Synthesis and self-assembly of an oligonucleotide-modified cyclobutadiene complex

Article abstract of DOI:10.1021/om9909791

An oligonucleotide-functionalized cyclobutadiene complex and its hybridization to a double-stranded organometallic DNA-object are reported here.

Full text of DOI:10.1021/om9909791

368
Organometallics 2000, 19, 368-370
Syn th esis a n d Self-Assem bly of a n
Oligon u cleotid e-Mod ified Cyclobu ta d ien e Com p lex
Shane M. Waybright, Chainey P. Singleton, J ames M. Tour,†
Catherine J . Murphy, and Uwe H. F. Bunz*
Department of Chemistry and Biochemistry, The University of South Carolina,
Columbia, South Carolina 29208
Received December 10, 1999
Summary: An oligonucleotide-functionalized cyclobuta-
diene complex and its hybridization to a double-stranded
organometallic DNA-object are reported here.
integral part of the DNA strand during its synthesis.
Bergstrom’s work provides an example of a nonbiogenic
organic molecule being connected to DNA by an oligo-
nucleotide synthesizer;8 however, that approach was
limited in that the organic was attached to the CPG
resin upon which the oligonucleotide was synthesized.
While several groups have incorporated coordination
compounds9 and other synthetic moieties10 into oligo-
nucleotides, we desired a conjugate that was robust,
whose moiety did not interfere with hybridization, and
which did not require postsynthetic modification. Thus,
we sought to exploit the well-established phosphor-
amidite chemistry.11 Phosphoramidites are valuable in
the construction of oligonucleotides by treating the
organic or inorganic molecule as a modified nucleotide.12
The groups of Kool,13 Thuong,14 and Letsinger15 have
incorporated molecules into the sugar phosphate back-
bone using this method for a variety of applications;
however, it has yet to be linked to constructing oligo-
nucleotide-directed architectures. We generalize this
concept by proposing the use of any diol, providing a
virtually limitless library of available organic moieties
for addition onto any oligonucleotide. Thus, we can
We describe, in this communication, a proof of concept
for creating nanoscale architectures. We utilize oligo-
nucleotides to self-assemble organic or organometallic
modules into precise arrangements. Here, we lay the
groundwork with the incorporation of a cyclobutadiene
complex into the sugar-phosphate backbone of DNA
and its self-assembly into a hybridized dimer. The
ability to control the design of novel nanoscale archi-
tectures encompasses the ultimate goal of materials
chemistry.1 Assembly of molecular building blocks
utilizing noncovalently programmed recognition sites
offers a biological solution to a synthetic problem. The
programmability of DNA, in particular, makes it an
exceptionally useful tool in assembling organic and
inorganic modules into predefined orientations with
respect to each other.2 The groups of Mirkin,3 Schultz,4
Tour,5 Rubin,6 Ihara,7 and Bergstrom8 have successfully
employed the concept of utilizing DNA in this manner.
A commonly overlooked, and limiting, aspect of DNA
nano-architectural design is the covalent attachment of
DNA to an organic or inorganic module. Both Mirkin3
and Schultz4 use a thiol linker to attach oligonucleotides
to gold nanoparticlessa highly effective postsynthetic
method for gold. A more desirable method would be one
in which the organic moiety of interest is directly fed
into the oligonucleotide synthesizer, making it an
(9) (a) Barton, J . K.; Kumar, C. V.; Turro, N. J . J . Am. Chem. Soc.
1986, 108, 6391. (b) Hurley, D. J .; Tor, Y. J . Am. Chem. Soc. 1998,
120, 2194. (c) Telser, J . Cruickshank, K. A.; Schanze, K. S.; Netzel, T.
L. J . Am. Chem. Soc. 1989, 111, 7221. (d) Telser, J .; Cruickshank, K.
A.; Morrison, L. E.; Netzel, T. L.; Chan, C.-L. J . Am. Chem. Soc. 1989,
111, 7226. (e) Bannwarth, W.; Schmidt, D. Tetrahedron Lett. 1989,
30, 1513. (f) Manchanda, R.; Dunham, S. U.; Lippard, S. J . J . Am.
