Ru(II)-carbohydrate dendrimers as photoinduced electron transfer lectin biosensors

Article abstract of DOI:10.1039/b814146k

Three tris(bipyridine)ruthenium dendrimers bearing either six or eighteen mannose units have been synthesized; encapsulation of the Ru(bipy)₃ core alters the rate of energy and electron transfer as well as the lectin biosensing abilities. The R

Full text of DOI:10.1039/b814146k

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Ru(II)–carbohydrate dendrimers as photoinduced electron transfer lectin
biosensorsw
a
b
a
´
´
Raghavendra Kikkeri, Ines Garcıa-Rubio and Peter H. Seeberger*
Received (in Cambridge, UK) 14th August 2008, Accepted 13th October 2008
First published as an Advance Article on the web 26th November 2008
DOI: 10.1039/b814146k
Three tris(bipyridine)ruthenium dendrimers bearing either six or
eighteen mannose units have been synthesized; encapsulation of
the Ru(bipy)3 core alters the rate of energy and electron transfer
as well as the lectin biosensing abilities.
Significant effort has been directed at utilizing the unique
combination of optical and electrochemical properties of
ruthenium tris(bipyridine) in various materials and biological
applications.1 Photo-excitation of ruthenium tris(bipyridine)
results in electron and/or energy transfer processes. These
events have been exploited to generate biosensors, establish
artificial photosynthesis systems and to induce the generation
of free radicals to photoxidize DNA, RNA and cell surfaces.2
In order to target specific biological events, ruthenium com-
plexes need to be equipped with recognition molecules. One
possible strategy involves the fusion of ruthenium(II) com-
plexes with a carbohydrate to target specific cells and
tissues via lectin–carbohydrate interactions. Lectins are
carbohydrate-binding proteins that mediate important bio-
logical processes such as cell growth, the inflammatory re-
sponse and viral infections.3 Since monosaccharide–lectin
binding affinities are usually quite weak, carbohydrate den-
drimers4 that increase the avidity and surround the ruthenium(II)
core have been explored. Homogenous branching of the
dendrimers produces a microenvironment that encapsulates
the ruthenium(II) core and modifies its photochemical and
electrochemical properties.1,5 Recently, the way in which
dendrimers encapsulate the core and alter its properties has
been studied in detail. Electron transfer on ferrocenyl redox
cores was shown to depend on the molecular orientation.6
Similar results were observed with divalent viologen and
Fig. 1 Structures of Ru(II)-complexes 1, 2 and 3 and BBV.
application, we present the study of the electron transfer mecha-
nism of these dendrimers used to detect lectins. We demonstrate
that increasing the dendrimer density around the Ru(bipy)3 unit
does indeed influence lectin sensing by photoinduced electron
transfer (PET) between the ruthenium(II) core and BBV mole-
cules and, in general, we show that covalent attachment of
carbohydrates to ruthenium tris(bipyridyl) based dendrimers
paves the way to new molecules capable of sensing lectins with
very low detection limits.
8
Ru(bipy)3 complexes,7 as well as tetravalent Fe4S4 and
porphyrin core redox units.9 However, a systematic investiga-
tion of the core activities for different carbohydrate densities
during biosensing processes has not been reported.
Mannose-capped dendrimers 1–3 (Scheme 1) were prepared
from bipyridine sugar derivatives 6 and 13 and reaction with
RuCl3 or cis-Ru(bipy)2Cl2 respectively. Deprotection of
the tert-butoxycarbonylamino group of 5 and coupling to
bipyridine 4,40-dicarboxylic acyl chloride yielded bipyridine
derivative 6. Refluxing 6 with cis-Ru(bipy)2Cl2 and RuCl3 in
ethanol followed by deacetylation yielded dendrimers 1 and 2
respectively. Reaction of tri-mannose substituted 5 with tripod
active ester 4 furnished 12. Then, 12 was reacted with
bipyridine 4,40-dicarboxylic acyl chloride, followed by
complexation with cis-Ru(bipy)2Cl2 and deacetylation to
obtain dendrimer 3.
Here, we report on investigations concerning the rate of
electron and energy transfer from the Ru(II)–carbohydrate den-
drimers to N,N0-4,40-bis(benzyl-3-boronic acid)-bipyridinium
dibromide (BBV, Fig. 1) and tetramethyl piperidine (TEMP),
respectively. Additionally, as an example of
a potential
a Laboratory for Organic Chemistry, Swiss Federal Institute of
Technology (ETH) Zurich, Wolfgang-Pauli-Str., 10, 8093 Zurich,
Switzerland. E-mail: seeberger@org.chem.ethz.ch;
Fax: +41 44 633 21 03; Tel: +41 44 633 12 35
b Physical Chemistry Laboratory, Swiss Federal Institute of
Technology (ETH) Zurich, Wolfgang-Pauli-Str., 10, 8093 Zurich,
Switzerland
The rate of electron transfer in complexes 1–3 was investi-
gated using PET between photo-excited Ru(II)-templates and a
quencher. Several quenchers were reported for the MLCT
(metal–ligand charge transfer) excited state of the [Ru(bpy)3]2+
w Electronic supplementary information (ESI) available: Full experi-
mental details for synthesis of metal–dendrimers and turbidity studies.
See DOI: 10.1039/b814146k
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Chem. Commun., 2009, 235–237 | 235

