CH2402 Ligand Design for Imaging Techniques Imaging agents are a very important aspect of everyday healthcare, the

e early and accurate diagnosis of many diseases can have a significant impact on the efficacy of treatments. There are several imaging modes available (Table 1) but typically those capable of scanning the entire body lack resolution and those with high resolution are only practical in vitro.
Table 1 - Imaging modalities

Modality Computed Tomography (CT) Magnetic Resonance Imaging (MRI) Positron Emission Spectroscopy (PET) Single-Photon Emission Computed Tomography (SPECT) Fluorescence microscopy Ultrasound

Agents None Gd, Dy Radioisotopes Radioisotopes

Penetration Deep Deep Deep Deep

Resolution m m mm mm

Fluorochromes, rhodamines, BODIPYs Microbubbles

Shallow (mm using visible light, cm using NIR) Deep

nm m

Regardless of the modality the issues encountered while developing an imaging agent are often similar; Toxicity the agent must be biologically safe at the required levels. This is true even of agents used in in vitro diagnoses, the cell functions must not be effected by the agent in order to provide a clear result Stability the agent must survive the conditions found in vivo Uptake the agent must be able to access the active site through cell membranes, the gut wall or the blood-brain barrier as necessary Localisation and distribution the agent must have a high specificity to limit background noise Circulation/residence time the agent must remain in vivo for long enough to perform a scan but for as little additional time as possible to prevent any harmful reaction Penetration of signal

The Physical Properties Required for Imaging There are several common traits in cell morphology worth considering when designing an imaging agent. Cells are wrapped in membranes; these are made of phospholipids and glycolipids and are essentially organic, non-polar structures. To cross these membranes the agent must be either lipophilic itself or able to take part in naturally occurring protein transfer processes used to shuttle important molecules across. There is also a potential barrier across the cell membrane, cations are, specifically, pumped out of the cell and so the inside of the cell is slightly negatively charged, large cations will become trapped within cells, this has implications on the toxicity and circulation of an agent. Within the cell there are further membranes surrounding organelles, one organelle of

particular interest is the nucleus, where DNA is found, others include mitochondria and endoplasmic reticulum. The cell itself is full of cytoplasm, this is a water gel containing some salts and macromolecules (overall solute levels are ~ 500 g L-1), any agent must therefore be built to exploit and survive this aqueous environment. Fluorescence Imaging of Biological Systems Advantages; High sensitivity for a relatively low cost, 4000 cf. 5mil for PET imaging Able to achieve specific staining with multi-channel detection Fluorescence agents provide information on the environment, they are responsive, they can provide information on conditions such as pH or oxygen levels Offer a clearer image with less background noise than found in typical microscopy

Figure 1 - The principle behind confocal microscopy, the preferred technique for fluorescence microscopy

Disadvantages; Background emission via autofluorescence is possible due to the natural fluorescence of many large, highly conjugated biomolecules Self-quenching Photobleaching, the photochemical loss of agent molecules Biocompatibility and toxicity, this technique often requires heavy metals Transport-membrane permeability and active uptake can be difficult to achieve Tissue damage and penetration are inversely proportional, UV light offers the best penetration but will also trigger cell death due

The photophysics of a fluorescence imaging agent are of utmost importance, of particular importance are the Stokes shift and the luminescence lifetime . The Stokes shift (Figure 2) is the difference, in wavelength, between the absorption and emission of a fluorescent molecule. The larger this shift, the easier it is to avoid autofluorescence (typically,

autofluorescence has a Stokes shift of <10nm), with values of 20-30nm considered typical for a monochromator.

abs abs

emit

emit

Figure 2 - Diagram showing a large Stokes shift (top) and a small Stokes shift (bottom)

