A cationic tetrahedral chromophore for amplified DNA detection

Article abstract of DOI:10.1016/j.tetlet.2005.11.080

The tetrahedral cationic chromophore, tetrakis [4-(9,9-bis(6′-(N,N,N- trimethylammonium)hexyl)-2-fluorenyl)phenyl]methane (1) shows better fluorescence resonance energy transfer (FRET) to the fluorescein (Fl) attached to the 5′-terminus of double-stranded

Full text of DOI:10.1016/j.tetlet.2005.11.080

Tetrahedron
Letters
Tetrahedron Letters 47 (2006) 437–439
A cationic tetrahedral chromophore for amplified DNA detection
a,c,
Parameswar K. Iyera,b, and Shu Wang
*
*
aDepartment of Chemistry and Institute of Polymers and Organic Solids, University of California, Santa Barbara, CA 93106, USA
bDepartment of Chemistry, Indian Institute of Technology Guwahati, North Guwahati-781039, Assam, India
cKey Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, PR China
Received 24 September 2005; revised 9 November 2005; accepted 16 November 2005
Available online 1 December 2005
Dedicated to Professor Guillermo C. Bazan, University of California, Santa Barbara
Abstract—The tetrahedral cationic chromophore, tetrakis [4-(9,9-bis(60-(N,N,N-trimethylammonium)hexyl)-2-fluorenyl)phenyl]-
methane (1) shows better fluorescence resonance energy transfer (FRET) to the fluorescein (Fl) attached to the 50-terminus of dou-
ble-stranded DNA (dsDNA-Fl) as compared to the linear oligomers 2 and 3 and also provides efficient DNA hybridization
detection.
Ó 2005 Published by Elsevier Ltd.
-
Advances in DNA sequence identification and detec-
tion-based technology allow high-throughput gene
expression profiling and nucleotide polymorphism
detection, which have profound implications in medical
sciences.1 Although fluorescence-based detection meth-
ods are well established, efforts to enhance the sensitivity
by developing simplified protocols are still continuing.2
Water-soluble conjugated polymers have shown large
quenching efficiencies in the presence of oppositely
charged acceptors and the resulting optical amplification
of fluorescent signals has been used for devising strand-
specific DNA, RNA and protein detection.3–6
I
N
+
-
I
+
N
-
N
I
-
I
+
N
+
+
-
N
-
+
I
I
N
N
I
+
-
N
+
-
In this contribution, we report the synthesis of a novel
water-soluble tetrahedral chromophore, tetrakis [4-(9,9-
bis(60-(N,N,N-trimethylammonium)hexyl)-fluorenyl)-
phenyl]methane (1) (Fig. 1). The key step in the synthesis
(Scheme 1) of chromophore 1 is the Pd-catalyzed
coupling of 2-bromo-9,9-bis(60-(N,N-dimethylamino)-
hexyl)-fluorene6 with tetrakis-[4-(40,40,50,50-tetramethyl-
10,30,20dioxaborolanephenyl)]methane (5) to yield7
tetrakis[4-(9,9-bis(60-(N,N-dimethylamino)hexyl)-2-fluo-
renyl)phenyl]methane (6). The quarternization of 6 with
excess methyl iodide gave the title compound 1 in good
yield.8 Our interest to develop tetrahedral molecular
I
1
Br-
Br-
N
Br-
N
Br-
N
N
+
+
+
+
n
n
n = 1, 2. n = 2, 3.
Figure 1. Structures of oligomers 1–3.
Keywords: Tetrahedral chromophores; FRET; DNA detection.
assemblies stems from the fact that these materials exhi-
bit useful optical and electronic properties and can be
purified to a very high degree using conventional organic
*
Corresponding authors. Tel.: +91 361 258 2314; fax: +91 361 269
0762 (P.K.I.); e-mail addresses: pki@iitg.ernet.in; wangshu@iccas.
ac.cn
0040-4039/$ - see front matter Ó 2005 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2005.11.080

