Synthesis of cationic water-soluble light-harvesting dendrimers

Article abstract of DOI:10.1021/ol047674p

(Chemical Equation Presented) Four generations of phenylenefluorene (1F)- and phenylenebis(fluorene) (2F)-terminated polyamidoamine (PAMAM) dendrimers were synthesized by coupling activated esters with commercially available PAMAM precursors. Treatment of

Full text of DOI:10.1021/ol047674p

ORGANIC
LETTERS
2005
Vol. 7, No. 10
1907-1910
Synthesis of Cationic Water-Soluble
Light-Harvesting Dendrimers
Shu Wang,† Janice W. Hong,‡ and Guillermo C. Bazan*,‡
Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100080, P. R. China, and Departments of Materials and Chemistry &
Biochemistry, Institute for Polymers and Organic Solids, UniVersity of California,
Santa Barbara, California 93106-9510
bazan@chem.ucsb.edu
Received November 11, 2004
ABSTRACT
Four generations of phenylenefluorene (1F)- and phenylenebis(fluorene) (2F)-terminated polyamidoamine (PAMAM) dendrimers were synthesized
by coupling activated esters with commercially available PAMAM precursors. Treatment of Boc-terminated pendant groups on the optically
active units with 3 M HCl in dioxane yields cationic water-soluble dendrimers. Fluorescence resonance energy transfer (FRET) experiments
with the cationic dendrimers as the donor and double stranded DNA containing a fluorescein label as the acceptor reveal cooperative optical
behavior.
Dendrimers are hyperbranched organic macromolecules with
well-defined three-dimensional architectures and a large
number of terminal groups that can be varied to control
properties such as size, molecular weight, topology, and
surface reactivity.1 They can be used to generate a highly
dense collection of chromophores with overall properties
similar to those of natural photosynthetic systems, which
absorb light and very efficiently transfer the resulting
excitations to lower energy sites.2 Recent interest has grown
in utilizing the light-harvesting (or antenna-like) properties
of dendrimers to achieve sensory signal amplification in the
presence of suitable energy or electron acceptors.3
reporter dyes has been used for devising strand-specific DNA
or RNA detection methods.4,5 The positive charges in these
conjugated polyelectrolytes are important for water solubility
and for orchestrating electrostatic interactions with the
negatively charged DNA (or RNA).6 However, structural
uncertainties in these polymers (i.e. molecular weight
distribution, variations in average molecular weight, and
(2) (a) Newkome, G. R.; Moorefield, C. N.; Vo¨gtle, F. Dendrimers and
Dendrons; Wiley-VCH: Weinheim, Germany, 2001. (b) Gilat, S. L.;
Adronov, A.; Fre´chet, J. M. J. Angew. Chem., Int. Ed. Engl. 1999, 38,
1422. (c) Balzani, V.; Campagna, S.; Denti, G.; Juris, A.; Serroni, S.;
Venturi, M. Acc. Chem. Res. 1998, 31, 26. (d) Maus, M.; De, R.; Lor, M.;
Weil, T.; Mitra, S.; Wiesler, U.-M.; Herrmann, A.; Hofkens, J.; Vosch, T.;
Mu¨llen, K.; De Schryver, F. C. J. Am. Chem. Soc. 2001, 123, 7668. (e)
Devadoss, C.; Bharathi, P.; Moore, J. S. J. Am. Chem. Soc. 1996, 118,
9635. (f) Yeow, E. K. L.; Ghiggino, K. P.; Reek, J. N. H.; Crossley, M. J.;
Bosman, A. W.; Schenning, A. P. H. J.; Meijer, E. W. J. Phys. Chem. B
2000, 104, 2596.
Light-harvesting properties and efficient fluorescence
energy transfer (FRET) can also be attained with water-
soluble cationic conjugated polymers and oligomers.4 The
resulting optical amplification of emission intensities from
(3) (a) Adronov, A.; Fre´chet, J. M. J. Chem. Commun. 2000, 1701. (b)
Schenning, A. P. H. J.; Peeters, E.; Meijer, E. W. J. Am. Chem. Soc. 2000,
122, 4489. (c) Hahn, U.; Gorka, M.; Vo¨gtle, F.; Vicinelli, V.; Ceroni, P.;
Maestri, M.; Balzani, V. Angew. Chem., Int. Ed. 2002, 41, 3595. (d) Balzani,
V.; Ceroni, P.; Gestermann, S.; Kauffmann, C.; Gorka, M.; Vo¨gtle, F. Chem.
Commun. 2000, 853.
† Chinese Academy of Sciences.
‡ University of California.
(1) (a) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A., III Angew. Chem.,
Int. Ed. Engl. 1990, 29, 138. (b) Bosman, A. W.; Janssen, H. M.; Meijer,
E. W. Chem. ReV. 1999, 99, 1665. (c) Ballauff, M. Top. Curr. Chem. 2001,
212, 177. (d) Fre´chet, J. M. J.; Tomalia, D. A. Dendrimers and Other
Dendritic Polymers; Wiley: New York, 2001.
(4) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 10954.
10.1021/ol047674p CCC: $30.25
© 2005 American Chemical Society
Published on Web 04/12/2005

