Self-assembled oligonucleotide-polyester dendrimers

Article abstract of DOI:10.1039/b209029e

A new approach to the controlled synthesis of multicomponent dendrimers is presented, in which three oligonucleotide-dendron conjugates were synthesized using solid phase techniques and hybridized to create a second generation polyester dendrimer with DNA

Full text of DOI:10.1039/b209029e

Self-assembled oligonucleotide-polyester dendrimers†
Sarah L. Goh, Matthew B. Francis and Jean M. J. Fréchet*
Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.
E-mail: frechet@cchem.berkeley.edu; Fax: +510 643 3079; Tel: +510 643 3077; http://www.frechet.com/
Materials Science Division, E. O. Lawrence Berkeley National Laboratories, Berkeley, CA 94720, USA.
E-mail: frechet@cchem.berkeley.edu; Fax: +510 643 3079; Tel: +510 643 3077; http://www.frechet.com/
Received (in Cambridge, UK) 17th September 2002, Accepted 29th October 2002
First published as an Advance Article on the web 13th November 2002
A new approach to the controlled synthesis of multi-
component dendrimers is presented, in which three oligonu-
cleotide–dendron conjugates were synthesized using solid
phase techniques and hybridized to create a second genera-
tion polyester dendrimer with DNA as a core and bearing
two types of peripheral functional groups.
Dendrimers have emerged as exceptionally versatile scaffolds
for the construction of well-defined nanoscale materials.
Through the selective introduction of diverse functionality at
the core and periphery of these macromolecules, components
have been prepared for a variety of applications, including light
Fig. 1 Three-component oligonucleotide–polyester dendrimer.
harvesting systems,1 drug delivery vectors,2,3 and lithographic
resists.4 A key next step in this field is the integration of
solubility when substituted with hydrophilic peripheral
multiple dendritic components into complex, multifunctional
groups.
molecular structures; however, the high synthetic demand of
These dendritic components, derived from 2,2-bis(hydroxy-
these materials places limitations on the quantity and types of
methyl)propionic acid, were synthesized by first reacting two
functional groups that can be incorporated, as orthogonal
equivalents of isopropylidene-protected monomer 114 with one
equivalent of dicyclohexylcarbodiimide (DCC) to generate
protecting group strategies or statistical coupling reactions are
required to introduce multiple functional domains.5–8 To
anhydride 2 (Scheme 1). Subsequent acylation of the terminal
address this objective, many research groups have explored
self-assembly as an efficient strategy to combine preformed
hydroxys of benzyl 2,2-bis(hydroxymethyl)propionate14 with 2
afforded doubly protected dendron 3. The benzyl ester core was
selectively unmasked under hydrogenolysis conditions to yield
dendritic molecules, typically through electrostatic, hydrogen
bonding, and metal–ligand binding interactions.8–10 However,
4, a second generation building block bearing a carboxylic acid
few of these strategies are capable of combining disparate
focal point.
components in a programmable fashion.
The oligonucleotide–dendron conjugates were synthesized
directly on a controlled pore glass (CPG) solid support.
Deoxyribonucleic acid (DNA) is an attractive framework for
the construction of self-assembling nanoscale assemblies due to
Oligonucleotide sequences were first prepared using an Expe-
its hybridization fidelity and well-defined double helical
dite 8909 DNA synthesizer (Applied Biosystems) with the
structure.11 The sequence specificity of DNA hybridization
terminal 5A dimethoxytrityl (DMT) protecting group retained.
The CPG beads were subsequently transferred to a fritted tube
allows several strands to be linked in a predictable fashion,
leading to complex, highly functional networks. This potential
(BioRad) and thoroughly rinsed before removal of the DMT
has resulted in the use of DNA scaffolds for the selective
protecting group with three exposures to 3% trichloroacetic acid
arrangement of nanoparticles12 and proteins.13 In an effort to
(TCA) in dichloromethane, Fig. 2. A simple protocol was next
developed for the acylation of the resulting 5A hydroxy group
with 4; a 2 h exposure to N-ethyl-NA-(3-dimethylaminopro-
pyl)carbodiimide (EDC) and dimethylaminopyridine (DMAP)
in CH2Cl2 was found to be optimal in this regard, affording the
supported isopropylidene protected conjugates, 5b–7b (Scheme
2). To generate a conjugate with an alternate peripheral
functionality, the acetal periphery was deprotected through
explore the use of DNA scaffolds for the organization of
dendrimer-based materials through self-assembly, we describe
herein a new synthetic strategy for the preparation of DNA–
dendron conjugates that retain their hybridization capabilities,
and the subsequent construction of multicomponent den-
drimers.
As an initial target, a three component system was designed
and synthesized to create a dendrimer bearing multiple
exposure to 10% TCA in ethylene glycol to yield 5c. These
functional moieties at the periphery (Fig. 1). Since DNA
oligomers of any sequence are readily available through the use
of automated solid phase synthesis, we sought to develop a
similarly convenient synthetic protocol to attach dendritic
components before cleaving the oligonucleotides from the
controlled pore glass support. The oligonucleotide sequences
were chosen so that the two 16 base conjugates, 5d and 6d, are
fully complementary to the third, 7d, which is composed of 32
nucleotides. Although many dendritic building blocks are
available, the polyester dendrons depicted in Fig. 1 were chosen
due to the their facile synthesis and high degree of water
† Electronic supplementary information (ESI) available: experimental
section and MALDI-TOF MS. See http://www.rsc.org/suppdata/cc/b2/
b209029e/
Scheme 1 Synthesis of a polyester dendron building block. (a) DCC,
CH2Cl2, 97%. (b) Benzyl 2,2-bis(hydroxymethyl)propionate, DMAP,
pyridine, CH2Cl2, 94%. (c) 10% Pd/C, H2, ethyl acetate, 95%.
2954
CHEM. COMMUN., 2002, 2954–2955
This journal is © The Royal Society of Chemistry 2002

