present in a given
sample. Serial dilution of as little as 200 ng of a DNA sample
containing 160 chemiluminescent abasic site labels per 106
nucleotides with unmodified calf thymus DNA provides a
preliminary sensitivity determination of ∼0.1 abasic sites
detected per 106 nucleotides.
(15) Nedospasov, A. A.; Khomutov, R. M. IzV. Akad. Nauk SSSR, Ser.
Khim. 1978, 2397-2400.
(16) Analytical data are as follows. Compound 2: 1H NMR (CDCl3) δ
3.96 (2H, q, J ) 7.1 Hz), 3.89 (2H, t, J ) 4.0 Hz), 2.70 (2H, t, J ) 5.0
Hz), 1.91 (3H, s), 1.65 (2H, m), 1.53 (2H, m), 1.26 (3H, t, J ) 7.7 Hz);
ESI/MS m/z 175 (M + H)+. Compound 3: 1H NMR (DMSO-d6) δ 9.00
(2H, m), 8.44 (2H, m), 8.22-7.63 (6H, m), 7.17 (2H, m), 5.63 (2H, m),
4.30 (1.3H, m), 3.60-1.60 (15.7H, m); ESI/MS m/z 683 (M + H)+; anal.
HPLC (Waters µBondapak C18; 30/70 AcCN/0.1% aqueous formic acid)
retention time 4.34 min, 97%. Compound 4: 1H NMR (CD3OD) δ 8.97-
8.90 (2H, m), 8.48-8.40 (2H, m), 8.19 (0.5H, d, J ) 8.4 Hz), 8.00-7.93
(2H, m), 7.88-7.76 (2H, m), 7.63 (0.5H, d, J ) 8.1 Hz), 7.14 (3H, s), 5.74
(2H, m), 4.25 (1.5H, m), 4.06 (1.5H, t, J ) 5.9 Hz), 3.94 (0.5H, t, J ) 6.7
Hz), 3.48-1.66 (4H, m), 1.54-1.42 (3.5H, m), 1.22 (0.5H, m); ESI/MS
m/z 671 (M + H)+, 693 (M + Na)+; anal. HPLC (Waters µBondapak C18;
30/70 AcCN/0.05% aqueous TFA) retention time 3.79 min, 98%.
(17) Adamczyk, M.; Chen, Y.-Y.; Mattingly, P. G.; Pan, Y.; Rege, S. J.
Org. Chem. 1998, 63, 5636-5639.
(18) Sakaibara, S.; Inukai, N. Bull. Chem. Soc. Jpn. 1965, 38, 1979-
1984.
780
Org. Lett., Vol. 1, No. 5, 1999
Adamczyk, Maciej