Chem. Soc. 1996, 118, 5144. (g) Chen, C.-H. B.; Sigman, D. S. J . Am.
Chem. Soc. 1988, 110, 6570. (h) Modak, A. S.; Gard, J . K.; Merriman,
M. C.; Winkeler, K. A.; Bashkin, J . K.; Stern, M. K. J . Am. Chem. Soc.
1991, 113, 283. (i) Magda, D.; Miller, R. A.; Sessler, J . L.; Iverson, B.
L. J . Am. Chem. Soc. 1994, 116, 7439.
(10) (a) Khan, S. I.; Grinstaff, M. W. J . Am. Chem. Soc. 1999, 121,
4704. (b) Wiederholt, K.; McLaughlin, L. W. Nucleic Acids Res. 1999,
27, 2487.
(11) (a) Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278. (b)
Caruthers, M. H. Science 1985, 230, 281.
* To whom correspondence should be addressed. E-mail: bunz@
psc.sc.edu.
† Current address: Department of Chemistry, Rice University,
Houston, TX.
(1) See the J uly 1999 issue of Chem. Rev.
(2) For reviews see: (a) Storhoff, J . J .; Mirkin, C. A. Chem. Rev.
1999, 99, 1848. (b) Niemeyer, C. M. Angew. Chem., Int. Ed. Engl. 1997,
36, 585.
(3) (a) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J . J .
Nature 1996, 382, 607. (b) Elghanian, R.; Storhoff, J . J .; Mucic, R. C.;
Letsinger, R. L, Mirkin, C. A. Science 1997, 277, 1078. (c) Mucic, R.
C.; Storhoff, J . J .; Mirkin, C. A.; Letsinger, R. L. J . Am. Chem. Soc.
1998, 120, 12674. (d) Storhoff, J . J .; Elghanian, R.; Mucic, R. C.; Mirkin,
C. A.; Letsinger, R. L. J . Am. Chem. Soc. 1998, 120, 1959.
(4) Alivisatos, A. P.; J ohnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth,
C. J .; Bruchez, M. P., J r.; Schultz, P. G. Nature 1996, 382, 609.
(5) Cassell, A. M.; Scrivens, W. A.; Tour, J . M. Angew. Chem., Int.
Ed. 1998, 37, 1528.
(12) (a) Coleman, R. S.; Dong, Y.; Arthur, J . C. Tetrahedron Lett.
1993, 34, 6867. (b) Coleman, R. S.; Kesicki, E. A. J . Am. Chem. Soc.
1994, 116, 11636. (c) Coleman, R. S.; Pires, R. M. Nucleic Acids Res.
1997, 25, 4771. (d) Coleman, R. S.; Arthur, J . C.; McCary, J . L.
Tetrahedron 1997, 53, 11191.
(13) Kool, E. T.; Breslow, R. Angew. Chem., Int. Ed. Engl. 1992, 31,
1617.
(14) (a) Thuong, N. T.; Chassignol, M. Tetrahedron Lett. 1988, 26,
5905. (b) Durand, M.; Chevrie, K.; Chassignol, M.; Thuong, N. T.;
Maurizot, J . C. Nucleic Acids Res. 1990, 18, 6353. (c) Giovannangeli,
C.; Montenay-Garestier, T.; Rougee, M.; Chassignol, M.; Thuong, N.
T.; Helene, C. J . Am. Chem. Soc. 1992, 113, 7775.
(6) An, Y.-Z.; Chen, C.-H. B.; Anderson, J . L.; Sigman, D. S.; Foote,
C. S.; Rubin, Y. Tetrahedron 1996, 52, 5179.
(7) (a) Ihara, T.; Nakayama, M.; Murata, M.; Nakano, K.; Maeda,
M. J . Chem. Soc., Chem. Commun. 1997, 1609. (b) Ihara, T.; Maruo,
Y.; Takenaka, S.; Takagi, M. Nucleic Acids Res. 1996, 24, 4273.