With a small Stokes shift it is possible for a fluorescence probe to self-quench, there will be some reabsorption of the emitted light by neighbouring molecules resulting in a loss of intensity. Autofluorescence, as well as having a low Stokes shift, also typically has a short lifetime, <10ns. By using agents with a longer lifetime it is possible to acquire data over a later and longer period, circumventing the issue of autofluorescence within the sample. Both a large Stokes shift and a longer luminescence lifetime are common among triplet emitters, this is due to the energy loss involved in the change from a singlet to a triplet as well as the spinforbidden nature of the relaxation. Singlet to triplet conversion is possible due to spin orbit coupling, a process favoured by heavy d/f block systems. d-block Lumophores Iridium, rhenium and rhodium lumophores are the most common, all are triplet emitters with a large component of the emission being 3MLCT (triplet metal ligand charge transfer). Charge transfer from the filled d6 t2g to a conjugated ligand, *1MLCT absorption, is also allowed. d-block lumophores depend on a readily oxidised metal coupled with a readily reduced ligand. The Jablonski diagram (Figure 3) for a 3MLCT d-block lumophore shows the origin of the longer lifetime and larger Stokes shift as well as providing some criteria for the ligand choice. In order to promote the desired transitions and remove sources of energy loss, the eg* orbitals must be higher in energy than the MLCT and so strong-field ligands are preferable. Lumophores of this class are d6 and kinetically inert, preventing free coordination sites on the metal from causing heavy metal toxicity, i.e. through coordination with DNA base pairs.

N.B. the emit is of a lower energy as some energy is lost during fluorescence, also the process is not necessarily 100% efficient, efficiency is described in terms of quantum yield

Singlet excited * d-d transition forbidden ISC Triplet Stokes Shift

d
3

pi

Figure 3 - The Jablonski diagram for a MLCT fluorescence process

Iridium Lumophores

Figure 4 - The structures of two Iridium lumophores

Tris-cyclometallated ppy complexes have a high luminescence intensity but are more difficult to tune and so bis-cyclometallates, using ppy and N^N complexes, that allow the introduction of groups to tune the fluorescence and importantly the biology are preferred. These systems are already cationic which is appropriate for transfer across cell membranes. Ir-bis-cyclometallates are synthesised from iridium chloride (Figure 5) and go via a bridged bis-ppy bis-( 2)chloro complex, the benefit of this approach being that in the final step the R group can be chosen to tune the properties as desired. By changing the ppy unit (Figure 6) the photophysics can be tuned with emission possible from the entire range of the spectrum, the lifetime is proportional to the energy gap and ranges from 3000 400ns. There are over 20 examples in the literature of iridium lumophores that show general cytoplasmic staining with a limited number of these showing poorly controlled localisation in the nucleus. There are also two examples of analogous rhodium systems that both show cytoplasmic staining.

Figure 5 - The synthesis of Ir-bis-cyclometallates

max = 450nm

max = 506nm max = 597nm

Figure 6 - Example iridium lumophores and their emission wavelengths

Ruthenium Lumophores Ruthenium dyes often take the same form as iridium dyes, only the synthesis requires an additional precaution. The reaction is saturated with lithium chloride to prevent the formation of mixed products, in Figure 7 X is the tuneable moiety.

Figure 7 - Synthesis of a ruthenium lumophore

Rhenium Lumophores Rhenium lumophores are built around a similar general design (Figure 8), but with only one bipy or phenathroline ligand capable of MLCT, substituted pyridine is again used to tune the localisation and uptake of these lumophores providing rapid access to a library of agents. Where R is a large lipophilic moiety (e.g. a C13 fatty acid), the imaging agent is likely to accumulate in membranes, smaller lipophilic units (e.g. cyclohexyl) are capable of entering the nucleolus.

Figure 8 - The general structure of a rhenium lumophore

With only one large unit complexed to the rhenium metal the core is smaller than that of iridium imaging agents.

Figure 9 - Synthesis of a rhenium lumophore

The AgPF6 used in the synthesis of rhenium complexes (Figure 9) is weakly coordinating and precipitates silver chloride, this is to increase the efficiency of the reaction, in turn limiting the waste of any pyridine. The reaction can be monitored via the emission as the electronegativity of the nitrogen, which impacts upon the energetics of the conjugated system (donation of electrons raises the energies involved), changes upon coordination although this effect may be weak. This also has some uses in vivo although these are limited. A particularly important class of rhenium agents are those that target the mitochondria. This organelle is protected by an internal membrane with a high potential barrier but being able to study and image the mitochondria is vital as it can be central in many diseases. Mitochondria have a high thiol concentration, a fact that rhenium agents take advantage of MitoTracker (Figure 10) is a cationic, lipophilic and thiol reactive molecule that, once inside the organelle, will form a fixed adduct with glutathione, a polar membrane-impermeant, thus holding the imaging agent within the mitochondria.

Figure 10 - MitoTracker Red and glutathione

There is one report of a dimeric rhenium agent (Figure 11) that shows MLCT, conjugates with peptide nucleic acids (PNAs) and shows nuclear accumulation as well as having an emission wavelength sensitive to the environment.