438
P. K. Iyer, S. Wang / Tetrahedron Letters 47 (2006) 437–439
a
b
O
C
C
I
B
O
33%
83%
4
4
4
5
-
-
I
I
N
N
N
N
+
+
c
C
C
93%
4
4
1
6
Figure 3. The integrated emission intensity of Fl for 1/dsDNA-Fl (blue
line), 2/dsDNA-Fl (black line) and 3/dsDNA-Fl (red line) as a
function of concentration.
Scheme 1. Synthesis of tetrahedral chromophore 1. Reagents and
conditions: (a) Bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, DMSO,
100 °C; (b) 2-bromo-9,9-bis(60-(N,N-dimethylamino)hexyl)fluorene,
Pd(PPh3)4, Na2CO3, toluene, water, reflux; (c) Ch3I, THF, water.
tures of complimentary strands in buffer solution at
57.5 °C (2 °C below its melting point) for 25 min and
then cooling to room temperature in phosphate buffer
solution (pH = 7.4, 50 mM). Fluorescein was chosen
since it is one of the ubiquitous dyes used in FRET
experiments and because it can be attached to DNA
structures using well-established protocols.11
chemistry techniques. Tetrahedral molecules of this type
offer distinct advantages over conjugated polymers. They
have well-defined structures and relative to their polymer
counterparts, there is no statistical distribution of chain
lengths and no negative contribution from unreacted
end groups. Furthermore, these tetrahedral molecules
form stable homogeneous films9 enabling their use in
various optoelectronic and sensor applications. The
absorption and fluorescence data for compounds 1–3
(Fig. 1) are shown in Figure 2. Both the absorption
and emission spectra of 1 are blue shifted compared to
those of 2 and 3. The absorption spectra of fluorescein
(Fl) overlaps with the emission spectra of chromophores
1–3, which is necessary for efficient Fo¨rster energy trans-
fer between these molecules.2 Fluorescence quantum effi-
ciencies (QEs) were determined in buffer solution against
quinine sulfate in 0.1 M H2SO4 (QE = 57%) as standard.
The QE of 1 was 39%, which is lower than those obtained
for 2 and 310 (93% and 76%), respectively.
Figure 3 establishes that the interactions of 1, 2 and 3
with ds-DNA-Fl are size and structure specific and sen-
sitive to the oligomer structure. Buffered conditions were
chosen in all our experiments as they stabilize DNA–
DNA interactions and would be included in any DNA
hybridization assay.12 The tetrahedral chromophore 1
resulted in better FRET efficiencies with dsDNA-F1 as
compared to the linear oligomers 2 and 3. This impor-
tant result strongly favors the hypothesis that electro-
static and hydrophobic interactions between negatively
charged DNA and cationic chromophores are size and
structure specific and are consistent with the literature
reports.13,14
A dsDNA-Fl from the probe having the sequence 50-
AGC ACC CAC ATA GTC AAG AT-30 with its com-
plimentary ssDNA (50-AGC ACC CAC ATA GTC
AAG AT-30) was chosen for our experiments. The dou-
ble stranded DNAs were obtained by annealing mix-
Since 1 provides better FRET to dsDNA-Fl, we further
studied its use for DNA hybridization detection. Figure
4 shows that the FRET to dsDNA-Fl is more efficient
relative to the non-complementary DNA (50-CGT
ATC ACT GGA CTG ATT GG-30). It is known that
dsDNA has a larger charge density than ssDNA, thus
stronger electrostatic interactions between
1 and
dsDNA result in more efficient FRET. In the presence
of non-hybridized DNA, there is no formation of
dsDNA and the non-complementary ssDNA shows
competition for interaction with 1 to ssDNA-Fl and
hence the energy transfer is less efficient. The integrated
emission intensity of Fl by energy transfer for dsDNA-
F1 is three times higher than that for the non-hybridized
DNA. This result shows that 1 can be used as a platform
for DNA hybridization detection.
In summary, we have reported the synthesis of the new
cationic tetrahedral chromophore 1. Tetrahedral chro-
mophore 1 demonstrates improved FRET to dsDNA-
Fl compared to linear oligomers 2 and 3, which proves
that the hydrophobic and electrostatic interactions are
size and structure specific. Preferred FRET by chromo-
Figure 2. The absorption (UV) and fluorescence (PL) spectra of 1–3 in
phosphate buffer solution (pH = 7.4, 50 mM).

