Introduction 1 1.0 General Introduction: The lanthanide ions have no known biological use, and only trace amounts are found in whole body analysis. However, they show biological activity. For instance, they may act as enzyme activation or deactivation and in in-vivo studies, as nerve impulse stimulators (1). The unique similarities in terms of coordination and binding characteristics between the paramagnetic lanthanides with calcium makes the lanthanides to act as an ―absorption probes‖ in understanding the biochemical reactions and for structural studies of biomolecule compounds and functions involving the isomorphous substitution of Ca(II) by Ln(III) (2-6). 1.1 Chemical reactivity One property of the Lanthanides that affect how, they will react with other elements is called the basicity. Basicity is a measure of the ease at which an atom will lose electrons. In another words, it would be the lack of attraction that a cation has for electrons or anions. In simple terms, basicity refers to have much of a base a species is. For the Lanthanides, the basicity series is in the following order: La(III) > Ce(III) > Pr(III) > Nd(III) > Pm(III) > Sm(III) > Eu(III) > Gd(III) > Tb(III) > Dy(III) > Ho(III) > Er(III) > Tm(III) > Yb(III) > Lu(III) In other words, the basicity decreases as the atomic number increases. Basicity differences are shown in the solubility of the salts and the formation of the complex species. Another property of the Lanthanides is their magnetic characteristics. The major magnetic properties of any chemical species are a result of the fact that each moving electron is a micro-magnet. The species are either diamagnetic, meaning they do not have unpaired electrons, or paramagnetic, meaning that they do have some unpaired electrons (7- 10).
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Introduction
1
1.0 General Introduction:
The lanthanide ions have no known biological use, and only trace
amounts are found in whole body analysis. However, they show
biological activity. For instance, they may act as enzyme activation or
deactivation and in in-vivo studies, as nerve impulse stimulators (1). The
unique similarities in terms of coordination and binding characteristics
between the paramagnetic lanthanides with calcium makes the
lanthanides to act as an ―absorption probes‖ in understanding the
biochemical reactions and for structural studies of biomolecule
compounds and functions involving the isomorphous substitution of
Ca(II) by Ln(III) (2-6).
1.1 Chemical reactivity
One property of the Lanthanides that affect how, they will react with
other elements is called the basicity. Basicity is a measure of the ease
at which an atom will lose electrons. In another words, it would be the
lack of attraction that a cation has for electrons or anions. In simple
terms, basicity refers to have much of a base a species is. For the
Lanthanides, the basicity series is in the following order:
However, Gd(III) emits in the UV, and it is not very useful as
luminescent probe for bio-analyses because its luminescence
interferes with either emission or absorption processes in the organic
part of the complex molecules. On the other hand, it can efficiently
transfer energy onto Eu(III) upon vacuum-UV excitation, resulting in
the emission of two red photons (the so-called quantum cutting or
down-conversion effect) (143).
Gd(III) is therefore, a potential phosphor component for mercury-free
fluorescent lamps. The sizeable energy gap displayed by Eu(III) and
Tb(III), explains why luminescent probes containing these ions have
been so popular during the last decades. Nevertheless, development of
dual luminescent time-resolved immunoassays has also stirred
interest for Sm(III) (∆E =7400, 4G5/2 → 6F11/2) or Dy(III) (7850 cm-1,
4F9/2 → 6F3/2) (144-149). The other ions have very low quantum yield
Introduction
19
in aqueous solutions and appear to be less useful with respect to
similar applications. Pr(III) emits both in visible and NIR ranges and is
often a component of solid state optical materials, in view of its ability
of generating up-conversion that is blue emission from 3P0 upon two
or three photon pumping of the 1G4 or 1D2 states (150). Thulium is a
blue emitter from its 3P0, 1D2 and 1G4 levels and is used as such in
electroluminescent devices; it is the first Ln(III) ion for which up-
conversion has been demonstrated (150); several other ions [Nd(III),
Dy(III), Ho(III) and Er(III)] present up-conversion processes as well. In
addition, Nd(III), Ho(III), Er(III) and Yb(III) have special interest in that
they emit in the NIR spectral range and are very useful in the design
of lasers (especially Nd(III) with its line at 1.06 mm) and of
telecommunication devices (151). The partial energy level diagram for
various Ln(III) ions is depicted in the Fig. 1.2
Introduction
20
Most of the electronic transitions of the trivalent Ln(III) ions involve a
redistribution of electrons within the 4f sub-shell. Electric dipole
selection rules forbid such transitions but these rules are relaxed by
several mechanisms. An important one is the coupling with
vibrational states, where a molecular vibration temporarily changes
the geometric arrangement around the metal ion and therefore, its
symmetry. Other mechanisms, which cause a breakdown of the
selection rules are the J-mixing and the mixing with opposite-parity
wave functions such as 5d orbital, ligand orbital or charge transfer
states. The coupling between these vibrational and electronic states
and the 4f wave functions depends on the strength of the interaction
between the 4f orbital and the surrounding ligands; in view of the
shielding of the 4f orbital, the degree of mixing remains small, and so
Fig. 1.2 Partial energy diagrams for the lanthanide aqua ions. The main luminescent levels are drawn in red, while the fundamental level is indicated in blue.