structural defects) limit the determination of detailed rela-
tionships between molecular structure and optical properties.7
It occurred to us that cationic dendrimers with precise
structures could be used in similar sensing schemes. In this
contribution, we report the synthesis of a series of cationic
polyamidoamine (PAMAM) dendrimer derivatives with
chromophores on the macromolecular surface. The positive
charges, located primarily on the exterior of the dendrimer,
yield water-soluble materials. For reference, the structures
of the zero- and second-generation PAMAM cores are shown
in Scheme 1.
Scheme 3. Synthesis of G0-4-1F
Scheme 1. Molecular Structures of the Zero (G0) and Second
(G2) Generations of PAMAM Dendrimers
Scheme 3 shows the synthetic entry into the G0-4-1F
series. Reaction of 2-bromo-9,9-bis(6′-bromohexyl)fluorene
(1)5d with sodium azide in DMSO provides 2-bromo-9,9-
bis(6′-azidohexyl)fluorene (2) in 86% yield. Reduction by
using lithium aluminum hydride and subsequent amine
protection with di-tert-butyldicarbonate (Boc2O) yields 2-bro-
mo-9,9-bis(6′-tert-butoxycarbonylaminohexyl)fluorene (4) in
65% yield. Suzuki cross-coupling9 of 4 with 4-carboxyphen-
ylboronic acid using Pd(PPh3)4 in 2 M aqueous sodium
carbonate gives 2-(4′-carboxyphenyl)-9,9-bis(6′-tert-butoxy-
carbonylaminohexyl)fluorene (5) in 44% yield. After reaction
with pentafluorophenol in the presence of dicyclohexylcar-
bodiimide (DCC), the activated pentafluorophenol 4-(9′,9′-
bis(6′′-tert-butoxycarbonylaminohexyl)-2′-fluorenyl)ben-
zoate (6) was obtained in 65% yield. Four different generations
of Boc-protected phenylene-fluorene terminated dendrimers
(Boc-G0-4-1F) were obtained by coupling the activated ester
6 with commercially available amine terminated PAMAM
cores. Treatment with 3 M HCl in dioxane yields the cationic,
water-soluble dendrimers G0-4-1F.
We chose as synthetic targets four generations of PAMAM
dendrimers with either phenylenefluorene (G0-4-1F, where
the subscript refers to the generation number and 1F is
phenylenefluorene, see Scheme 2) or phenylenebis(fluorene)
Scheme 2. Molecular Structures of G0-4-1F and G0-4-2F
The series G0-4-2F was prepared by the sequence of
reactions in Scheme 4. In a procedure similar to that for 2,
(5) (a) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc.
2003, 125, 896. (b) Wang, S.; Bazan, G. C. AdV. Mater. 2003, 15, 1425.
(c) Wang, S.; Gaylord, B. S.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126,
5446. (d) Wang, S.; Liu, B.; Gaylord, B. S.; Bazan, G. C. AdV. Funct. Mater.
2003, 13, 463. (e) Liu, B.; Gaylord, B. S.; Wang, S.; Bazan, G. C. J. Am.
Chem. Soc. 2003, 125, 6705.
(6) Pinto, M. R.; Schanze, K. S. Synthesis 2002, 1293.
(7) Liu, B.; Wang, S.; Bazan, G. C.; Mikhailovsky, A. J. Am. Chem.
Soc. 2003, 125, 13306.
(8) Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin,
S.; Roeck, J.; Ryder, J.; Smith, P. Polym. J. 1985, 17, 117.
(9) Miyaura, N.; Suzuki, A. Chem. ReV. 1995, 95, 2457.
(G0-4-2F, where 2F is phenylenebis(fluorene)) at the surface
of the macromolecules. At near neutral pH only the amine
groups on the terminal chromophores are protonated; the
PAMAM interior is neutral.8
1908
Org. Lett., Vol. 7, No. 10, 2005

