Thermostable and monoconjugable gold clusters with a dodecadentate thioether ligand gripper

Article abstract of DOI:10.1002/anie.200502370

(Figure Presented) Get a grip: A tailor-made ligand bearing 12 thioether groups can act as a "gripper" for the Au₅₅ cluster to give thermally stable, monofunctionalized labels for biomolecules (see scheme). Conjugates of the gold cluster with D

Full text of DOI:10.1002/anie.200502370

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nanoscale thioether-based “gripper”. This method allows
nanoparticles to be singly functionalized without the other-
wise necessary step of purification of mixtures by HPLC.[25]
We demonstrate that “gripped” clusters in oligonucleotide
conjugates can survive the temperature conditions of poly-
merase chain reaction (PCR) and hybridization experiments.
The concept behind our ligand is sketched in Figure 1. The
Au55 cluster was proposed to
possess a cuboctahedral core,
the surface of which is com-
posed of eight corner-sharing
triangles (111) and six squares
(110).[1] Although there is an
ongoing debate[26,27] about the
cluster geometry, we decided to
adopt this proposal as a work-
inghypothesis. Au 55 is expected
Figure 1. General concept of
a gold cluster gripper. Four
tripodal units (1,3,5-tris(thio-
methyl)benzene groups)
DNA Nanotechnology
DOI: 10.1002/anie.200502370
to bind four tripodal ligands
provided that the binding
mode of the ligands is compa-
rable to that of the Schmid
cluster, in which the 12 triphe-
nylphosphanes most likely
occupy the corners of the
cuboctahedron. If the four tri-
podal ligands are connected by
occupy four (111) faces of the
cuboctahedral cluster. The tri-
podal units are linked (bold
lines) to give a dodecadentate
ligand, which also bears a
functional group (FG) suita-
ble for conjugation to a bio-
molecule.
Thermostable and Monoconjugable Gold Clusters
with a Dodecadentate Thioether Ligand
Gripper**
Wolf Matthias Pankau, Sven Mönninghoff, and
Günter von Kiedrowski*
suitable linkers, a dodecadentate ligand will result. The
synthesis was also designed so that the final material carries
a single functional group to allow conjugation to (bio)mol-
ecules.
The Schmid cluster,[1] [Au55(PPh3)12Cl6], has stimulated many
different areas of technology ranging from catalysis research[2]
to the concept of quantum electronics.[3,4] Monofunctional-
ized water-soluble derivatives of this cluster[5] have become
commercially available and found numerous applications.[6–14]
Recently, gold clusters have been employed as universal
fluorescence quenchers in molecular beacons[15] and as nano-
scale antennas for the reception of radio radiation in the GHz
frequency region, which causes local and selective inductive
heatingof cluster-labeled biomolecules. [16] These applications
as well as current experiments in DNA nanotechnology[17–23]
point to the need for the increased thermostability of these
gold clusters. We have shown that replacement of the
monopodal triphenylphosphane ligands in such clusters with
water-soluble tripodal thioethers based on 1,3,5-tris(thiome-
thyl)benzene leads to gold clusters with improved stability.[24]
Herein, we report a new generation of biocompatible gold
clusters that are surrounded by a single dodecadentate,
Scheme 1 shows the synthesis of the target ligand 8. We
selected a monomaleimido group as the monoconjugable unit
to ensure functional comparability with the commercially
available variant of the Schmid cluster. Thioether 2 was
generated from trisbromide 1 as a key precursor that allowed
the synthesis of 8 by a divergent–convergent route in only six
steps with an overall yield of 31%. The gold cluster was
synthesized from ligand 8 by phase-transfer synthesis, as
previously described for the tristhioether derived from l-
cysteine.[24] High-resolution transmission electron microscopy
revealed a narrow size distribution of the clusters with a mean
diameter of 1.4 nm, which is as expected for Au55 particles.
The cluster was conjugated with a 5’-thiol-modified 27-mer
oligonucleotide, the complement of which was labeled with
fluorescein in the 3’ position.
To study the thermostability of the label, a thermal-stress
experiment was performed in which the fluorescence signal of
a mixture of both oligonucleotides was monitored during 100
cycles of temperature variation. Each cycle consisted of a
period of heatingfrom 20 to 95 8C at 10 KminÀ1, a thermal-
stress period for 6 min at 958C, and a period of coolingat a
rate of 20 KminÀ1, which corresponds to an average temper-
ature of 70.58C. The process of the temperature cycling
experiment is depicted in Figure 2. At low temperatures, the
duplex holds the gold cluster and the fluorescent dye within
spatial proximity. Quenchingof fluorescence takes place,
which results in low fluorescence intensity. At high temper-