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Scheme 1 Synthesis of Ru(II)-dendrimers 1, 2 and 3. (a) Triethylamine
(TEA)/DCM/8; (b) 4, TEA, DCM; (c) TFA, 2,2’-bipyridine
4,4’-dicarboxylic acyl chloride, DCM, TEA; (d) RuCl3, EtOH;
(e) cis-Ru(bipy)2Cl2, EtOH; (f) NaOMe, MeOH.
Fig. 2 Kinetic profile of singlet oxygen formation upon irradiation of
complexes 1 (’), 2 (~), 3 (J) and Ru(bipy)3 (n).
RuðIIÞ ! RuðIIÞꢁ
model.5 Among them, methyl viologen dication (MV2+) shows a
high quenching constant.9 Moreover, MV2+ does not possess
excited states below the MLCT level of the [Ru(bipy)3]2+
chromophoric unit, but is readily reduced. As a consequence,
quenching can only take place by electron transfer. Since the
boronic acid derivative of MV2+ (BBV) has a high affinity for
sugar, BBV has been used extensively in the development of sugar
sensors.10 All compounds exhibited different quenching constants
(kq), that were obtained with the Stern–Volmer equation,
energy transfer
ꢂ!
ꢁ
3
1
ð2Þ
RuðIIÞ þ O2
RuðIIÞ þ O2
1O2 þ TEMP ! TEMPO
Continuous irradiation of the Ru-complexes (2 mM) in the
presence of TEMP (100 mM) yielded a nitroxide triplet in the
EPR spectrum (see ESIw) typical for the TEMPO radical. The rate
of appearance of the TEMPO signal decreases from 1 to 3, in
support of the notion that carbohydrate encapsulation of the
Ru(II)-template stops effective energy transfer to dissolved oxygen.
After gaining insight into how the energy and electron transfer
processes are affected by the dendrimer structure, we studied how
selective and sentitive these processes can be by illustrating the
capabilities of dendrimers 1–3 as lectin biosensors. Concanavalin
A (ConA) and galanthus nivilis agglutinin (GNA) that recognize
mannose were selected as lectins.11 Both, ConA and GNA are
tetramers that contain one and three mannose binding sites per
subunit respectively. The sensors are based on the specific and
strong binding between mannose-dendrimers and the lectins
(Fig. 3). This tight interaction segregates the quencher (BBV)
from the Ru(II)-complex, thus reconstituting the fluorescent
signal (Fig. 4).
to/t = Io/I = 1 + kqto[Q]
(1)
where, to and Io are the excited-state lifetime and quantum
yield in the absence of quencher, and t and I are the same
quantities measured in the presence of a certain concentration
of quencher [Q]. The experimental values of I0, to and kq of
complexes 1, 2 and 3, together with the standard Ru(bipy)3 are
given in Table 1. In the absence of quencher, there is a
difference in the quantum yield between the complexes with
six (1) and eighteen mannose units (2 and 3) that must be
related to the different density of dendritic encapsulation.
Moreover, the difference in carbohydrate densities has a
dramatic effect on the quenching constant, which decreases
from complex 1 to 3 by almost an order of magnitude. These
results indicate that a high degree of carbohydrate density
around the ruthenium core allows for efficient encapsulation
and modification of the core properties.
To evaluate the rate of energy transfer by 1–3, we studied
the formation of molecular oxygen in the singlet state upon
photoexcitation of the ruthenium tris(bipyridine) complexes
(Fig. 2). Tetramethyl piperidine (TEMP) was used as a trap for