Introduction
21
are the oscillator strengths of the f–f transitions. As a consequence,
even if many lanthanide containing compounds display a good
quantum yield, direct excitation of the Ln(III) ions rarely yields highly
luminescent materials. Indirect excitation (called sensitization or
antenna effect) (152-153)has to be used and is proceeds in three
steps (154-156), as shown in following Fig 1.3
First, light is absorbed by the immediate environment of the Ln(III) ion
through the attached organic ligands (chromophores). Energy is then
transferred onto one or several excited states of the metal ion and
finally, the metal ion emits light as shown in Fig 1.4. Sensitization of
trivalent lanthanide ions is an exceedingly complex process involving
numerous rate constants (157-160).
Fig. 1.4 Simplified diagram showing the main energy flow paths during sensitization of
Lanthanide luminescence via its surroundings (ligands).
Fig. 1.3 Antenna effect of Ln 3+
Introduction
22
In aqueous solution, interaction with water (both in the inner and
outer coordination sphere of the Ln(III) ion) lead to a severe quenching
of the metal luminescence via O–H vibrations (161). Although
disadvantageous to the design of highly luminescent edifices, this
phenomenon can be used to assess the number of water molecules (q)
interacting in the inner coordination sphere. Several approximate
phenomenological equations have been proposed based on the
assumptions that O–D oscillators do not contribute to de-activation
and that all the other de-activation paths are the same in water and in
deuterated water and can henceforth be assessed by measuring the
lifetime in the deuterated solvent (162-164).
The emission from lanthanides has proven useful as a sensitive
detection method in biological systems and has facilitated their
understanding. The changes in the intensity of the Eu(III)
luminescence upon binding to proteins and enzymes have been
utilized to examine the ligation sphere within the active site, whereas
distance and conformational information under physiological
conditions has been obtained from energy transfer studies either
between two lanthanide ions or from the protein‘s residues to Eu(III)
or Tb(III) bound to the active site (165-166). Luminescence
enhancement of a given probe in the presence of nucleic acids can in
principle yield such detection, with marked safety and environmental
advantages over radioactive labelling. Owing to the emissive properties
of Tb(III) and Eu(III), including their luminescence enhancement
through energy transfer and their ability to bind single stranded
regions of proteins.
One initial landmark was the discovery in year 1942 of what is known
today as the antenna effect by Weissman (20), who demonstrated that
energy transfer occurs from the bound ligands to the metal ion
providing an excellent way for the sensitization of the Ln(III) ion
luminescence.
Introduction
23
The use of lanthanide f-ions such as Eu(III), Tb(III), Sm(III), Yb(III),
Nd(III), etc. in such luminescent supramolecular systems, has become
a very active area of research (167-174). From the view of developing
luminescent chemical sensors, changes in various photo-physical
properties such as wavelength, lifetimes and quantum yield (the
outputs) can all be modulated as a result of external perturbation.
This phenomenon can also be employed to investigate the formation
and physical properties of complexes of supramolecular structures
and self-assemblies (175-178).
