Chemico-enzymatic synthesis of a new fluorescent-labeled DNA by PCR with a thymidine nucleotide analogue bearing an acridone derivative

Article abstract of DOI:10.1016/j.bmcl.2006.10.072

Triphosphate of a thymidine analogue bearing acridone was prepared and incorporated as a substrate for PCR using KOD Dash DNA polymerase forming a new fluorescent-labeled DNA that is useful as a DNA probe.

Full text of DOI:10.1016/j.bmcl.2006.10.072

Bioorganic & Medicinal Chemistry Letters 17 (2007) 776–779
Chemico-enzymatic synthesis of a new fluorescent-labeled
DNA by PCR with a thymidine nucleotide analogue bearing
an acridone derivative
Atsushi Shoji, Tomoya Hasegawa, Masayasu Kuwahara,
Hiroaki Ozaki and Hiroaki Sawai*
Department of Applied Chemistry, Gunma University, Kiryu, Gunma 376-8515, Japan
Received 11 July 2006; revised 23 October 2006; accepted 25 October 2006
Available online 28 October 2006
Abstract—Triphosphate of a thymidine analogue bearing acridone was prepared and incorporated as a substrate for PCR using
KOD Dash DNA polymerase forming a new fluorescent-labeled DNA that is useful as a DNA probe.
ꢀ 2006 Elsevier Ltd. All rights reserved.
DNAs tagged with a fluorescent label are important
tools for biological and biochemical studies and for
diagnostic use as a DNA probe. Various methods have
been reported for the synthesis of fluorescent-tagged
DNA.1–3 DNA with high density of labels could increase
detection sensitivity. Polymerase chain reaction is suit-
able for simultaneous labeling and amplification of the
DNA with a fluorophore, if a fluorescent-labeled nucle-
otide can be a substrate for DNA polymerase during
PCR. Taq and KOD Dash DNA polymerases can ac-
cept some 50-triphosphates of 20-deoxyuridine deriva-
tives bearing fluorescein or cyanine dyes via an amino
linker arm at the C5 position yielding the corresponding
fluorescent-labeled DNA during PCR, although co-exis-
tence of natural substrate TTP is required for the reac-
tion.3–7 Complete displacement of the natural substrate
TTP with the fluorescent-tagged 20-deoxyuridine deriva-
tives resulted in loss of the formation of the fluorescent-
tagged PCR product. Although fluorescein and cyanine
dyes are widely used as a fluorescent-labeling agent for
various biomolecules such as DNA or protein, these
molecules have disadvantages, such as sensitivity to
photobleaching and fluorescent quenching in acidic
and basic conditions. Moreover, they are large bulky
molecules with negative or positive charges, which re-
sults in loss of the substrate activity of the labeled nucle-
otide in PCR.
The acridone moiety is highly fluorescent and stable
against photo-degradation, oxidation, and heat.8–10
Further, acridone is a rather small molecule. Several
types of acridone derivatives have been prepared and
used as a fluorescent label for peptides,11 antibody,12,13
and amino acids.14 However, to our knowledge, no re-
port has appeared on the conjugate of DNA and an
acridone derivative as a fluorescent DNA probe. Previ-
ously, we reported that triphosphate of a thymidine ana-
logue bearing a methylene group at the C5 a-position
with an amino-linker arm could be accepted by KOD
Dash DNA polymerase as a substrate in PCR.15 The tri-
phosphate of the thymidine analogue with an amino-
linker was further reacted with an acridone derivative
yielding a new thymidine analogue tagged with acri-
done. The thymidine analogue was found to be a good
substrate for KOD Dash DNA polymerase without
co-existence of a natural substrate. Here, we report the
preparation of the acridone-tagged thymidine analogue
and its enzymatic incorporation into the DNA by
PCR using KOD Dash DNA polymerase, along with
the fluorescent properties of the resulting DNA.
We first undertook preparation of 2-acridone acetic acid
(3) to introduce the acridone moiety to the modified
thymidine or DNA bearing an amino-linker via an
amide-bond formation. Compound 3 was prepared as
a possible anti-inflammatory agent by Taraporewala
and Kaufman.16 We prepared 3 by modification of their
method as shown in Scheme 1. Ullmann–Goldberg
condensation of o-bromo benzoic acid with methyl
Keywords: Acridone; Fluorescent-labeled DNA; PCR; DNA probe.
*
Corresponding author. E-mail: sawai@chem.gunma-u.ac.jp
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.072

