Glycoconjugated porphyrin dimers as robust ratiometric temperature sensors

Article abstract of DOI:10.1039/c4cc03367a

We report the properties of glycoconjugated porphyrin dimers behaving as highly sensitive ratiometric temperature sensors in water. This effect results from interactions between carbohydrate and water altering molecular relaxation kinetics leading to temperature sensitive dual emission. These dimers are robust ratiometric fluorescent probes over a large temperature window (20-90 °C). This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc03367a

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Glycoconjugated porphyrin dimers as robust
ratiometric temperature sensors†
Cite this: Chem. Commun., 2014,
50, 9529
Fabien Hammerer,*a Guillaume Garcia,b Pauline Charles,b Aude Sourdon,b
Sylvain Achelle,c Marie-Paule Teulade-Fichoub and Philippe Maillardb
Received 5th May 2014,
Accepted 29th June 2014
DOI: 10.1039/c4cc03367a
www.rsc.org/chemcomm
We report the properties of glycoconjugated porphyrin dimers classical methods cannot be used. The design and development of
behaving as highly sensitive ratiometric temperature sensors in molecular thermometers at the nanometric scale have received
water. This effect results from interactions between carbohydrate increasing attention.9 Several fluorescent nanomaterials, such as
and water altering molecular relaxation kinetics leading to tempera- semiconductor quantum dots (QDs),10 gold nanoclusters,11 rare
ture sensitive dual emission. These dimers are robust ratiometric earth-doped nanoparticles,12,13 polymer-based nanogels14 and
fluorescent probes over a large temperature window (20–90 8C).
temperature dye indicators such as rhodamine B,15 have already
shown great potential for nanothermometry in biological systems.
Over the last few years, the development of fluorescent sensors Thermally sensitive fluorescent indicators for molecular thermo-
has been the subject of significant interest. Fluorescence allows metry have been proposed to monitor temperature changes in
easy, sensitive and non-invasive detection of a specific substance tumor cells which have a perturbed metabolism implying lower or
(metal ions, pollutants, and biomolecules)1 as well as measure- higher physiological temperatures than normal ones.16
ment of physical parameters like pH,2 viscosity3 or electric field.4
For this purpose, some devices have been developed among
A wide range of mechanisms have been discovered that allow which polymer based sensors have shown superior properties
the detection of specific stimuli both at molecular5 and supra- in terms of precision17 or photobleaching.18 However the
molecular levels.6 Among the local quantities that can be transition involved in temperature sensing often takes place
monitored by fluorescence sensing is temperature. Some of the within a few degrees thereby limiting their performance.19–22 In
reactions taking place in living cells are exothermic, among which this particular aspect, molecular approaches seem to be more
adenosine triphosphate (ATP) hydrolysis is the most frequent.7 promising with ‘‘slower’’ transitions (spin transition, ‘‘Twisted
This reaction leads to a slightly higher temperature in mitochon- Intramolecular Charge Transfer’’ or intramolecular rotation)
dria compared to the other organelles. In addition, cancer cells allowing accuracy over a larger temperature range.23,24
are known to have a significantly higher temperature than healthy
Conjugated porphyrin dimers were first reported by Anderson
ones.8 These observations contribute to make temperature mon- et al. because of their superior two-photon absorbing properties
itoring a strategic stake in biological media and biocompatible and later allowed strategic breakthroughs in the development
fluorescent probes could prove useful in several domains where of two-photon activated Photodynamic Therapy (2PA-PDT), a
promising technique in oncology.25–29 These authors showed that
the fluorescence emission and singlet oxygen production were
both controlled by the intramolecular rotation of the dimers30,31
and that both could be modulated by the viscosity of the media,
opening the way to the imaging of micro-viscosity in cells.32
The rotation of the dimers along the butadiynyl axis leads to
a dual fluorescence of the plane conformation (l 4 700 nm)
and of the twisted one (around l = 650 nm) and the ratio
a Laboratoire de Chimie Bioorganique et Bioinorganique, Institut de Chimie
´
´
ˆ
Moleculaire et des Materiaux d’Orsay, Bat 410-420, Centre Universitaire,
F-91405 Orsay cedex, France. E-mail: fabien.hammerer@gmail.com;
Fax: +33 1 69 15 72 81
b
ˆ
CSVB-UMR176, Institut Curie, Bat 110-112, Centre Universitaire,
F-91405 Orsay cedex, France
c UMR CNRS 6226, IUT de Lannion, Institut des Sciences Chimiques de Rennes,
rue Edouard Branly, BP 30219, F-22302 Lannion cedex, France
† Electronic supplementary information (ESI) available: All methods and pro- between the two intensities indicates the local viscosity. Such
ducts used for the synthesis of compound 1. UV/Vis spectra were recorded using
probes, called ratiometric, avoid the dependence of the signal
on the local concentration and are therefore of great interest.