Figure 11 - Dimeric rhenium imaging agent

Figure 12 - A summary of the use of d-block lumophores as fluorescence imaging agents

f-block Lumophores Lanthanides behave as Lewis acids, they form a sphere of positive charge and, due to how diffuse the f-orbitals are, there is no overlap with the ligand lone pairs. They are therefore hard ions and prefer hard donors, the ligands are organised entirely due to stearic interactions and the bonds are mostly electrostatic and, consequently, labile. Stability is achieved in lanthanide chemistry through the use of large chelating systems. The crystal field splitting in lanthanide ions is negligible. Unlike d-block elements where it is responsible for the spectra we see, in f-block chemistry the emission is due to spin-coupling interactions. An interesting result of this is emission spectra that show peak wavelengths dependent only upon the metal ion in question, i.e. Eu3+ will always have emissions at characteristic frequencies. The intensity of the emission is affected by the nature of the solvent and ligand system, and different systems will provide different fine splitting and so, using these fingerprints, f-block lumophores can provide useful information on the environment. To optimise the intensity of the emitted light an f-block lumophore will usually contain an antennae molecule (Figure 13). Since the f-f transition is forbidden an organic antenna that first absorbs energy before passing it to the metal triplet state greatly enhances the practicality of using an fblock lumophore. The antenna used may also be built on d-block (Figure 14) functionality as opposed to purely organic antenna in which the transfer of energy is d to f triplet-triplet exchange. The result is a lumophore with a large Stokes shift and a very long lifetime, however, designing an antenna can be complex as it must have a higher energy singlet and triplet state than the lanthanide and so care must be taken in designing these systems. Design is further complicated by biological conditions, in which the large amount of environmental Ca2+ ions can lead to ion exchange resulting in toxicity from the lanthanide, this is a factor that is easy to overlook in the laboratory.
1

An
3

An

energy transfer
Ln*

An

Ln

Antenna

Lanthanide

Figure 13 - Simple Jablonski diagram showing the action of an antennae in f-block lumophores

f-block agents are much more suitable than d-block counterparts for time-gated experiments as a result of the long lifetime. They are also often near-infrared (NIR) emitters, ideal for use in vivo as NIR wavelengths easily penetrate the body while being at a low enough energy to not harm cells or interfere with cellular processes. There is a slight trade-off common to photophysics, in like systems the lifetime of the fluorescence will decrease as the NIR character of the emission increases, even further complicating ligand design.

The lanthanide complexes used in imaging are often not cationic but neutral, instead of passive diffusion across the potential barrier at the cell membrane they are instead taken into the cells by active transfer processes. The importance of active processes is proven by a simple experiment, by incubating the imaging agent with cells at 4oC, a temperature at which active transfer processes will be almost entirely stopped, and then raising the temperature back to 37 oC. If it can be shown that the agent enters the cell at body temperature, and if at 4 oC there is very little, if any, transfer the agent is being transported by active processes.

Figure 14 - The synthesis of an f-block imaging agent that utilises a d-block antenna

The Synthesis of f-block Imaging Agents The synthesis of these agents is often complicated and step-wise (Figure 15), although the coordination chemistry itself is simple, often involving little more than addition of the lanthanide halide and heating the solution.

Figure 15 - Synthesis of an f-block agent

In order to guarantee a ligand system that will successfully retain the lanthanide ion and provide suitable photophysics while maintaining tuneable biological properties there is often a need to protect and deprotect different moieties. The ligand design will often incorporate several carboxylates or other hard donors to saturate the coordination at the metal; this is in order to

prevent the complexation and subsequent vibration of water molecules that would otherwise quench fluorescent processes. Frster Resonance Energy Transfer (FRET) If two species have overlapping absorption/emission spectra it is possible for non-radiative, distance dependent (typically <10nm, much shorter than the wavelengths emitted) energy transfer via dipole-dipole coupling. This process can be used to probe the structure and folding arrangement of proteins due to the short-distance nature of the phenomenon. Radioimaging Positron Emission Tomography Radioimaging provides a much more realistic route to full-body scans than fluorescence imaging while also providing lower risk of toxicity, due to the incredibly small concentrations used, at a slightly poorer resolution, mm as opposed to nm. The emission of a positron (Figure 16), + decay, followed by annihilation with an electron, will release two collinear photons. The positron range will depend on the energy which is unique to each positron-emitter and is the reason for poorer resolution.