1.6 Applications of Lanthanide ions
1.6.1 Therapeutic application
Lanthanide complexes are of considerable interests for their
therapeutic utility providing strong impetus to explore their biological
activities (179-181). There is fast moving research on lanthanides and
their interrelations with bio-systems to understand their functional
roles in biology and medicine (182-184).
The coordinating chemistry of lanthanides, relevant to the biological,
biochemical and medical aspects, makes a significant contribution to
understanding the basis of application of lanthanides, particularly in
biological and medical systems. The importance of the applications of
lanthanides, as an excellent diagnostic and prognostic probe in
clinical diagnostics, and an anticancer material, is remarkably
increasing. Lanthanide complexes based X-ray contrast imaging and
lanthanide chelates based contrast enhancing agents for magnetic
resonance imaging (MRI) are being excessively used in radiological
analysis in our body systems. Conjugation of antibodies and other
tissue specific molecules to lanthanide chelates has led to a new type
of specific MRI contrast agents and their conjugated MRI contrast
agents with improved relaxivity, functioning in the body similar to
drugs (185).
Introduction
24
In 1931, Maxwell and coworkers used an aqueous solution of
lanthanum chloride for treating cancer by administering LaCI3
solution intraperitoneally. However, it was only the work of Anghileri
and coworkers, which could successfully demonstrate the strong
inhibitory effects of LaCl3 and other lanthanide compounds on the
growth of sarcoma tumors in rats. Excellent work has come out of
Anghileri‘s laboratory, on lanthanide compounds and complexes in
cancer research as a diagnostic and prognostic probe. These workers
used Ln(III) as an adjunct to the distraction of tumors by using a
combination of the complexes of two different lanthanides specially
derived form hydroxy carboxylic acids for treating animals and in
some cases involving humans suffering from Yoshida Sarcoma. The
results were found astounding. The complexes of lanthanides are
getting more and more applications in cancer therapy and the most
important of these are those derived from poly (aminocarboxylic)
acids. These days‘ diagnostic imaging procedures are a routine part of
modern medicine and are useful in performing the initial diagnosis,
the planning of the treatment and post treatment evaluation (185-
195).
Lanthanide ions are trivalent and similar in their chemical and
biological properties to the alkaline earth elements. In the past, some
lanthanide compounds were used in the treatment of tuberculosis, as
anticoagulant agents for prevention of thrombosis, and as anti nausea
agents during early pregnancy. More recently, lanthanides have been
used in dentistry for cancer treatment as anti-inflammatory agents,
and as antivirus agents (196).
Alpha- and flaviviruses contain class II fusion proteins, which form
ion-permeable pores in the target membrane during virus entry. The
pores generated during entry of the alphavirus Semliki Forest virus
have been shown previously to be blocked by lanthanide ions. Here,
analyses of the influence of rare earth ions on the entry of the flavi
viruses, West Nile virus and Uganda S virus revealed an unexpected
Introduction
25
effect of lanthanide ions. The results showed that a 30 s treatment of
cells with an appropriate lanthanide ion changed the cellular
chemistry into a state in which the cells no longer supported the
multiplication of flaviviruses. This change occurred in cells treated
before during or after infection, did not inhibit multiplication of
Semliki Forest virus and did not interfere with host-cell multiplication.
The change was generated in vertebrate and insect cells, and was
elicited in the presence of actinomycin D. In vertebrate cells,
specifically La(III), Ce(III), Pr(III) and Nd(III) elicited the change. In
insect cells, additional lanthanide ions had this activity. Further
analyses showed that lanthanide ion treatment blocked the ability of
the host cell to support the replication of flavivirus RNA. These results
open two areas of research: the study of molecular alterations induced
by lanthanide ion treatment in uninfected cells and the analysis of the
resulting modifications of the flavivirus RNA replicase complex. The
findings possibly open the way for the development of a general
chemotherapy against flavivirus diseases such as Dengue fever,
Japanese encephalitis, West Nile fever and yellow fever (197).
Lanthanum and the fourteen elements following it (together, called the
lanthanide series) are used in medical image visualisation. At a
certain concentration, the cerium compounds could be possibly
involved in the control of cell proliferation and inhibiting the growth of
cancer cells (198-199).
