Synthesis of ODNs containing 4-methylamino-1,8-naphthalimide as a fluorescence probe in DNA

Article abstract of DOI:10.1016/S0960-894X(02)00404-3

Synthesis and fluorescence properties of oligodeoxynucleotides containing 4-methylamino-1,8-naphthalimide (NI) have been described. NI was successfully incorporated into DNA without significant destabilization of DNA whilst retaining its high fluorescence quantum yield. The attachment site of the NI greatly affected its property as an energy acceptor in FRET analysis.

Full text of DOI:10.1016/S0960-894X(02)00404-3

Bioorganic & MedicinalChemistry Letters 12 (2002) 2363–2366
Synthesis of ODNs Containing 4-Methylamino-1,8-naphthalimide
as a Fluorescence Probe in DNA
Kiyohiko Kawai,* Kazuhiro Kawabata, Sachiko Tojo and Tetsuro Majima*
The Institute of Scientific and Industrial Research (SANKEN), Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan
Received 15 April2002; accepted 27 May 2002
Abstract—Synthesis and fluorescence properties of oligodeoxynucleotides containing 4-methylamino-1,8-naphthalimide (NI) have
been described. NI was successfully incorporated into DNA without significant destabilization of DNA whilst retaining its high
fluorescence quantum yield. The attachment site of the NI greatly affected its property as an energy acceptor in FRET analysis.
# 2002 Elsevier Science Ltd. All rights reserved.
Fluorescence resonance energy transfer (FRET) is a
process by which the excited state energy of one fluor-
escent dye is transferred to another dye. Since the
FRET efficiency is dependent upon the distance between
the donor and acceptor dyes, this technique has been
The NI group was conjugated to 50-end of ODN
by converting the N-(3-propanol)-4-methylamino-1,8-
naphthalimide (NIOH) to the corresponding phosphoro-
amidite derivative (4, Scheme 1).16 For incorporation of
the dye at the desired position inside the DNA, Yamana
et al. have reported that 20-sugar position of ODNs
is a suitable site for covalent attachment of several
fluorophores.14,17ꢀ21 According to their procedure, a
nucleoside derivative containing an NI group was syn-
thesized by the reaction of 20-amino-20-deoxyuridine
with 2-(4-methylamino-1,8-naphthalimide)acetic acid
(UNI, Scheme 2).22 UNI was converted to the phosphoro-
amidite derivative (10) and NI-containing ODNs were
synthesized by DNA synthesizer according to the stan-
dard procedure.23
widely used to determine the structure1ꢀ4 and conforma-
5ꢀ7
tionaltransition of nucelic acids,
and the interaction
of the biomolecules even at the single molecule level.8,9
Recently, by incorporating three dye molecules in
DNA, multiple FRET resulting from irradiation at a
single wavelength has been successfully applied to detect
single nucleotide polymorphism.10 Thus, the usage of
severalcombinations of fluorescent dyes with different
photochemical properties would be useful for multiple
FRET analysis.11ꢀ13 However, since fluorescence quan-
tum yield (ꢀF) of the dye molecules often becomes low
upon attachment to DNA, the dye molecules used for
DNA labeling in FRET analysis were restricted to
fluorescein, N,N,N0,N0-tetramethyl-6-carboxylrhodamine,
cyanine dyes, coumarin,14,15 and their derivatives. Here,
in order to expand the dye library for FRET analysis, we
have synthesized oligodeoxynucletotides (ODNs) con-
taining 4-methylamino-1,8-naphthalimide (NI). It has
been demonstrated that NI can be introduced into
DNA without significant loss of the duplex stability and
its high ꢀF.
First, to check the duplex stability of the NI-containing
ODNs, melting temperatures were measured for repre-
sentative ODNs studied here (Table 1). NIOH con-
jugated ODN (INIOH/II) showed higher stability
compared to the corresponding unmodified ODN (I/II).
On the other hand, incorporation of UNI (IUNI/II)
resulted in slight destabilization of ODN compared with
that of I/II. Thus, NI was incorporated into DNA
without large disturbance of the duplex stability.
Although NI has a high ꢀF (0.8 in methanol),24 attach-
ment of dye to DNA often causes a significant decrease
of ꢀF. ꢀF values for INIOH/II and IUNI/II were measured
to be 0.46 and 0.40, respectively. Thus, NI retained the
ꢀF upon incorporation into DNA. To obtain the infor-
mation on the energy accepting properties of NI, we
synthesized a series of ODNs containing NI as an
energy acceptor and pyrene (UPy) as an energy donor,25
*Corresponding authors. Tel.: +81-6-6879-8496; fax: +81-6-6879-
8499; e-mail: kiyohiko@sanken.osaka-u.ac.jp (K. Kawai). Tel.: +81-
6-879-8495; fax: +81-6-6879-8499; e-mail: majima@sanken.osaka-u.
ac.jp (T. Majima).
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00404-3