(8) Shi, J .; Bergstrom, D. E. Angew. Chem., Int. Ed. Engl. 1997, 36,
111.
(15) (a) Manikrao, S.; Wu, T.; Letsinger, R. L. J . Am. Chem. Soc.
1992, 114, 8768. (b) Letsinger, R. L.; Wu, T. J . Am. Chem. Soc. 1994,
116, 811. (c) Letsinger, R. L.; Wu, T. J . Am. Chem. Soc. 1995, 117,
7323. (d) Lewis, F. D.; Helvoigt, S. A.; Letsinger, R. L. J . Chem. Soc.,
Chem. Commun. 1999, 327.
10.1021/om9909791 CCC: $19.00 © 2000 American Chemical Society
Publication on Web 01/21/2000

Communications
Organometallics, Vol. 19, No. 4, 2000 369
Sch em e 1a
a
Legend: (a) PdCl2(PPh3)2/CuI/PPh3/NEt3; (b) CpCo(CO)2/p-xylene; (c) DMTCl/pyr; (d) 2-cyanoethyl diisopropylchlorophos-
phoramidite/diisopropylethylamine/THF.
tion,18 and the isolated monoprotected product (8) was
activated with 2-cyanoethyl diisopropylchlorophosphor-
amidite.11,18 The target molecule 2 was isolated and
stored desiccated in the freezer.
The cyclobutadiene complex 2 was attached to the 5′-
end of an oligonucleotide with a palindromic sequence
of 5′ GTAGCGCTAC 3′. The sequence was synthesized
on an automated oligonucleotide synthesizer and al-
lowed to begin a cycle for the addition of another base.
F igu r e 1. Schematic illustration of a duplex oligonucleo-
Following the activation step, a THF solution of the
cyclobutadiene complex was syringed through the col-
umn containing the growing oligonucleotide. After a 2
min addition, the column was placed back online to
complete the capping and oxidation steps before workup.
Additionally, an oligonucleotide of the same sequence
was synthesized without an organic for use as a control.
tide-modified organic (OMO).
construct a wide variety of oligonucleotide-modified
organic (OMO) molecules with tailored oligonucleotide
sequences as well as tailored organic moieties for the
construction of novel architectures applicable in materi-
als chemistry.
Purification of the cyclobutadiene OMO by HPLC
allowed the isolation of two components. The UV-vis
spectrum of the less retained component was charac-
teristic of DNA with a broad band at 260 nm, while the
UV-vis spectrum of the second component consisted of
the broad 260 nm band with a shoulder at 310 nm,
suggesting that this was the fraction containing the
cyclobutadiene OMO. Figure 2 depicts the spectrum of
the cyclobutadiene OMO compared with the spectra of
the oligonucleotide control and the free cyclobutadiene
complex (6).19 Comparing the control spectrum with the
cyclobutadiene spectrum, one can easily see that the
additional shoulder at 310 nm must be due to the
cyclobutadiene complex, as this feature is present in the
spectrum of 6. Electrospray MS of the cyclobutadiene
OMO provided further evidence for covalent attach-
ment. A single component of m/z 3657 was observed for
the cyclobutadiene OMO, where as the control had a
single component of m/z 3027sa difference of 640 Da,
which can be accounted for by the cyclobutadiene
As a first experiment, we desired to construct a dimer
hybrid (1; Figure 1) of an OMO with a palindromic
recognition sequence where the sphere can be any
organic/organometallic molecule. In this specific case a
(tetraphenylcyclobutadiene)cyclopentadienylcobalt com-
plex was chosen. Thus, complex 2 was the target
molecule. It contains a DMT protecting group and a
phosphoramidite activating group, making it conducive
for automated oligonucleotide synthesis. Preparation of
the complex (Scheme 1) began with the Pd/Cu coupling16
of phenylacetylene (3) with 4-bromophenethyl alcohol
(4). Cyclization of the resulting substituted tolane (5)
with cyclopentadienylcobalt dicarbonyl (CpCo(CO)2)
gave a mixture of isomers designated ortho and para
with regard to substitution around the cyclobutadiene
ring. These isomers were separable by chromatogra-
phy.17 The para isomer was subjected to DMT protec-
(16) Zhang, Y.; Schuster, G. B. J . Org. Chem. 1997, 59, 11855.