A big stimulus arose in the mid 1980‘s when a small Finnish
company, Wallac Oy from Turku, marketed bioassays based on
timeresolved luminescence of Eu(III) (200).
In the literature, very few Yb(III) complexes have been reported to display
promising anti-cancer activities without photoactivation or conjugation
to cytotoxic counterparts/radionuclides (201).
The La(III), Eu(III) and Yb(III) complexes 1La, 1Eu and 1Yb,
respectively, from cyclen by incorporating four amino esters
(the‗pseudo‘ dipeptide GlyAla) as pendant arms into cyclen. From an
Introduction
26
applications point of view, such lanthanide ion based ribonuclease
mimics are highly desirable as therapeutics in gene therapy, as
antisense agents, and as tools in molecular biology and genetics (202-
203).
Several years ago, however, the remarkable catalytic activity of the
lanthanide ions was discovered, and both DNA and RNA were for the
first time hydrolysed at reasonable rates under physiological
conditions. The Ce(IV) ion is the most active for DNA hydrolysis,
whereas Tm(III), Yb(III) and Lu(III) (the last three lanthanide ions) are
quite effective for RNA hydrolysis (204-206). Lanthanide have been
used as biochemical probes to study calcium transport in
mitochondria and other organelles (207). Lanthanide complexes have
an increasingly important role in medicine, where they are employed
as diagnostic as well as therapeutic agents (208). The peculiar
electronic properties of lanthanide ions, in fact, are exploited for the
development of powerful NMR probes for medical application (209);
Gd(III) complexes are in current clinical use for magnetic resonance
imaging (210-211) and lutetium compounds have shown a great
potential as radio sensitizer for the treatment of certain types of
cancers (212). The study of the remarkable catalytic activity of the
rare earth metals for the hydrolysis of nucleic acids is another active
field of research, mainly because it is essential for further
developments in biotechnology, molecular biology, therapy and related
fields (213-216). The biological properties of the Ln(III) ions, primarily
based on their similarity to calcium, have been the basis for research
into potential therapeutic applications of Lns since the early part of
the twentieth century (217-219).
However, lanthanides might be clinically valuable for blocking gene-
specific transcription for antibiotics or chemotherapeutic applications
or for customized targeting of oncogenic mutations (220). The
gadolinium concentration in the new biomaterial is relatively high;
this flux is becoming to be close to the necessary therapeutic values.
Introduction
27
This means that the new biomaterial opens an opportunity to carry
out the effective irradiation by thermal neutrons using smaller and
safer machines with a smaller flux and to reduce harmful effects of
the irradiation (221).
Rare earth elements (REEs) are widely used in industry and medicine.
As an example, radioactive REEs can be used in the diagnosis and
treatment of cancer. This therapeutic aspect attracts increasing
interest and inspires many researchers to investigate REE effects on
tumour development and growth. There is substantial evidence
showing that REEs inhibit proliferation and induce apoptosis in
certain cancer cell lines (222-228).
1.7 Biochemistry of Ln(III) ion
Lanthanides are known to form complexes with many functional
groups found in biological molecules, especially with donor groups
that posses a lone pair of electrons, such as oxygen atoms in the
carboxylic groups of amino acids. Lanthanides can bind at the active
sites of biomolecules, replacing various ions that include Ca(II), Zn(II),
Mg(II), Mn(II), Fe(II) and Fe(III).
The ability of lanthanide ions to luminescence at room temperature
and their specific spectroscopic characteristics made them a versatile
probe for the study of the biochemical processes that take place in
metal binding proteins and metalloenzymes.
The effect of coordination compounds of lanthanides with DTPA on the
phase behavior of DPPC liposomes is smaller than that of their
chlorides. La(III), Gd(III), and Yb(III), can displace Ca(II) binding on
DPPC liposomes, but there coordination compounds of DTPA can
hardly displace Ca(III) (229).
Information regarding the composition and structure of the metal
binding site can be obtained from the emission and excitation spectra
of Tb(III) and Eu(III)-protein complexes. Furthermore, binding
Introduction
28
constants of metal ions such as Ca(II) and Zn(II) to proteins can also
be calculated using Eu(III)-metal competition titration method (230).