functionalization). In Boc-G2-1F, the theoretical ratio is 0.77
and the observed ratio is 0.77 (>99% functionalization). For
higher generation dendrimers (G3 and G4), the NMR data
were consistent with 90-95% surface functionalization.
Incomplete coverage is attributed to the nonquantitative yield
of the coupling steps and known imperfections of the com-
mercial PAMAM dendrimers, together with difficulties in
the purification of the larger structures.
Scheme 4. Synthesis of G0-4-2F
The optical properties of the G0-4-1F and G0-4-2F are
summarized in Table 1. Measurements were performed in a
Table 1. Summary of Optical Properties of G0-4-1F and
G0-4-2F (pH 6.0), Where PL is Photoluminescence
ꢀ
ꢀ/OU
UV
PL
(M-1‚cm-1
× 105
)
(M-1‚cm-1
× 104
)
QE
(%)
(λmax, nm) (λmax, nm)
G0-1F
320
320
320
322
345
346
345
344
403
400
400
395
426
426
423
423
0.9
3.7
7.0
13
1.6
5.6
9.1
18
2.3
2.3
2.2
2.0
3.9
3.5
2.8
2.8
35
29
24
20
40
35
33
26
G2-1F
G3-1F
G4-1F
G0-2F
G2-2F
G3-2F
G4-2F
50 mM potassium phosphate buffer at pH 6 to ensure
protonation of only the surface basic sites. The absorption
maxima are observed at 320 and 345 nm for G0-4-1F and
G0-4-2F, respectively, corresponding to the π-π* transition
of the conjugated units. For both G0-4-1F and G0-4-2F, the
extinction coefficient (ꢀ) of the molecules increases as a
function of generation, while the ꢀ value contributed by each
optical unit (OU) decreases. This drop in ꢀ/OU ratio is
consistent with interchromophore contacts that become more
pronounced as the number of surface OUs increases. Slight
hypsochromic shifts and reductions in fluorescence quantum
efficiencies (QEs) are also observed for the larger genera-
tions, consistent with the increase in chromophore-chro-
mophore interactions.10 For a given generation, the absorption
and fluorescence maxima and the extinction coefficient for
G0-4-2F are larger than those of G0-4-1F, which reflect the
more extended conjugation length in the G0-4-2F series.
The function of G0-4-1F and G0-4-2F as donors in FRET
processes was examined.11 Fluorescein (Fl) was chosen as
the acceptor, since its absorption spectrum overlaps with the
emission spectra of 1F and 2F. Fluorescein was attached to
(negatively charged) double stranded DNA (dsDNA-Fl) to
ensure association with the cationic G0-4-1F and G0-4-2F
by electrostatic forces. Double stranded DNA (dsDNA-Fl)
was obtained by hybridization of ssDNA-Fl (Fl-5′-ATCT-
TGACTATGTGGGTGCT-3′) with a complementary strand
of equal length.
2-bromo-7-iodo-9,9-bis(6′-bromohexyl)fluorene (7)5d was
converted to 2-bromo-7-iodo-9,9-bis(6′-azidohexyl)fluorene
(8) by reaction with sodium azide in DMSO in 92% yield.
Reduction by using triphenylphosphine in THF/water (v/v
7:1), followed by protection of the amino groups with Boc2O
yields 2-bromo-7-iodo-9,9-bis(6′-tert-butoxycarbonylamino-
hexyl)fluorene (9). Selective Suzuki cross-coupling of com-
pound 10 with the iodide site in 9 gives 4-[9′,9′;9′′,9′′-
tetra(6′′′-tert-butoxycarbonylamino-hexyl)-7′,2′′-bisfluoren-
2′-yl]benzoic acid (12).5d The activated pentafluorophenyl
ester 13 was obtained starting with 12 by a similar method
for the preparation of 6. The G0-4-2F series was synthesized
by coupling the activated ester 13 with the appropriate
PAMAM dendrimer, followed by HCl treatment.
The 1H NMR spectra in CDCl3 of Boc-protected dendrim-
ers can be used to calculate the degree of surface function-
alization. For the Boc-protected dendrimers, the internal
dendrimer methylene protons are observed from 3.2 to 3.6
ppm and from 2.3 to 2.7 ppm. The peaks from 7.2 to 8.0
ppm are due to proton signals on the aromatic terminal
groups. The ratio of the integrations of aromatic/CH2 proton
signals provides the degree of surface functionalization. In
Boc-G0-1F, with 44 aromatic and 36 CH2 protons, the
theoretical ratio is 1.22. The observed ratio is 1.23 (>99%
(10) Although the ꢀ/OU was not evaluated for the Boc-protected
dendrimers, previous work has shown that interchromophoric coupling exists
with other organic-soluble dendrimers. See ref 3b.
(11) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer
Academic/Plenum Publishers: New York, 1999.
Org. Lett., Vol. 7, No. 10, 2005
1909

