Preparation of terbium complexes to sense the metal


During last 15 years there has been a remarkable progress in the use of fluorescence in the biological sciences. Few years ago, fluorescence spectroscopy was used as primary tool in the biophysics and biochemistry. But now a day it is used in the clinical chemistry, DNA sequencing, genetic analysis and environmental monitoring. Because of the difficulties of handling radioactive substances, the sensitivity of fluorescence detection and the expenses, there is development of medical tests based on the fluorescence.

Emission of a light from any substance, which occurs from the electronically excited states, is termed as luminescence. It is formally divided into two categories, phosphorescence and fluorescence, which depends on the excited states. In excited (singlet) states, the electron present in the excited orbital is paired (opposite spin) with the second electron in the ground state. As electron returns to the ground state there is emission of a photon. Emission rates of fluorescence are 108 s-1, so typical fluorescence lifetime is near to 10 ns (10 x 10-9 s).

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Phosphorescence is the emission of light from the triplet-excited states, which has the electron in the excited orbital with the same spin orientation as the ground-state electron. Transitions to the ground state are prohibited and also the emission rates are slow (103 -100 s-1), thus the lifetime of phosphorescence are typically from milliseconds to seconds. Phosphorescence is not usually observed in the fluid solutions at room temperature, because of existence of many deactivation processes that complete with the emission, such as quenching processes and nonradioactive decay. It is also noted that the distinction between phosphorescence and fluorescence is not always clear. Following Jablonski diagram shows the phenomenon of both Phosphorescence and fluorescence.

Fluorescence is widely used in biology and medicine. The measurement of fluorescence spectrum, polarisation and lifetime are powerful methods in study of different fields. It is highly sensitive to the biochemical environment of fluorophore. Fluorophore has been modified such that their spectra change as a function of concentration of substances, such as calcium and pH. In the determination of the important reporters of protein structure and folding fluorescent spectra plays important role. Fluorescence resonance energy transfer (FRET) spectrally monitors the protein motion and domain structure on the subnanometer scale. It is a nonradioactive process where energy transfers between two fluorophores.

Fluorescence lifetime gives the complementary information to the spectral measurement. Number of flurophores may respond to the environmental changes with the lifetime variations. These fluorescence lifetime measurements are also useful in distinguishing static and dynamic quenching mechanism.

Fluorescence polarization is used to measure the rotational diffusion rate of macromolecules. This rotational diffusion contains the information related to the protein shape. On the basis of the polarization measurement, diffusional restrictions of molecules in macrostructures, such as cytoskeleton can be quantified. The combination of the polarization measurement and lifetime allows the quantification of rotational rate and which as been used to study the protein domain motion.

Fluorometer is widely used in various analytical procedures, usually with a single detection wavelength and single exciting wavelength. Fluorescent molecule can be concentrated as low as 1 part per trillion, which can be further measured because of its sensitivity. Fluorescence in various wavelengths can be measured by an array detector, which is used in the detection of compounds from HPLC flow.

Light, lanthanides and luminescence are fascinating words, because lanthanides originate from the Greek word lanthaneien, which means, "lying hidden" and also it is associated with the life and its big-bang origin. Luminescence has been the instrumental in the discovery of the lanthanides elements, and, in turn, these elements have played a leading role in light conversion technology such as plasma displays, lasers, light-emitting diodes, and cathode-ray.

Lanthanides show a number of features in their chemistry, which make them to differentiate them from the d-block metals. The reactivity of the lanthanides is greater than that of the transition metals. The coordination numbers of the elements are in the range from 3 to 12, which depends on the steric demand of ligand. The coordination number between 8 to 9 is the most frequently observed. Their coordination geometrics are determined by the ligand steric factors rather than the crystals field effects. Lanthanides form liable 'ionic' complexes, which undergo the facile exchange of ligand. The 4f orbitals in Ln3+ ion do not play a part directly in the bonding, being well shielded by the 5p6 and 5s2 orbitals. Their magnetic and spectroscopic properties are thus largely uninfluenced by the ligand. Lanthanides have very sharp electronic spectra and small-crystal field splittings, when compared with the d-block metals. They generally prefer anionic ligand with the donor atoms of higher electronegativity. Lanthanide readily forms the hydrated complexes because of the high hydration energy of the small Ln3+ ion, and thus causes uncertainty in assigning the coordination numbers. Lanthanides do not form multiple bonds Ln=N or Ln=O known for certain actinides and many transition metals. They do not form stable carbonyls and have no chemistry in 0 oxidation state.