Table 1 MALDI-TOF MS Data for dendrimer building blocks
Conjugate
MW (theory)
MW (exptl.)
Error
5d
6d
7d
5130.5
5383.8
10277.0
5126.3
5380.3
10271.1
0.08%
0.07%
0.06%
To demonstrate the self-assembly ability of the obtained
conjugates, the three compounds were combined in equimolar
ratios in aqueous 50 mM NaCl solution. The solutions were
allowed to anneal at room temperature for 12 h, and the
resulting structures were analyzed by non-denaturing poly-
acrylamide gel electrophoresis (Fig. 2). The single strand
conjugate 7d is shown in lane 2. Upon hybridization with either
5d or 6d, the mobility of the duplex band is reduced (lanes 3 and
4, respectively). Complexation of 7d with both 16-mers
produces a trimer of 32 base pairs, as can be seen in lane 5. The
migration of the trimer is slower than expected for an
unmodified 32-mer due to the presence of the three dendrons on
the duplex. The distinct major band in this sample indicates
hybridization of the conjugates to give the desired dendrimer
target in high yield.
Fig. 2 Native polyacrylamide gel electrophoresis of self-assembled
dendrimers. Lane 1: Double stranded DNA ladder (20, 30, 40 bp). Lane 2:
7d. Lane 3: 5d + 7d. Lane 4: 6d + 7d. Lane 5: 5d + 6d + 7d.
Oligonucleotides were visualized using GelStar stain (BioWhittaker).
The self-assembly of dendronized oligonucleotides stream-
lines the synthetic demands required to incorporate multiple
functional groups on the periphery of a single molecular target.
Through variation of the sequences and lengths of the DNA
strands, the structure and functional properties of the assembled
dendrimers can be tuned. Furthermore, due to the solid-phase
nature of the synthesis, dendrimers bearing more complex
functionality can be envisaged, and indeed, we have already
successfully attached higher generation dendrimers, chromo-
phores, and polymers to the periphery of these molecules.
Current efforts involve the exploration of the physical proper-
ties of these materials and the continued use of this strategy for
the construction of multicomponent nanoscale materials
through self-assembly.
The authors would like to thank the US Department of
Energy (DE-AC03-76SF00098) and Lawrence Berkeley Na-
tional Laboratory’s Molecular Design Institute II, funded by the
office of Naval Research (Grant No. N0001498F0402) for
support. Fellowship support (MBF) from the Miller Institute for
Basic Research in Science is gratefully acknowledged.
Scheme 2 Solid phase synthesis of oligonucleotide–dendron conjugates. (i)
3% TCA/CH2Cl2. (ii) 4, DMAP, EDC, CH2Cl2 (iii) 10% TCA/ethylene
glycol. (iv) Morpholine/methanol (1+1), 60 °C. 5a–d: DNA (5A-3A)
TTCTCTTCAGTTCACA. 6a–d: DNA (5A-3A) GCAGACGGTAATGACG.
7a–d: DNA (5A-3A) CGTCATTACCGTCTGCTGTGAACTGAAGAGAA.
The shaded sphere represents the controlled-pore glass support.
