Synthesis and properties of purine-type base-discriminating fluorescent (BDF) nucleosides: Distinction of thymine by fluorescence-labeled deoxyadenosine derivatives

Article abstract of DOI:10.1016/j.tetlet.2005.08.157

Novel base-discriminating fluorescent (BDF) nucleoside, 8-fluorescence-labeled adenosine derivative (^8PyA), was developed for the detection of thymine base on a target DNA. The BDF nucleoside was incorporated into oligodeoxynucleotides by post-

Full text of DOI:10.1016/j.tetlet.2005.08.157

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 7605–7608
Synthesis and properties of purine-type
base-discriminating fluorescent (BDF) nucleosides: distinction
of thymine by fluorescence-labeled deoxyadenosine derivatives
Yoshio Saito,a Kazuo Hanawa,a Kaori Motegi,a Kenji Omoto,b
Akimitsu Okamotob and Isao Saitoa,*
aNEWCAT Institute, School of Engineering, Nihon University and SORST, Japan Science and Technology Agency, Tamura,
Koriyama 963-8642, Japan
bDepartment of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, Kyoto 615-8510, Japan
Received 21 July 2005; revised 24 August 2005; accepted 24 August 2005
Available online 16 September 2005
Abstract—Novel base-discriminating fluorescent (BDF) nucleoside, 8-fluorescence-labeled adenosine derivative (8PyA), was devel-
oped for the detection of thymine base on a target DNA. The BDF nucleoside was incorporated into oligodeoxynucleotides by
post-synthetic modification. BDF probes containing 8PyA selectively emit fluorescence only when the base opposite BDF nucleoside
is thymine and act as effective reporter probes for homogeneous SNP typing.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Single nucleotide polymorphisms (SNPs) are the most
abundant genetic variations in the human genome and
appear on average once every 1.0–1.9 kb.1 They are of
medical and pharmacological interests in studies of dis-
ease susceptibility and drug response. Various tech-
niques for SNP typing have been developed, such as
50-exonuclease assay using TaqMan,2 molecular bea-
con,3 the Invader assay,4 and the DNA microarray
method.5 However, the single base discrimination in
nearly all reported methods is directly or indirectly
based on the different hybridization efficiency between
matched and mismatched duplexes. Such DNA probes
have inherent limitations of selectivity because the differ-
ences in the hybridization efficiency vary with the
sequence context and are often very small for the
detection of a single base mismatch in a long target
DNA. Particularly, to attain enough signal to noise
(S/N) ratio when using the DNA microarray method,
the hybridization and washing conditions must be care-
fully selected to minimize undesirable responses from
the mismatched hybridization probes. Considering these
limitations, alternative probes that do not rely on
hybridization events are highly desired.
Recently, we reported a new homogeneous assay method
to discriminate a single base alteration by base-discrimi-
nating fluorescent (BDF) probes.6 Particularly, we
reported novel pyrenecarboxamide-labeled pyrimidine-
type BDF nucleosides PyU (1) and PyC (2) and indicated
that these BDF probes selectively emit fluorescence only
when the complementary bases are perfectly matched.7
The use of PyU- and PyC-containing BDF made it possible
to discriminate adenine and guanine at specific sites on
the target DNA by simple mixing. However, discrimina-
tion of pyrimidine bases by purine-type BDF nucleosides
has not yet been achieved. In this paper, we report novel
adenosine-type BDF probes for the discrimination of thy-
mine base, the first thymine selective fluorophore (Fig. 1).
The synthesis of ODN containing an aminopropyl
linker (ODN(NH2A), 50-d(CGCAATNH2ATAACGC)-
30), is outlined in Scheme 1. 8-Bromo-20-deoxyadenosine
4 was coupled with a pyrene substituted propargylamine
under Sonogashira conditions by using Pd(PPh3)4 to
afford 6, which was then hydrogenated on 10% Pd/C
in MeOH to give 7. Protection of the amino group with
DMF acetal and the 50-hydroxyl group of deoxyribose
with the 4,40-dimethoxytrityl group resulted in forma-
tion of 9. After conversion to phosphoramidite, 10 was
incorporated into oligonucleotides by an automated
DNA synthesizer.8
*
Corresponding author. Tel.: +81 24 956 8911; fax: +81 24 956
8924; e-mail: saito@mech.ce.nihon-u.ac.jp
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.08.157