A. Shoji et al. / Bioorg. Med. Chem. Lett. 17 (2007) 776–779
777
O
O
O
OMe
O
OH
OH
Cu,K2CO3
dry-DMF
OMe
H2N
+
N
H
Br
1
O
O
OH
OMe
NaOH aq
MeOH
O
O
PPA
N
H
N
H
2
3
O
N
H
N
H2N
NH
O
O
O
O
O
O
O
HO P O P O P O
OH OH OH
O
O
H
N
N
H
NH
O
OH
O
N
H
N
4
O
O
O
HO P O P O P O
OH OH OH
O
HBTU,DIPEA
dry-DMF
OH
5
Scheme 1. Synthetic procedure of acridone acetic acid 3 and acridone-tagged thymidine analogue 5.
p-aminophenyl acetate in dry DMF in the presence of
potassium carbonate and copper powder catalyst17 gave
N-phenylanthranilic acid derivative (1) in 65% yield.
Cyclodehydration of 1 was carried out with polyphos-
phoric acid at 85 ꢁC for 1 h. Usual workup of the reac-
tion mixture and chromatography on silica gel using 5%
methanol/dichloromethane as an eluent gave 2 as a
white solid in 61% yield. The methyl ester of 2 was
hydrolyzed with sodium hydroxide in aqueous methanol
to yield 3 in 66% yield. Its structure was confirmed by
NMR and ESI-mass spectra and spectroscopic proper-
ties are listed in the Note.18 Compound 3 displayed high
fluorescence intensity in aqueous solution, quantum
yield of 3 was 0.894 when excited at 365 nm. 50-Triphos-
phate of 5-aminohexylcarbamoylmethyl-20-deoxyuri-
dine (4) was prepared by the procedure described
previously.15,19 The acridone derivative 3 (0.8 mg,
3 lmol) was coupled with 4 (10 ODU260, 1.0 lmol) in
the presence of HBTU, diisopropylethylamine, and tri-
butylamine in dry DMF for 1 h at room temperature.
The purified acridone-tagged thymidine analogue 5
was obtained in 5% yield (1.0 ODU260) after purification
by HPLC on an ODS-silica gel column, and confirmed
by an ESI-mass spectrum.18
Figure 1. PCR assays of the acridone-tagged TTP analogue using
KOD Dash DNA polymerase. The mixture (20 ll) for PCR contained
0.5 ng/ll DNA template (pUC18, 2686 bp), 0.4 lM of each primer,
modified dNTP mix (0.2 mM of each nucleotide), and 0.5 U/10 ll of
DNA polymerase in the buffer supplied by the maker for the DNA
polymerase reaction. PCR assays were carried out at 94 ꢁC/1 min, 30
cycles of 94 ꢁC/30 s, 52 ꢁC/30 s, 74 ꢁC/1 min, and 74 ꢁC/5 min. The
reaction mixture was quenched by addition of formamide-dye solution
and PCR products were separated by 2% agarose gel electrophoresis.
The gel was visualized after staining with ethidium bromide. lanes 1
and 10: marker DNA (100–1200 bp); lane 2: negative control without
TTP; lane 3: positive control 4; lane 4: 4 + 5 (9:1); lane 5: 4 + 5 (7:3);
lane 6: 4 + 5 (1:1); lane 7: 4 + 5 (3:7); lane 8: 4 + 5 (1:9); and lane 9: 5.
All reaction mixtures contained dNTP (dATP+dCTP+dGTP) in
addition to TTP analogues.
We examined the incorporation of the acridone-labeled
thymidine triphosphate 5 in place of TTP during PCR
using KOD Dash DNA polymerases. pUC18 plasmid
DNA, and DNA A, 50-GGAAACAGCTATGACCAT
GATTAC-30, and DNA B, 50-CGACGTTGTAAAAC