the sensitized excitation of the dendrimer at 330 nm, relative
to the direct excitation of Fl at its absorption maximum (480
nm). As a control, no FRET takes place when Fl unattached
to DNA is added. These results demonstrate that higher Fl
emission intensities are attained by the overall larger optical
cross section of the dendrimer structure and efficient FRET
to the Fl, than by direct excitation of the Fl. Furthermore,
the negative charge of the DNA is responsible for complex-
ation to the cationic dendrimer and satisfying the distance
requirement for FRET. It should be noted that the sensitized
Fl emission intensity by G0-4-2F is approximately twice that
obtained with G0-4-1F, when the same generation is com-
pared. Although the number of chromophores in this
comparison is equal, the G0-4-2F OUs have better spectral
overlap with Fl, a larger extinction coefficient, and a larger
quantum yield, relative to the G0-4-1F series.
In summary, new cationic light-harvesting dendrimers
were designed, synthesized, and characterized. FRET experi-
ments demonstrate that higher generation dendrimers exhibit
better signal amplification of DNA-bound fluorescent report-
ers. These water-soluble dendrimers may prove useful in
biosensor applications, due to their collective behavior and
well-defined structures.
Figure 1. The emission spectra of dsDNA-Fl in the presence of
G
0-4-1F in potassium phosphate buffer solution (50 mM, pH 6.0);
[OU] ) 6.4 × 10-7 M, [dsDNA-Fl] ) 1.0 × 10-8 M, excitation
wavelength is 330 nm.
As shown in Figure 1, excitation of G0-4-1F at 330 nm
results in successful FRET to the dsDNA-Fl ([dsDNA-Fl]
) 1.0 × 10-8 M, [OU] ) 6.4 × 10-7 M). The spectra also
reveal a more effective “antenna effect” for the higher
generation dendrimers.12 FRET efficiency reaches a limiting
value for the third and fourth generation structures. This limit
is consistent with the lower extinction coefficient (per optical
unit) and QE, compared to the third generation dendrimers.
For G3-1F, a 6-fold increase in Fl emission is observed by
Acknowledgment. The authors are grateful to the NIH
(GM62958-01) and the Institute for Collaborative Biotech-
nologies for financial support.
Supporting Information Available: Detailed experi-
mental procedures and characterization data of all intermedi-
ates and dendrimer products. This material is available free
of charge via the Internet at http://pubs.acs.org.
(12) The “antenna effect” arises from a larger ꢀ and a higher binding
constant due to multivalency of the higher generation dendrimers.
OL047674P
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Org. Lett., Vol. 7, No. 10, 2005