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K. Kawai et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2363–2366
ꢁ
.
Scheme 1. (a) 3-Amino-1-propanol, EtOH, reflux, 8 h, 94%; (b) CH3NH2, CuSO4 5H2O, DMA, 80 C, 2 h, 32%; (c) P(N-iPr2)(OCH3)Cl, DIEA,
pyridine, 1.5 h.
ꢁ
ꢁ
.
Scheme 2. (d) Glycine, DMA, 80 C, 17 h, 77%; (e) CH3NH2, CuSO4 5H2O, DMA, 80 C, 3 h, 47%; (f) DCC, HOBt, DMF, 20 h, 68%; (g) DMTr-Cl,
pyridine, 21 h, 56%; (h) P(N-iPr2)(OCH3)Cl, DIEA, pyridine, 16 h.
with those of unmodified ODNs (I/II), and their corre-
sponding singly labeled ODNs (INIOH/II and IUNI/II).
Since NI has a very low molar extinction coefficient at
351 nm (<1500 Mꢀ1 cmꢀ1), selective excitation of pyr-
ene is achieved at this wavelength (Fig. 1). The emission
spectra of INIOH/IIPyn and IUNI/IIPyn (n=1–4), excited at
351 nm, are shown in Figure 2a and b, respectively. The
fluorescence intensity of NI at 540 nm was reduced and
that of pyrene at 380 nm was increased with increasing
distance between two dyes, demonstrating the occur-
rence of the FRET between these two dyes. The effi-
ciency of energy transfer (E) was obtained from the
excitation spectrum of the NI at the given wavelength
351 nm (I351) according to the equation,
Table 1. Melting temperatures of pyrene and NI modified ODNs
ODN
Tm (ꢁC)a
I/II
37.2
40.3
36.2
41.6
44.9
41.3
44.3
39.0
40.8
INIOH/II
IUNI/II
I/IIPy1
I/IIPy4
INIOH/IIPy1
INIOH/IIPy4
IUNI/IIPy1
IUNI/IIPy4
aUV melting measurements were carried out in a pH 7.0 Na phosphate
buffer (20 mM) at a totalstrand concentration of 8 mM.
I351 ¼ "A þ E"D
ð1Þ
in which the distance between NI and pyrene was varied
by placing A-T base pairs between pyrene and NI
resulting in ca. 10–21 A distance between UPy and
where "A and "D are the extinction coefficient of the
energy acceptor and energy donor at 351 nm, respec-
tively.26 The FRET efficiencies measured for duplexes
INIOH/IIPyn and IUNI/IIPyn are shown graphically in Fig.
2c, which shows a clear dependence of E on the distance
between two dyes. Interestingly, E was significantly low
NIOH, and 14–24 A distance between UPy and UNI
,
respectively. The incorporation UPy resulted in the
increase of Tm values (I/IIPy1 and I/IIPy4); thus the
doubly labeled ODNs showed higher stability compared

K. Kawai et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2363–2366
2365
Figure 1. Absorption spectra of INIOH/II (solid line), IUNI/II (dashed
line), and I/IIPy1 (dotted line). The measurement conditions were the
same as in Table 1.
for IUNI/IIPyn compared to INIOH/IIPyn. Since the same
energy donor is used in both cases, this difference is
considered to rely on the properties of the energy
acceptor. For the energy transfer occurring via the
Forster mechanism, E is expressed as follows,
ꢀ
ꢁ
6
E ¼ 1= 1 þ ðR=R0Þ
ð2Þ
ð3Þ
ꢀ
ꢁ1=6
R0 ¼ 8:8 ꢀ 10ꢁ28JꢀDnꢁ4ꢁ2
ð
ð
J ¼ "AðlÞfDðlÞl4dl= fDðlÞdl
ð4Þ
where J is the spectraloveralp of the donor emission
spectrum and the acceptor absorption spectrum, ꢀD is
the ꢀF for energy donor, n is the refractive index of the
medium, and ꢁ is relative orientation of the emission
dipole of the donor and the excitation dipole of the
acceptor. As for the explanation of the observed low E
for IUNI/IIPyn, the absorption maximum for IUNI/II (459
nm) is red-shifted by 8 nm relative to that for INIOH/II
(451 nm), which may result in a decrease of J. However,
J was found to be 1.6ꢂ1031 and 1.2ꢂ1031 nm6 molꢀ1 for
INIOH/IIPyn and IUNI/IIPyn, respectively, only partly
explaining the lower E for IUNI/IIPyn. As for the other
factor, rotation of the NI attached at 20-sugar position
inside the DNA is likely to be restricted compared to NI
Figure 2. Fluorescence spectra (lex=351 nm) for (a) INIOH/II, INIOH
IIPyn (n=1–4) and (b) IUNI/II, IUNI/IIPyn (n=1–4). The measurement
conditions were the same as in Table 1. (c) FRET efficiency in DNA
/
duplexes INIOH/IIPyn and IUNI/IIPyn
.
labeled at the 50-end, leading to the low ꢁ2 value for UNI
.
Thus, in this case, the difference of ꢁ2 may significantly
contribute to this observed large difference of E between
these two systems (Scheme 3).8,27,28
In conclusion, NI was incorporated into DNA as a
fluorescence probe through two attachment procedures.
It has been demonstrated that NI can be incorporated
into DNA without large alternation of the duplex
stability and its high ꢀF. Though low E was observed
for UNI, rotationalproperties of the NI may provide
fruitfulinformation on the conformationalfreedom of
the ODN around UNI.
Scheme 3.

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K. Kawai et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2363–2366
(m, 2H, OCH2CH2), 6.71 (d, 1H, J=8.4 Hz, naphthalene H3),
Acknowledgements
7.68 (dd, 1H, J=7.3 and 8.6 Hz, naphthalene H6), 7.82 (br,
1H, NHCH3), 8.29 (d, 1H, J=8.4 Hz, naphthalene H2), 8.44
(d, 1H, J=7.3 Hz, naphthalene H5), 8.61 (d, 1H, J=8.6 Hz,
naphthalene H6). 31P NMR (162 MHz, DMSO-d6) d 134.88.
EIMS (positive ion) m/z 446 (M+1).
This work has been partly supported by a Grant-in-Aid
for Scientific Research from Ministry of Education,
Science, Sport and Culture of Japan.
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,
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