atures, the fluorescent oligonucleotide exists in its single-
strand form, and an increase in fluorescence signal is observed
[*] Dr. W. M. Pankau, S. Mönninghoff, Prof. Dr. G. von Kiedrowski
Lehrstuhl für Organische Chemie I
Bioorganische Chemie
Ruhr-Universität Bochum
Universitätsstrasse 150, NC 2/173, 44780 Bochum (Germany)
Fax: (+49)234-32-14355
E-mail: kiedro@ruhr-uni-bochum.de
[**] This work has been supported by the Deutsche Forschungsge-
meinschaft and the BMBF. We thank M. Wüstefeld for carrying out
the oligonucleotide syntheses, G. Schmid for a sample of
[Au55(PPh3)12Cl6], and Degussa AG for the generous donation of
tetrachloroauric acid.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2006, 45, 1889 –1891
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Communications
because the gold cluster is no longer in proximity to act as a
quencher. If the gold cluster escapes the grip of the ligand
duringa cycle, or if the conjugation with the complementary
strand breaks, no fluorescence quenchingwill be observed in
the followingcycle. Thus, kinetic information about the
stability of clusters and their oligonucleotide conjugation can
be derived from the increase in low-temperature fluorescence
as a function of the cycle number (and thus duration of
thermal stress). Figure 3 depicts the development of the
Figure 3. Fluorescence intensity (I) during 100 temperature cycles of a
solution containing 1 mm 5’-ATGCACCCATTGGACATAACCGGGAAT-3’-
FAM, 3 mm 5’-SH-modified complementary oligonucleotide conjugated
with a mixture of gold-filled and unfilled grippers 8, 300 mm NaCl, and
100 mm 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.5.
temperature-driven fluorescence oscillations as a function of
time. Fittingof the fluorescence minima to a model involving
first-order decomposition of the cluster yielded a half-life of
t1/2 = 55 Æ 5 cycles, which corresponds to 900 min for an
average temperature of T= 70.58C. The increase of the
maxima of fluorescence oscillations in Figure 3 is due to a
slight evaporation of water from the cuvette, which could not
be avoided completely at the high temperatures employed.
Control experiments (see the SupportingInformation)
allow us to draw the followingconclusions: There is evidence
Scheme 1. Structure and synthesis of the dodecadentate ligand 8
bearing a monomaleimido functional group. Boc=tert-butyloxycar-
bonyl; DBU=1,8-diazabicyclo[5.4.0]-7-undecene;
EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
TFA=trifluoroacetic acid; NHS=N-hydroxysuccinimide.
Figure 2. Chemical and physical processes underlying the determination of the cluster stability in the thermal-stress experiment. The fluorescence
of the 3’-fluorescein labeled oligonucleotide in the presence of the complementary 5’-gold cluster is measured during the heating/cooling cycles.
FAM=fluoroscein amino modifier.
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Chemie
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is as expected in view of the UV-absorption properties of the
ligand. Moreover, the fluorescence of both the duplex without
the gripper and the labeled single strand decreases with the
temperature; the latter, however, shows a higher level of
fluorescence intensity, which is attributable to the poorer
stackingof the 3 ’-fluorescein with the single strand. The
different baseline courses for the single- and double-stranded
species also explain why a slight increase in fluorescence is
observed at the meltingtransition for the duplex with the
empty gripper.
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Any conceivable application of gold clusters in DNA bio-
and nanotechnology in which their remarkable properties are
employed will require compatibility of the label with at least
the basic procedures of molecular biology. To the best of our
knowledge, the gripped cluster presented here is the first
example of an Au55 monolabel survivingthe temperature
conditions of PCR and hybridization protocols. A typical
PCR experiment may be equivalent to around 100 min of the
heatingexperiments described above, consideringthat the
exposure to a temperature of 958C lasts for only around 1 min
in PCR whereas each of these heatingcycles lasted 6 min at
958C. The average temperature in our experiment is close to
that of PCR, so that one cycle in our experiment may be
equivalent to four to six typical PCR cycles. It is evident from
Figure 3 that only a small fraction (less than 10%) of the gold
nanocrystals did not survive the first 100 min of treatment. It
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Received: July 7, 2005
Revised: December 12, 2005
Published online: February 22, 2006
Keywords: cluster compounds · DNA · fluorescence · gold ·
.
nanotechnology
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