778
A. Shoji et al. / Bioorg. Med. Chem. Lett. 17 (2007) 776–779
Figure 2. (A) Fluorescence spectrum of the acridone-tagged DNA produced by PCR. Fluorescence spectra were taken with excitation at 388 nm. (B)
HPLC profiles of the digested products of the acridone-tagged DNA by nuclease P1 and alkaline phosphatase. HPLC condition: Wakosil 5C18
(4 · 25 cm); linear gradient elution of acetonitrile (2.1–37.1% in 35 min) in 50 mM triethylammonium acetate (pH 7.2) at flow rate of 1.0 ml/min.
HPLC was monitored by UV absorption at 260 nm. Only peak of Tacr was observed when HPLC was monitored by fluorescence at 450 nm with
excitation at 390 nm (data not shown).
GACGGCCAGT-30, were used as a template and prim-
ers, respectively, for the PCR assay. PCR was carried
out according to the procedure shown in the legend of
Figure 1. The PCR products were analyzed by 2% aga-
rose gel electrophoresis and visualized with ethidium
bromide. PCR with the thymidine derivative 4 demon-
strated formation of the 108 base-pair DNA product
in accordance with the previous report (Fig. 1, lane
3).15 Combined use of 4 and 5 in 9:1, 7:3, 5:5, 3:7, and
1:9 molar ratio as a substrate yielded the full-length
PCR product, as expected (lanes 4–8). Complete dis-
placement of 4 with 5 also gave the corresponding 108
base-pair DNA with acridone as a fluorescent label (lane
9). Successful PCR with this template and primers re-
quires the incorporation of 40 thymidine analogues with
a single stretch of four successive thymidine residues.
Thus, multi-labeling of DNA with acridone could be
accomplished by using the substrate 5 and KOD Dash
DNA polymerase. It is reported that no fluorescent-
labeled DNA could be obtained by PCR without com-
bined use of natural TTP and a fluorescent-labeled thy-
midine analogue, when cyanine dyes or fluorescence was
used as a labeling agent.3–7 A bulky fluorescent molecule
in these thymidine analogues suppresses the successive
incorporation of the modified substrate.
Fluorescence spectrum of the resulting modified DNA is
shown in Figure 2A, which showed the incorporation of
acridone moiety. Figure 2B shows a HPLC profile of
digested products of the modified DNA by nuclease
P1 and alkaline phosphatase. The acridone-tagged thy-
midine was confirmed by comparing the retention time
with that of the authentic sample synthesized by cou-
pling of 3 and the corresponding thymidine analogue,
5-aminohexylcarbamoylmethyl-20-deoxyuridine.20 The
composition of the normal nucleosides and acridone-
tagged thymidine nearly corresponded to the desired
composition of the 108 mer DNA (A/G/C/T/TAcr
49:59:59:9:40).
=
In conclusion, we prepared 50-triphosphate of new acri-
done-tagged thymidine. PCR using this modified nucle-
otide with KOD Dash DNA polymerase provides a new
entry for the synthesis of a DNA probe with a high den-
sity of fluorescence, and complements the previous
method of the DNA probe synthesis, because acridone
has strong fluorescence intensity and is stable against
photo-degradation.
Acknowledgments
We prepared the modified DNA by PCR in large scale,
and the resulting acridone-tagged DNA was character-
ized by fluorescence spectrum and nuclease digestion.
In brief, 10 portions of 0.1 ml reaction mixture contain-
ing 1 nM DNA template, 1 lM of each primer, modified
dNTP mix (0.2 mM of each nucleotide), and 5 U of
DNA polymerase in the buffer supplied by the maker
for the DNA polymerase reaction were put in reaction
tubes and set on a PCR thermal cycler. The PCR was
carried out under the same conditions as described
above. All PCR products were collected, passed through
a spin column (cut-off, 10 k), and separated by 2% aga-
rose gel. The modified DNA on the gel was cut out,
extracted with TBE buffer, and passed through a spin
column for desalting. The modified DNA (0.70
OD260 nm, 0.18 nmol) was obtained in a pure form.
The molar absorption coefficient of the modified DNA
was estimated from the sum of those of the DNA and
acridone.
This work was supported in party by Grant-in-Aid for
Scientific Research from the Japan Society for Promo-
tion of Science and by PRESTO from the Japan Science
and Technology Agency.
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18. Compound 3; ESI-Mass (m/z, negative mode): 252.2.
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7.29 (t, 1H), 7.50–7.54 (m, 2H), 7.68–7,75 (m, 2H), 8.26 (d,
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407 nm (e 4900). Fluorescence: kmax 423 nm, 448 nm
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