P. K. Iyer, S. Wang / Tetrahedron Letters 47 (2006) 437–439
439
(b) Stork, M.; Gaylord, B. S.; Heeger, A. J.; Bazan, G. C.
Adv. Mater. 2002, 14, 361; (c) Gaylord, B. S.; Heeger, A.
J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
10954; (d) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. J.
Am. Chem. Soc. 2003, 125, 896; (e) Wang, S.; Bazan, G. C.
Adv. Mater. 2003, 15, 1425; (f) Wang, S.; Gaylord, B. S.;
Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 5446; (g) Liu,
B.; Gaylord, B. S.; Wang, S.; Bazan, G. C. J. Am. Chem.
Soc. 2003, 125, 6705.
7. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem.
1995, 60, 7508.
8. Tetrakis[4-(40,40,50,50tetramethyl-10,30,20dioxaborolane)-
1
phenyl]methane 5: H NMR (400 MHz, CDCl3, ppm): d
7.67 (d, 8H), 7.29 (d, 8H), 1.31 (s, 48H). 13C NMR
(125 MHz, CDCl3, ppm): d 149.56, 134.24, 130.37, 83.82,
65.99, 25.12. MS (Fast atom bombardment-FAB): 824(M).
Elemental analysis: calculated for C49H64B4O8: C, 71.40;
H, 7.83. Found: 70.80, 7.13. Tetrakis[4-(9,9-bis(60-(N,N-
dimethylamino)hexyl)-2-fluorenyl)phenyl]methane 6: 1H
NMR (400 MHz, CDCl3, ppm): d 7.69 (m, 28H), 7.51
(m, 8H), 7.32 (m, 8H), 2.12 (m, 64H), 2.00 (m, 16H), 1.26
(m, 16H), 1.07 (m, 32H), 0.67 (m, 16H). 13C NMR
(125 MHz, CDCl3, ppm): d 151.39, 150.96, 145.92, 140.92,
140.59, 139.48, 139.19, 131.68, 127.20, 126.99, 126.44,
125.99, 122.98, 121.29, 120.13, 119.89, 64.42, 59.95,
55.23, 45.63, 40.55, 30.09, 27.76, 27.25, 23.89. MS (Fast
atom bombardment-FAB): 1995(M). Tetrakis[4-(9,9-bis-
(60-(N,N,N-trimethylammonium)hexyl)-2-fluorenyl)phenyl]-
Figure 4. The integrated emission intensity of Fl for 1/dsDNA-Fl
(blue), 1/ssDNA-Fl + non-hybridized ssDNA (green) and 1/ssDNA-
Fl (red) as a function of concentration.
phore 1 to ds-DNA-F1 than to ssDNA-Fl + non-
hybridized ssDNA and ssDNA-Fl provides an opportu-
nity for using 1 in DNA-hybridization assays. Chromo-
phore 1 presents a simple, homogeneous and sensitive
platform for detecting DNA hybridization.
1
methane 1: H NMR (500 MHz, DMSO-d6, 90 °C, ppm):
d 7.85 (m, 24H), 7.45 (m, 20H), 3.38 (m, 32H), 2.96 (m,
72H), 1.47 (m, 16H), 1.13 (m, 32H), 0.65 (m, 16H). 13C
NMR (125 MHz, DMSO, 90 °C, ppm): d 151.95, 151.29,
146.42, 141.09, 139.23, 138.97, 138.05, 131.97, 128.15,
127.93, 126.91 126.49, 123.92, 121.67, 121.12, 120.73,
66.75, 64.83, 55.77, 53.54, 31.29, 29.32, 26.13, 24.18, 22.81.
MS (TOF-ESI-electrospray ionization): 1438.2 (M-2I),
916.1 (M-3I), 655.4 (M-4I), 499.1 (M-5I), 394.6 (M-6I),
320.2 (M-7I), 265.2 (M-8I).
Acknowledgements
This work was supported by Dupont Displays, Santa
Barbara (SB030014), NIH (GM62958-01) and the
NSF (DMR-0097611).
References and notes
9. (a) Odham, W. J., Jr.; Lachicotte, R. J.; Bazan, G. C. J.
Am. Chem. Soc. 1998, 120, 2987; (b) Wang, S.; Odham, W.
J., Jr.; Hudack, R. A.; Bazan, G. C. J. Am. Chem. Soc.
2000, 122, 5695; (c) Robinson, M. R.; Wang, S.; Bazan, G.
C.; Cao, Y. Adv. Mater. 2000, 12, 1701; (d) Robinson, M.
R.; Wang, S.; Heeger, A. J.; Bazan, G. C. Adv. Funct.
Mater. 2001, 11, 413.
10. Wang, S.; Liu, B.; Gaylord, B. S.; Bazan, G. C. Adv.
Funct. Mater. 2003, 13, 463.
11. (a) http://www.probes.com; (b) Bioconjugate Techniques;
Hermanson, G. T., Ed.; Academic Press: San Diego, CA,
1996.
1. Balakin, K. V.; Korshun, V. A.; Mikhalev, I. I.; Maleev,
G. V.; Malakhov, A. D.; Prokhorenko, I. A.; Berlin, Y. A.
Biosens. Bioelectron. 1998, 13, 771.
2. Lakowicz, J. R. Principles of Fluorescence Spectroscopy,
2nd ed.; Kluwer Academic/Plenum: New York, 1999.
3. McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. Rev.
2000, 100, 2537.
4. (a) Chen, L.; McBranch, D. W.; Wang, H. L.; Helgeson,
R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A.
2000, 96, 12287; (b) Wang, D.; Gong, X.; Heeger, P. S.;
Rininsland, F.; Bazan, G. C.; Heeger, A. J. Proc. Natl.
Acad. Sci. U.S.A. 2002, 98, 49.
12. Basic DNA and RNA Protocols; Harwood, A. J., Ed.;
Humana Press: New Jersey, 1996.
13. Wang, S.; Gaylord, B. S.; Bazan, G. C. Adv. Mater. 2004,
16, 2127.
5. Pinto, M. R.; Schanze, K. S. Synthesis-Stuttgart 2002, 9,
1293.
6. (a) Wang, J.; Wang, D.; Miller, E. K.; Moses, D.; Bazan,
G. C.; Heeger, A. J. Macromolecules 2000, 33, 5153;
14. Tang, M. X.; Szoka, P. C. Gene Ther. 1997, 4, 823.