Design of Lanthanides Luminescent complexes

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The photophysical, electronic and magnetic properties of lanthanides complexes strongly depend on the control of metals coordination sphere. Design of ligands need to be concentrated to obtain the optimize property of interest. A report by the Lehn 1973 describes the basic principles of design of the organic ligands for alkali-earth cations and alkali. In this review, the parameters that should be considered to control over structural, chemical and thermodynamical properties of the complex are clearly described. From these parameters the ligand topology (shape, size, dimensionality, chirality and connectivity), the binding sites (electronic properties, number, shape, arrangement, nature), the layer properties (flexibility/rigidity, thickness, and the ratios of lipophilicity/hydrophilicity), the counterions effect and environment properties are particularly important. These general rules are applied to any type of ligand, independently of metal cations. The complex formation is due to the attraction between a metal cation and a ligand and also associated with their total or partial desolvation. Simply, the surface of the metal cation interacts with the coordination sites of ligand thus replacing totally or partially the first solvation sphere.

In the complexation of lanthanides in aqueous medium, the dehydration step is endothermic. This represents a contribution of unfavourable energy to the variation in Gibbs energy, so that overall process is driven by entropy. This difficulty can be overcome with the use of polydentate ligands, which is convenient to use because of its chelate effect, and can afford for highly stable complexes in aqueous solution. Ligand-lanthanide interaction has to be maximized, in order to increase the thermodynamic stability. Due to their hard character, lanthanide cations shows priority for the hard binding sites, having greater electrostatic components. As luminescent Ln3+ complex is a multicomponent system, which contains active components, named as, the antenna, the coordination site, the metal cation, which are organised in a supramolecular structure. So in order to optimize the overall sensitization efficiency, the choice of this components and their respective position in overall structure needed to be consider during molecular design step.

Choice of lanthanide

Lanthanides cations have emission properties, which covers a wide spectral range that covers from the UV (Gd3+) to the visible: red (Eu3+), yellow (Dy3+), orange (Sm3+), blue (Tm3+) and green (Tb3+) to the NIR (Er3+, Nd3+, and Yb3+). Following energy diagram gives the idea of the emissive levels of Ln3+ cations emitting in the visible range with the energies of triplet (green) and singlet (blue) excited states of some regularly used chromophores.

Usually lanthanides possess relatively long-lived excited states, which can carry out energy transfer to vibrational oscillators of higher frequency such as NH, OH and, to lower extent, CH. As a result, the presence of these groups in the relation of the metal favours thermal dissipation of the energy i.e. vibronic coupling that gives rise to quenching of the luminescence. In precise, the lower the excited state energy of the lanthanide ions the more efficient will be the deactivation done by the vibronic coupling i.e. "energy gap rule".

Choice of antenna

The chromophore that evokes the sensitatization of lanthanide light emission is termed as "antenna" and it plays important role in determination of the emission intensity of the lanthanide complex. The antenna can be any hetro-aromatic or aromatic extremely -conjugated system featuring by high efficiencies of intersystem crossing and high efficiency of light absorption and energy transfer processes. The efficiency of the chromophore to act like sensitizer is related to the energy of its triplet excited state. This energy should be atleast 1850 cm-1 greater than the lowest emitting levels of the lanthanide cations. When the energy gap is higher, then the energy transferred from the triplet flows through the non-radioactive excited states of the metal till it achieves the emissive levels and thus metal centred emission occurs. In reverse, the lower energy gap limits the emission quantum yield, because of thermal deactivation due to O2-quenching towards the chromophore triplet level and due to back energy transfer. According to the above rule mentioned, it is unlikely that for the different lanthanide cations the same chromophore can efficiently act as antenna. This is can be complicated by the possible quantity of non-radioactive excited states, such as LMCT (ligand-to metal charge transfer), which can occur with suitable ligands, thus results in non-luminescent lanthanide complexes. Another important point to consider is that the excitation wavelength of the antenna should be more than ca. 350 nm, this is help to avoid the use of expensive excitation sources and to avoid expensive quartz optics in immunoassay applications. Energy transfer process of the chromphore depending upon the nature and position can be occurring by the Forster or Dexter mechanisms. In order to obtain the fast energy transfer a short distance between the lanthanide cations and the sensitizer is advantageous; the best results can be achieved when the antenna directly coordinates with the metal centre. Besides the nature of the binding sites, their relative position in whole structure of ligand also plays important role in satisfying the coordination properties of chromophores. Following diagram shows the possible ways to position the antenna within the ligand.