The binding constant of Eu(III) is first determined using the titration
curve of the intensity of the 5D0 → 7F0 (λem = 579nm) emission as a
function of total equivalents of Eu(III) added to the protein. The
intensity of the Eu(III) luminescence decreases as a known quantity of
the competing metal ion is added, which is proportional to the amount
of replaced Eu(III), making the relative binding constant measurement
possible (230-232).
The hydration number (the number of water molecules) in the inner
coordination sphere of the metal can also be determined by measuring
the differences of emission lifetime of Tb(III) and Eu(III) in H2O and
D2O. As discussed earlier, the excited state lifetime of Eu(III) is very
sensitive to the number of water molecules coordinated to the ion
thus, replacing H2O by D2O can cause lifetimes to increase
dramatically (by a factor of ~9). For example, two water molecules
were found at the Ca(II) binding site in the satellite tobacco necrosis
virus (233-236) and four waters were found to coordinate at the
catalytic Mg(II) site in the ATP dependent enzyme glutamine syntheses
(237). Knowledge of the number of water molecules leads to the direct
estimation of the total number of coordinating atoms supplied by the
protein, since, Ln(III) ions usually possess total coordination numbers
of 7 to 9.
Laser-excited Ln(III) ion has been widely used to identify the metal
binding site in a variety of proteins and metalloenzymes (238).
Analysis of the 5D0 → 7F0 band in the excitation spectrum and
luminescence titration of Eu(III) in a complex with bovin α-
lactalbumin, at least two different calcium binding sites and three
kinds of ligands have been determined (239).
Lanthanide Luminescent Bioprobes (LLBs) are amongst the most
sensitive luminescent probes because their excited states are long-
Introduction
29
Fig. 1.5 Lanthanide Luminescent Bioprobes (LLBs)
lived, which allows Time Resolved Detection (TRD) of their
luminescence. Applications of LLBs to immunoassays, DNA analysis,
ligand binding assays, analytes sensing, and cellular imaging has
been reviewed (240-245). Route mechanism of LLBs is represented in
the Fig. 1.5.
1.8 Aim & significance of the work
The main aim of this Ph.D. programme is to focus on application of
Ln(III) in mimicking the biochemical reaction, that involved Ca(II), one
of the most abundant metal ions in our body system.
To achieve the aim following objects are selected:
i) Study of the Ln(III) ions like Nd(III) and Pr(III) ions inleution with
biomolecules in conditions similar to physiological conditions,
using 4f-4f transitions.
ii) Study of the Ln(III) ions and their interaction with
Lysozyme/GSSG using electroanalytical tools.
iii) Study of the Eu(III) ion and their interaction with
Lysozyme/GSSG using Luminescence method.
Introduction
30
iv) Study the divalent states of Europium, Samarium,
Praseodymium, Neodymium and ytterbium in non-aqueous
medium.
v) Ultra sonic sound technique will be used for various acoustical
properties of Eu(III) and Eu(III) + Lysozyme in ternary system.
Adopted methodology in our lab, we have spectro fluorometer (1501
RF) from Shimadzu, UV-1800 from Shimadzu and One electro
chemical substation with US. UV-1800 will be used for study of 4f-
4f transition of Pr(III) and Nd(III). Spectro-Florometer 1501 RF will
be used for Luminescence study of Eu(III) and its in taking with
Lysozyme/GSSG. Electrochemical substation (CH 660B) will be
used for study of divalent states of Eu/Sm/Pr/Nd/Yb and their
interaction with Lysozyme/GSSG.
Introduction
31
References :
1) S J Lippard. Current Research Topics in BioInorganic
Chemistry. ISBN: 0-471-54088-9. Publication by John Wiley &
Sons, New York, London.
2) C Victory Devi and N S Rajmuhon. Calculation of Energy
Interaction and Electric Dipole Intensity Parameters to Explore
the Interaction between the Trivalent Praseodymium and Uracil
Using 4f-4f Transition Spectra as an Absorption Probe. Int. Conf.
Chem. and Chemi. Proc., 10 (2011).
3) N Sudhindra, G Ramchandria, A Minaz Gagnani, S Ram Shukla
and M Indira Devi. Absorption Spectral Studies Involving 4f-4f
Transitions as Structural Probe in Chemical and Biochemical