7606
Y. Saito et al. / Tetrahedron Letters 46 (2005) 7605–7608
O
O
NH2
N
O
H
N
O
N
NH2
N
N
H
N
N
H
HN
N
N
N
O
O
HO
O
HO
O
HO
O
OH
OH
OH
PyU (1)
PyC (2)
8PyA (3)
Figure 1. Structure of pyrenecarboxamide-labeled BDF nucleosicles.
NH2
NH2
TFAHN
TFAHN
HO
N
N
N
N
Br
5
N
N
N
N
a)
HO
O
O
4
6
OH
OH
N
NH2
N
N
TFAHN
N
N
TFAHN
N
N
b)
N
c)
N
N
HO
HO
O
O
8
7
OH
OH
N
N
N
N
N
N
TFAHN
N
N
TFAHN
DMTrO
N
N
d)
e)
N
DMTrO
N
O
O
O
N
P
OH
NC
10
9
O
Scheme 1. Reagents and conditions: (a) 5, Pd(PPh3)4, Cul, Et3N, DMF, rt, 13 h, 35%; (b) 10% Pd/C, MeOH, rt, 5 h, 88%; (c) DMF acetal, DMF,
50 ꢁC, 2h, 90%; (d) DMTrCl, pyridine, rt, 8 h, 75%; (e) ( i-Pr2N)2PO(CH2)2CN, 1H-tetrazole, CH3CN, rt, 1.5 h.
Table 1. Oliaonucleotides (ODNs) used in this study
NH2
N
NH2
N
O
O
H
N
N
H2N
N
O
Sequences
R
O N
N
N
N
ODN(8PyA)
ODN(8FIA)
ODN(N)
50-d(CGCAAT8pyATAACGC)-30
N
O
O
O
R
O
O
50-d(CGCAAT8FIATAACGC)-30
30-d(GCGTTANATTGCG)-50,
N = T, C, G, A
ODN(NH2A)
O
O
HO
O
O
ODNBRCA1
(
8PyA)
50-d(GGTACCA8PyATGAAATA)-30
30-d(CCATGGTTACTTTAT)-50
30-d(CCATGGTCACTTTAT)-50
CO2H
R =
ODNBRCA1(T)
ODNBRCA1(C)
8PyA
8FlA
Scheme 2. Reagents and conditions: succinimidyl ester, 1 M NaHCO3,
aq DMF.
duplexes (ODN(8PyA)/ODN(N); N = C, G, A) and
single-stranded ODN (ODN(8PyA)) showed weak fluo-
rescence emission (UF = 0.065, 0.017, 0.051, and 0.040,
respectively).9 In contrast, the fluorescence of the per-
fectly matched duplexes (ODN(8PyA)/ODN(T)) showed
strong emission at 406 nm (UF = 0.167) (Fig. 2). In melt-
ing temperature (Tm) measurements of the duplex, a
high duplex stability was observed for 8PyA-containing
duplex (ODN(8PyA)/ODN(T), Tm = 64.7 ꢁC, Table 2),
which was more stable than a natural A/T base pair
(Tm = 51.2 ꢁC).
The synthesis of BDF probes ODN(8PyA) and ODN-
(FlA) has been accomplished by using ODN(NH2A).
As shown in Scheme 2, each fluorophore was post-syn-
thetically incorporated into ODN(NH2A). A DMF
solution of the succinimidyl ester of each fluorophore
was added to ODN(NH2A) in 1 M NaHCO3, and this
was incubated at 37 ꢁC for 17 h. The resulting suspended
mixture was filtered off and evaporated to dry. ODNs
were purified by reverse phase HPLC. The newly synthe-
sized ODNs are summarized in Table 1.
It was reported that bulky substituents on purine
nucleosides at the C-8 position favor the syn conforma-
tion around the N-glycosidic bond in solution.10 When
Initially, the fluorescence of 8PyA-containing ODN was
examined. The fluorescence spectra of the mismatched