an Agilent Cary 300 spectrophotometer. Fluorescence spectra were recorded by
using a Cary Eclipse spectrophotometer. Both apparatus were equipped with a
Inspired by Anderson’s approach of 2PA-PDT sensitizers, we
Cary temperature controller. For every measurement, temperature was checked
developed a family of glycoconjugated porphyrin dimers (Fig. 1)
optimized for the targeted 2PA-PDT treatment of retinoblastoma
using a thermocouple. All curves were smoothed following the Savitzky–Golay
method (5 points). See DOI: 10.1039/c4cc03367a
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Fig. 2 Emission spectra of compounds 1–5 in water at 20 1C (1 mM,
lexc = 450 nm).
Dual emission was also observed in other polar solvents
such as MeOH, EtOH or DMF (see ESI,† S9) though to a lesser
extent for compounds 2–5 whilst emission of compound 1
displays a single band in all tested solvents. According to
Anderson’s interpretation, the glycosyl moieties alter the relaxa-
tion kinetics of the dimers, allowing emission from the twisted
excited state in all the tested polar solvents.
If viscosity was the driving parameter, an increase in tem-
perature should ease the transition rate and lead to the
decrease in the intensity of the blue band. This is the case in
polar organic solvents (see ESI,† S9). However, the fluorescence
emission of dimer 2 in water between 20 and 90 1C (shown in
Fig. 3) follows the opposite trend: high temperature favors
emission from the twisted state.
Under the same conditions, the absorption underwent weak
modifications in the Soret area. This suggests that the observed
phenomenon is mainly due to the evolution of the populations
in the excited state rather than in the ground state. Following
previous work on dimer analogs by Winters et al.,31 an inter-
pretation of this phenomenon is presented in Fig. 4. Excitation
at 450 nm leads to the promotion in the S2 states of both
Fig. 1 Structures of compounds 1–5.
through specific interactions between carbohydrates and specific
lectins overexpressed at retinoblastoma cell membranes.33–35
The glycosyl moieties also provide the aqueous solubility of the
otherwise highly aromatic and hydrophobic porphyrin dimers.
Despite a high two-photon cross section and high singlet oxygen
production in organic solvents, the dimers showed no photo-
toxicity toward cancer cells during in vitro assays due to poor cell
internalization.35
Water is known for being a highly structured solvent because
of the existence of numerous intermolecular H bonds.36 Carbo-
hydrates, thanks to their multiple hydroxyl functions, are excel-
lent H bond donors and acceptors, which allow them to insert
into the network created by water molecules resulting in their
excellent solubility. It has recently been shown that carbohydrate
molecules can influence neighbouring water molecules up to
twice the thickness of the solvatation layer.37,38 The dissolution
of dimers bearing six carbohydrate moieties will presumably
lead to a strong organization of water at their proximity. The
results described previously prompted us to investigate and use
these interactions.
This publication presents the influence of solvent and tem-
perature on the emission properties of the glycosylated dimers
2–5, previously designed as photosensitizers for one-photon and
two-photon excited photodynamic therapy,35 compared with the
properties of the non-reported non-glycosylated analog 1 bearing
six {2-[2-(2-methoxyethoxy)ethoxy]ethoxy}phenyl moieties as meso-
substituents (synthesis described in the ESI†). We now propose
these molecules as robust, ratiometric probes for temperature
sensing along with an interpretation of their peculiar properties.