Figure 16 - Positron emission and annihilation

Isotopes with a longer half-life (Table 2) provide further benefit in that they can be used to track the development of a tumour or treatment through repeat scans over a given time period. Heavy metal isotopes are toxic and highly polar and so still require coordination chemistry to be safely applied.

Figure 17 - A PET scanner Table 2 - Isotopes common to PET imaging

Isotope
11 18

life (hrs) 0.3 1.8 13 6 89, 17 67 15 10, 78, 1 73 78 160

C F 64 Cu 99m Tc
186/188

Re

111 86

In Y Ga

66/67/68 201

Tl 89 Zr
117

Lu

Bio-relevant oxidation states; typical ligand families in biomedical applications N/A N/A Cu+, Cu2+, ATSM, polymine, polycarboxylate Tc+, TcV; tren, dipicolylamine Re+, ReV; tren, dipicolylamine, CO (PET); polypyridyls, CO (confocal microscopy) In3+ polymine, polycarboxylate Y3+ polymine, polycarboxylate Ga3+ polymine, polycarboxylate Tl3+ polymine, polycarboxylate Zr4+; polyanion, polyamine polyol (PET); cyclopentadienyl derivatives (cytology) Lu3+ polymine, polycarboxylate

64

Cu PET Agents

64

Cu is a positron emiutter with a half-life, t1/2, of 13 hours. Since Cu2+ is d9 and therefore Jahn Teller active, most agents are designed to be square planar with the two axial sites left free for water to coordinate (the rate of ligand exchange at the axial sites is practically diffusion controlled), otherwise macrocycles can be used to contain the metal. Copper is best suited to medium donor ligands, e.g. sulphur and nitrogen in the form of imines, Schiff bases and pyridines, and many are based on Cu-ATSM (Figure 18). Derivatives of Cu-ATSM are able to localise in hypoxic cells such as tumour cells, which grow so fast they are unable to maintain the required blood flow. It is suggested that in hypoxic cells the Cu2+ is reduced and unlike in healthy cells there is little re-oxidation, the cationic Cu+ species is then retained in the cells. Protonation may also explain how the localisation occurs as tumour cells are usually acidic.

Figure 18 A Cu-ATSM derivative

These species are synthesised by transmetallation of luminescent zinc analogues, this is a rapid process ensuring little loss of radiochemical yield (RCY). The luminescence of the zinc complexes is due to the organic fluorophore, the zinc is only responsible for holding the fluorophore on a plane. Copper is even more effective at holding the surrounding ligand in a plane, however d 9 paramagnetic copper is far too effective at quenching fluorescence. The zinc analogues have the potential to perform bimodal imaging in conjunction with the copper complexes. Single Photon Emission Computed Tomography SPECT imaging is similar to PET except it employs agents that decay by -emission.
99m

Tc SPECT Agents There are two main families of 99mTc agent; TcI(CO)3 units TcV complexes, usually L4Tc=O where L= nitrogen, oxygen or sulphur

Technetium is often generated as pertechnetate, TcVII, which is then reduced to a biocompatible form in the presence of a ligand to form a membrane permeable complex. TcI complexes are often derived from bispicolylamine which can be incorporated into bioactive units using peptide coupling through the single amino acid chelate (SAAC), upon complexation with TcI(CO)3(aq)3 (generated from TcVII in situ via TcO4- (aq)+ HC(O)BH2 [Tc(CO)3(H2O)3]I) the active agent is formed (Figure 19).

Figure 19 - Synthesis of Tc (CO)3L3 agents

Bimodal Imaging Bimodal imaging is the use of a single probe that utilises two different imaging modalities. This would allow for one agent that can image both the whole body and at a cellular level without the fear of false results, with separate agents the localisation may not be congruent. Using combined radio and fluorescence imaging agents is perhaps the ideal bimodal approach since radio imaging works on the whole body but with a low resolution, perfectly complimenting the high resolution provided by fluorescence. This could be used to guide surgeons or to diagnose more specific biochemistry within a tumour. Both radio and fluorescence depend on photon detection and so potentially both could be detected simultaneously. Several issues arise, the ligands for the radioisotope and fluorophore must be robustly linked, and it may be difficult to define a doseage since both techniques rely on different concentrations in general. One example of a potential bimodal agent is a Cu-ATSM-pyrene complex (Figure 20) that, unusually, does not show fluorescence quenching by the d9 Cu2+.

Figure 20 - A Cu-ATSM-pyrene bimodal imaging agent