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Choice of coordination site

The coordination site is formed by groups arranged or number of donor atoms in a covalently ordered structure and ability of binding the metal cation strongly. Depending upon the dimensionality, the coordination site can be monodimensional, bidimensional and tridimensional. The factors to be considered for preparing highly luminescent lanthanide complexes are such as positioning of the antenna within the coordination site combined with its chemical and physical properties. This can be understood mainly in two ways: with the antenna subunits (i) integrated in to the coordination site structure (ii) covalently attach through a respective spacer.

Lanthanide Luminescent

The lanthanides luminescent properties are dominated by their low extinction coefficients. Under normal conditions also the lanthanide luminescent is quenched in non-radiative processes. In order to be detected by the time-resolved fluroscence (TRF), the lanthanide is sensitized by covalent attachment of organic chromophore named as "antenna" to the lanthanide chelate. This chromophore acts to absorb the excitation light, and then absorbed light is transferred from the excited singlet state of antenna to the lanthanide ion's triplet state, results in the emission of a photon. From the following diagram (a) the luminescence of lanthanide can be understood, the organic chromophore acts as an antenna, thus absorbs the light. This energy is transferred to the lanthanide-excited state and with a long lifetime a fluorescent signal is emitted. (b) The diagram explains the photoluminescence of lanthanide ions. (1) Antenna absorbs the light and transfers the energy from ground state to the excited state. (2) from the excited state of the antenna, energy is transferred to the emitting state of the lanthanide (3) a photon is emitted by the lanthanide ion, and thus returns to the ground state. (4) an emission band is obtained from the respective lanthanide ion.

(a) (b)

Application of the Lanthanides

When a lanthanide complex emits intense luminescene, applications like optical imaging, sensing drug concentration and in the field of biomedical become possible. This is based on the long-lived excited states of the lanthanide ions. Complexes such as europium and terbium can exhibit intense visible line-like emission, and thus these are mostly studied. Near infrared (NIR) lanthanide emitters such as Er3+, Sm3+, Dy3+, Pr3+, Ho3+, Nd3+, and Yb3+ have been less investigated in early times. However, in recent times more interest in these emitters have registered, which shows the possible use of these emitters in the field of various photonic applications and telecommunication fields and in biomedical. Its given that emitters with longer-wavelength are more able to penetrate in the human tissue than the visible light. So long-wave emitters can be useful in various medical diagnostic procedures. In the same way, NIR luminescence from ions such as Yb3+, Er3+ and Nd3+ proves very helpful in optical signal amplifier in telecommunication network. Emitters such Tb3+, Eu3+, Sm3+ and Dy3+ can be coupled with appropriate antennas, and can be incorporated in transparent and stable ligands. Following diagram shows the type of emission and their major areas of applications.

The phthalimides has broad emission in the visible region (400-600 nm), which overlaps with the lanthanides electronic absorption and phthalimides shows involvement in extended intermolecular interaction, which is appealing as potential conduits need for electronic excitation energy. Two compounds were prepared and were named as (1) & (2), which contains phthalimide chromophore. These compounds were then further hydrolyzed to form a complex with the Tb3+ ions. These hydrolyzed ligands were used to sensitize the lanthanide ions. These complexes were then studied with the help of phosphoresce spectroscopy. At different ph, the intensity of these complexes was recorded. As these complexes are very ph dependant, the highest and lowest intensity of the respective complex was observed. Following schemes shows the synthesis and hydrolysis of both the compound.

Scheme 1: Synthesis of Compound (1)

Scheme 2: Synthesis of Compound (2)