While all dimers showed similar absorption properties in
water (see ESI,† S8), glycosyl groups had a strong impact on
fluorescence emission. At 20 1C, compound 1 (red) shows a
single emission band at l = 730 nm whereas the glycosylated
compounds 2–5 exhibit a dual emission peak at 630 and 740–
800 nm (Fig. 2), which will be referred to as blue and red
transitions respectively.
Fig. 3 Fluorescence emission of dimer 2 in water between 20 and 90 1C
(1 mM, lexc = 450 nm). Inset: absorption spectra at 20 and 90 1C.
9530 | Chem. Commun., 2014, 50, 9529--9532
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Fig. 6 Temperature characteristics between 20 and 90 1C (left) and
accuracy between 30 and 45 1C (right) of 2 and 4.
Fig. 4 Relaxation kinetics of dimer 2 in water at low (blue) and high (red)
temperatures.
Further studies are underway to determine the exact nature of
twisted and planar conformations. Fast relaxation of the this phenomenon.
twisted conformation toward the planar form at 20 1C leads
The thermal stability and accuracy of the dimers were inves-
mainly to emission in the red band. At 90 1C, the rotation is tigated to determine the potential of these molecules as tem-
hindered and both emissions are observed.
perature probes. They were first subjected to heating–cooling
Similar behaviors were observed for dimers 3 to 5 but not for cycles between 20 and 90 1C (Fig. 5). The parameter r = Ib/Ir,
the non-glycosylated analog 1 which quickly degraded upon where Ib = intensity of the blue band, Ir = intensity of the red
heating thereby confirming the role of the carbohydrate moieties band, measured as a function of temperature was introduced.
that also provide a shielding effect.
It is weak at low temperatures and increases with temperature.
Although the exact origin of both effects has yet to be
The best reproducibility was observed for compounds 2 and
determined, we believe that H bonds between hydroxyl groups 4 for which a stable regime was observed after the first cycle.
and water are at the heart of the phenomenon. Temperature Compound 3 also reached a similar state but needed 2 to 3
modulates the number and strength of these bonds thereby additional cycles. Dimer 5 exhibits more or less the same
altering the relaxation kinetics. They might also form a bundle response after 4 cycles but its emission spectra showed impor-
around the fluorophore slowing or preventing the diffusion of tant drifting which could indicate significant degradation over
reactive species, such as oxygen, at its proximity. This explana- time. This result is consistent with the behavior of dimer 1 and
tion is consistent with the absence of photosensitization by the shows that longer polyethyleneglycol (PEG) linkers weaken the
dimers in water, even at high temperatures. To sustain this carbohydrate shield.
hypothesis, the behavior of the compounds is easily modified by
r values for compounds 2 and 4 were plotted between 20 and
the addition of biological components in the solution (see ESI†). 90 1C (Fig. 6, left). The slow evolution allows temperature
sensing over the whole temperature window. The dimers also
allowed precise measurement of the temperature between
30 and 45 1C (Fig. 6, right) with accuracies of Æ2 1C for
compound 2 and Æ1 1C for 4, making these compounds
promising candidates for biological applications after encapsu-
lation in nano-containers.
We report the influence of temperature on the spectroscopic
properties of a series of glycoconjugated porphyrin dimers and
hexa-PEG. These compounds, previously designed as photosen-
sitizers for one-photon and two-photon excited photodynamic
therapy,35 exhibit unexpected dual fluorescence in polar solvents
in general whose relative intensity depends on the temperature
of the solvent. Water leads to a peculiar behavior where the band
of highest energy is exalted whilst previous results would predict
the opposite. This was attributed to the specific nature of water
as an organized solvent due to the high number of H-bonds in
which carbohydrates insert remarkably well.39 These bonds
modify the intramolecular rotation of the dimer leading to the
dual emission and also provide an efficient shield against high
temperatures.
Fig. 5 Stability of the probes during heating–cooling cycles in water.
Evolution of the r parameter versus cycles.
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