Y. Saito et al. / Tetrahedron Letters 46 (2005) 7605–7608
7607
Figure 2. (a) Fluorescence spectra of 2.5 lM ODN (8pyA) hybridized
with 2.5 lM ODN(A), ODN (G), ODN (C), or ODN (T), and that of
single-stranded ODN(8py A) (50 mM sodium phosphate, 0.1 M sodium
chloride, pH 7.0. room temperature). Excitation wavelength was at
Figure 3. (a) Fluorescence spectra of 2.5 lM ODNBRCA1
(
8PyA)
hybridized with 2.5 lM ODNBRCA1(T) or ODNBRCA1(C), and that of
single-standard ODNBRAC1
8PyA) (50 mM sodium phosphate, 0.1 M
(
381 nm. (b) Comparison of the fluorescence for the bases opposite 8Py
A
sodium chloride, pH 7.0, room temperature). Excitation wavelength
was at 358 nm. (b) Comparison of the fluorescene for the bases
opposite 8PyA (50 mM sodium phosphate, 0.1 M sodium chloride,
pH 7.0, room temperature). ‘‘SS’’ denotes a single-standard BDF
(50 mM sodium phosphate, 0.1 M sodium chloride, pH 7.0, room
temperature), ‘‘ss’’ denotes a single-stranded BDF probe. The sample
solutions were illuminated with a 365 nm transilluminator.
probe. The sample solutions were illuminated with
transilluminator.
a 365 nm
Table 2. Melting temperature (Tm)
5'-d(CGCAAT 8PyATAACGC)-3'
3'-d(GCGTTA N ATTGCG)-5'
nated at 365 nm, and the fluorescence images were taken
by a digital camera. A strong fluorescence emission was
observed with the ODNBRCA1
duplex, whereas the mismatched duplex (ODNBRCA1
(
8PyA)/ODNBRCA1(T)
Sample (N)
Tm (ꢁC)
(
(
8PyA)/ODNBRCA1(C)) and single-stranded ODNBRCA1
-
T
C
G
A
64.7
55.9
59.5
56.4
8PyA) showed a weak emission (Fig. 3). The fluores-
cence quantum yield of the matched duplex (8PyA/T
base pair, UF = 0.280) was at least 2.3 times larger than
that observed for the mismatched duplexes.
The 8PyA-containing BDF probe facilitates the distinc-
tion of thymine on a target DNA by the drastic fluores-
cence change. These fluorescence properties of 8PyA are
favorable as an SNP typing probe. However, when BDF
probes containing a –C8PyAC– sequence were used, the
fluorescence emission was strongly quenched by the
flanking C/G base pairs. This indicates that there are
some sequence limitations for the use of 8PyA-containing
BDF probe.
pyrenecarboxamide fluorophore is attached to adenine
at the C-8 position, the polarity-sensitive fluorophore
is expected to be extruded to the outside of the groove,
a highly polar aqueous phase, due to base-pairing with
thymine (anti-conformation), and a strong fluorescence
emission should be observed. In contrast, the mis-
matched base pair containing 8PyA shows a weak fluo-
rescence emission probably because conformational
change from anti to syn occurs due to the lack of
base-pairing. Thus, the fluorophore relocates to a low-
polarity hydrophobic site within the groove, which
weakens the fluorescence emission. Therefore, 8PyA-con-
taining BDF probe is thought to emit a highly T-selec-
tive fluorescence.
In conclusion, we have developed a novel purine-type
BDF probe, which can clearly distinguish thymine base
on the complementary strand. The clear fluorescence
change observed here is very useful for SNP typing.
SNP typing using BDF probes serves as a powerful
alternative to current SNP typing methods.
Although 8PyA-containing ODN showed a clear T-selec-
tive fluorescence emission, completely no selectivity was
observed in the fluorescein-labeled BDF probe (ODN
(
8FlA)), possibly because fluorescein does not interact
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