(17) Conclusive ortho/para isomer assignments await X-ray crystal-
lography data. However, given that the ortho isomer should have a
greater dipole moment than the para isomer, the faster running spot
on TLC was assigned the para isomer. This was confirmed with 13C
NMR; the spectra of the DMT-protected/phosphoramidite-activated
forms of each isomer were compared. There were four cyclobutadiene
peaks distinguishable for the ortho isomer and only three for the para
isomer (C2 symmetry).
(18) Oligonucleotides and Analogues: A Practical Approach; Eck-
stein, F., Ed.; IRL Press: Oxford Press: New York, 1991.
(19) Note that the units of the y axis are not directly comparable,
since the oligonucleotide spectra were acquired in aqueous solvent
while the spectrum of 6 was taken in acetonitrile.

370 Organometallics, Vol. 19, No. 4, 2000
Communications
F igu r e 2. UV-vis spectra of DNA (A), cyclobutadiene
OMO (B), and cyclobutadiene complex (C). The shaded area
indicates the feature of the cyclobutadiene complex absent
in the DNA but present in the cyclobutadiene OMO. The
dotted baselines are added for clarity.
F igu r e 3. A 12% native polyacrylamide gel run at 600 V
for 3 h in 1X TBE buffer stained with ethidium bromide
(lanes 1 and 2) DNA control; (lane 3) cyclobutadiene OMO;
(lane 4) standard 25 bp PCR ladder (sizes indicated); (lane
5) standard pBR322 HAE III digest.
moiety. If the cyclobutadiene complex were nonco-
valently associated with the oligonucleotide, one would
expect to see a molecular ion for just the oligonucleotide,
which is not the case.
cyclobutadiene complex influences thus the size and
shape of the OMO.
This synthetic approach can be applied to any oligo-
nucleotide sequence and any organic or organometallic
module. The only prerequisite is the presence of the diol
moiety and sufficient solubility of the DMT-protected/
phosphoramidite-activated module in THF. The addition
of the module to the oligonucleotide is not limited to the
end of the sequence but, rather, can be incorporated site-
specifically via the deprotection of the DMT group and
continuation of sequence. Any available building block
of interest for complex nanoscale architectures can be
incorporated into DNA strands with minimum effort
and high reliability. Soon we will report upon self-
assembled nanostructures utilizing this concept.
The cyclobutadiene OMO was annealed to form a
double-stranded DNA hybrid, as shown in Figure 1. The
double-stranded OMO (1) was characterized by melting
temperature (Tm) and gel electrophoresis. The presence
of the cyclobutadiene complex on the 5′-end of the DNA
may influence duplex formation; thus, a melting-tem-
perature study of the OMO and the DNA was performed
from 20 to 80 °C. The melting temperatures as well as
the shape of the curves were similar for each sample,
demonstrating that the cyclobutadiene complex neither
contributes to nor prevents the duplex formation of the
double-stranded OMO.
A native polyacrylamide gel electrophoresis experi-
ment was performed to compare the structure of the
double-stranded OMO (1) with that of the duplex control
(Figure 3). The nondenaturing gel demonstrated that
the mobility of the OMO was less than that of the
identically sized fragment without the complex at-
tached. The mobility through the gel is a function of size,
shape, and charge on the molecule. Thus, when the size
of the OMO is compared with that of the unmodified
DNA and the size markers, the OMO is slightly larger
than the unmodified DNA. The covalently attached
Ack n ow led gm en t. We thank the NSF-EPSCoR and
USC start-up funds for funding.
Su p p or tin g In for m a tion Ava ila ble: Text giving experi-
mental details and spectroscopic data for 2-8, electrospray
MS data, annealing details, melting curves, and PAGE details
for dsOMO and dsDNA. This material is available free of
charge via the Internet at http://pubs.acs.org.
OM9909791