singlet oxygen to form a stable species (TEMPO) easily
detected by EPR (see eqn (2)).2
Table 1 Photophysical data of complexes 1–3. Quantum yield and
lifetime were measured by excited complex 1–3 at lmax = 450 nm and
emission at lmax = 645 nm
Compound
lmax/nm
kq/Mꢂ1 sꢂ1
to/ms
Io
1
2
645
648
648
613
9.8 ꢃ 108
1.8 ꢃ 108
1.1 ꢃ 108
2.5 ꢃ 109
0.61
1.31
1.26
0.54
0.072
0.102
0.112
0.062
3
Ru(bipy)3
Fig. 3 Schematic representation of the Ru(II)–carbohydrate dendrimer
lectin sensor.
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results indicate that PET biosensing is more effective with the
small dendrimer 1. Dendrimers 2 and 3 are sensitive bio-
markers to study lectin–carbohydrate interactions, owing
to their high quantum yield and high carbohydrate density.
The application of 2 and 3 to optical biosensing on
microarrays and for in vitro imaging is currently under
investigation.
We thank the ETH Zurich for financial support.
¨
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Fig. 4 Rate of fluorescence gain from Ru(II)–carbohydrate–BBV
upon addition of lectin; 1 (n), 2 ( ) and 3 (’) at lmax = 645 nm.
}
Using a donor/acceptor mixture of complex 1 and BBV, a
spontaneous gain in fluorescence upon the addition of 75 nM of
ConA and a further slow increase in the signal at 200–1000 nM
was observed. In contrast, for 0 to 100 nM of ConA, complexes
2 and 3 displayed much more modest gains in fluorescence
compared to complex 1 but a steady and linear increment upon
the addition of 100–600 nM. Similar experiments with the
higher valency lectin GNA were performed (see ESIw). The
detection limits for the Ru-complexes were calculated based on
these results (Table 2) and we found that complex 1 is notice-
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higher detection limit whereas the range of linear response is
broader. Complex 1 has the best compromise between encap-
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sensing. While complexes 2 and 3 bind much more strongly
to lectins than complex 1, the effective encapsulation of the
Ru(II)-core by the carbohydrate dendrimer results in low
quenching and weak PET, and thus is a less sensitive biosensor.
In conclusion, we have synthesized three new carbohydrate
dendrimers with a Ru(bipy)3 core unit. The electron and
energy transfer rates of the photoexcited state were established
using the quencher BBV and monitoring the formation of
singlet oxygen. The behavior of complexes 2 and 3 was typical
of an encapsulated Ru(bipy)3 core unit with one order of
magnitude decrease in the quenching rate compared to the
Ru(bipy)3 complex. The quenching constant decreases with
increasing number and size of the dendritic branches. Similar
results were also observed for the energy transfer process. The
influence in the rate of photoinduced electron transfer was also
visible in the lectin sensing process. Complex 1 showed more
sensitive detection compared to complexes 2 and 3. These
Table 2 Detection limit of lectins using different Ru–mannose
dendrimers
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Compound
ConA/nM
GNA/nM
1
2
3
28 ꢄ 3
340 ꢄ 12
347 ꢄ 14
25 ꢄ 4
328 ꢄ 9
331 ꢄ 12
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Chem. Commun., 2009, 235–237 | 237