conditions were uniquely effective for the complete removal of
the acetal protecting groups without leading to depurination of
the oligonucleotide strand. The peripheral hydroxy groups
liberated by this procedure provide reactive sites that can be
further functionalized using phosphoramidites or additional
acylating agents.
The final step in the conjugate synthesis was the cleavage of
the oligonucleotide from the solid support and concomitant
removal of the nucleotide base and phosphate protecting
groups. The typical ammonium hydroxide conditions used for
this purpose could not be employed, as they readily cleaved the
ester moiety linking the dendron to the DNA strand. However,
a 2 h exposure to a 1+1 mixture of morpholine/MeOH at 60 °C
was found to remove all of the protective groups from the
oligonucleotide strand and cleave the material from the
synthesis support without interfering with the pivaloyl ester
linkage. Using this protocol, conjugate 5d was obtained with
four hydroxy groups at the periphery of the dendron. Both 6d
and 7d were cleaved from the CPG beads prior to acetal
removal, providing conjugates with isopropylidene function-
alized dendrons.
Notes and references
1 A. Adronov and J. M. J. Fréchet, Chem. Commun., 2000, 18,
1701–1710.
2 H. Ihre, O. L. De Jesús, F. C. Szoka and J. M. J. Fréchet, Bioconjug.
Chem., 2002, 13, 443–452.
3 Y. Kim and S. C. Zimmerman, Curr. Opin. Chem. Biol., 2001, 2,
733–742.
4 D. C. Tully, K. Wilder, J. M. J. Fréchet, A. R. Trimble and C. F. Quate,
Adv. Mater., 1999, 11, 314–318.
5 C. Modrakowski, S. C. Flores, M. Beinhoff and A. D. Schlüter,
Synthesis, 2001, 14 , 2143–2155.
6 S. M. Grayson and J. M. J. Fréchet, J. Am. Chem. Soc., 2000, 122,
10335–10344.
7 W. Zhang, I. D. T. Nowlan, L. M. Thomson, W. M. Lackowski and E.
E. Simanek, J. Am. Chem. Soc., 2001, 123, 8914–8922.
8 F. Vögtle, S. Gestermann, R. Hesse, H. Schwierz and B. Windisch,
Progress in Polymer Science, 2000, 25, 987–1041.
9 J. M. J. Fréchet, Proc. Natl. Acad. Sci., 2002, 99, 4782–4787.
10 S. C. Zimmerman and L. J. Lawless, Top. Curr. Chem., 2001, 217,
95–120.
11 M. S. Shchepinov, K. U. Mir, J. K. Elder, M. D. Frank–Kamenetskii and
E. M. Southern, Nucleic Acids Res., 1999, 27, 3035–3041.
12 C. M. Niemeyer, Angew. Chem., Int. Ed., 2001, 40, 4128–4158.
13 J. M. Tomkins, B. K. Nabbs, K. Barnes, M. Legido, A. J. Blacker, R. A.
McKendry and C. Abell, ChemBioChem, 2001, 5, 375–378.
14 H. Ihre, A. Hult, J. M. J. Fréchet and I. Gitsov, Macromolecules, 1998,
31, 4061–4068.
Purification of the crude conjugates was accomplished by
reverse phase HPLC; the collected fractions were analyzed by
matrix assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectrometry (Table 1) for character-
ization. All obtained molecular weights agreed with the
calculated values to within 0.08%.
CHEM. COMMUN., 2002, 2954–2955
2955