Subha Viswanathan

Alternatives to Gadolinium-Based Metal Chelates for Magnetic Resonance Imaging†

Magnetic Resonance Imaging (MRI) has been immensely valuable in diagnostic clinical imaging over the last few decades owing to its exceptional spatial and anatomical resolution. The signal in MRI is generated by relaxation of the transverse component of the net magnetization of protons present in the body, predominantly from bulk water. Thus, any agent or process that affects the net magnetization of the water protons in body tissues will also influence image contrast. Gd3+-based contrast agents shorten both the longitudinal and transverse relaxation times (T1 and T2) of water protons to approximately the same extent, in essence by relaxing all nearby proton spins. This effect is detected as increased signal intensity in T1 weighted MRI images when the appropriate pulse sequence is applied. Over the past 25 years Gd3+-complexes have been spectacularly successful as extracellular or blood pool T1 agents but their relative insensitivity to changes in environment coupled with the fact that they are never completely silent limits their applicability in the design of responsive MRI agents. A conceptually different approach to contrast enhancement is based on chemical exchange saturation transfer (CEST). This technique relies on dynamic chemical exchange processes inherent in biological tissues to transfer saturated 1H spins into the bulk water proton pool, which leads to a decrease of net magnetization and is detected as a negative contrast (darkening of the image) in MRI. Originally exchangeable -NH and -OH protons of various biomolecules were used to generate CEST contrast (DIACEST). However, these agents suffer from a few drawbacks, particularly in association with the small, usually less than 6 ppm, chemical shift difference between the two exchanging pools. The great benefit of using paramagnetic hyperfine shifting lanthanide complexes as CEST agents (PARACEST) is that the chemical shift difference between the two exchanging pools can potentially be much larger, up to several hundred ppm, facilitating easy saturation of one of the exchangeable spin pools without partial saturation of the bulk water pool. Another advantage of PARACEST is that the exchangeable sites are not limited to -NH or -OH protons but sites with faster exchange rates such as a Ln3+-bound H2O molecule, in particular, can also be considered. Since the water exchange rate on lanthanide complexes is extremely sensitive to the chemical environment, this has created unprecedented opportunities in the design of responsive PARACEST agents. In addition, multi-frequency MRI imaging is inherent to PARACEST: multiple agents present in the body can be imaged in one experiment by selectively turning on and off each agent by applying the appropriate saturation frequency.

Para-Derivatized Pybox Ligands As Sensitizers in Highly Luminescent Ln(III) Complexes

New complexes of pyridine-bis(oxazoline) derivatized with -H, -OMe, and -Br at the para position of the pyridine ring with Eu(III) and Tb(III) have been isolated. These are highly luminescent in the solid state, regardless of the ligand-to-metal ratio. Several of the metal complexes were isolated and characterized by single crystal X-ray diffraction, showing the rich diversity of structures that can be obtained with this family of ligands. [Eu(PyboxOMe)(3)](NO(3))(3)·3CH(2)Cl(2), 1, crystallizes in the monoclinic space group P2(1)/n and has the cell parameters a = 14.3699(10) Å, b = 13.4059(9) Å, c = 25.8766(18) Å, β = 95.367(1)°, and V = 4963.1(6) Å(3). The isostructural [Tb(PyboxOMe)(3)](NO(3))(3)·3CH(2)Cl(2), 2, crystallizes with the parameters a = 14.4845(16) Å, b = 13.2998(15) Å, c = 25.890(3) Å, β = 94.918(2)°, and V = 4969.1(10) Å(3). 3, a 1:1 complex with the formula [Eu(PyboxBr)(NO(3))(3)(H(2)O)], crystallizes in the monoclinic P2(1)/c space group with a = 11.649(2) Å, b = 8.3914(17) Å, c = 20.320(4) Å, β = 100.25(3)°, and V = 1954.5(7) Å(3). 4, a product of the reaction of PyboxBr with Tb(NO(3))(3), is [Tb(PyboxBr)(2)(η(2)-NO(3))(η(1)-NO(3)](2)[Tb(NO(3))(5)]·5H(2)O. It crystallizes in the monoclinic space group P2(1) with a = 15.612(3) Å, b = 14.330(3) Å, c = 16.271(3) Å, β = 92.58(3)°, and V = 3636.5(13) Å(3). [Tb(Pybox)(3)](CF(3)SO(3))(3)·3CH(2)CN, 5, crystallizes in the triclinic space group P1̅ with a = 12.3478(2) Å, b = 15.0017(2) Å, c = 16.1476(4) Å, α = 100.252(1)°, β = 100.943(1)°, γ = 113.049(1)°, and V = 2594.80(8) Å(3). Finally, compound 6, [Tb(Pybox)(2)(NO(3))(H(2)O)](NO(3))(2)·CH(3)OH, crystallizes in the triclinic P1̅ space group with a = 9.7791(2) Å, b = 10.1722(2) Å, c = 15.3368(3) Å, α = 83.753(1)°, β = 78.307(1)°, γ = 85.630(1)°, and V = 1482.33(5) Å(3). In solution, the existence of 3:1, 2:1, and 1:1 species can be observed through absorption and luminescence speciation measurements as well as NMR spectroscopy. The stability constants in acetonitrile, as an average obtained from absorption and emission titrations, are log β(11) = 5.4, log β(12) = 8.8, and log β(13) = 12.8 with Eu(III) and log β(11) = 4.5, log β(12) = 8.4, and log β(13) = 11.7 for the Tb(III) species with PyboxOMe. Pybox displayed stability constants log β(11) = 3.6, log β(12) = 9.1, and log β(13) = 12.0 with Eu(III) and log β(11) = 3.7, log β(12) = 9.3, and log β(13) = 12.2 for the Tb(III) species. Finally, PyboxBr yielded log β(11) = 7.1, log β(12) = 12.2, and log β(13) = 15.5 for the Eu(III) species and log β(11) = 6.2, log β(12) = 11.0, and log β(13) = 15.4 with Tb(III). Photophysical characterization was performed in all cases on solutions with 3:1 ligand-to-metal ion stoichiometry and allowed determination of quantum yields and lifetimes of emission for PyboxOMe of 23.5 ± 1.6% and 1.54 ± 0.04 ms for Eu(III) and 21.4 ± 3.6% and 1.88 ± 0.04 ms for Tb(III). For Pybox these values were 25.6 ± 1.1% and 1.49 ± 0.04 ms for Eu(III) and 23.2 ± 2.1% and 0.44 ± 0.01 ms for Tb(III) and for PyboxBr they were 35.8 ± 1.6% and 1.46 ± 0.03 ms for Eu(III) and 23.3 ± 1.3% and a double lifetime of 0.79 ± 0.05/0.07 ± 0.01 ms for Tb(III). A linear relationship between the triplet level energies and the Hammett σ constants was found. Lifetime measurements in methanol as well as the NMR data in both methanol and acetonitrile indicate that all complexes are stable in the 3:1 stoichiometry in solution and that there is no solvent coordination to the metal ion.

Multi-Frequency PARACEST Agents Based on Europium(III)-DOTA-Tetraamide Ligands†

Magnetic resonance imaging (MRI) is one of the most versatile and powerful diagnostic tools in modern medicine. Recently, a conceptually different approach to contrast enhancement based on chemical exchange saturation transfer (CEST) has emerged that takes advantage of slow-to-intermediate exchange conditions between two or more pools of protons (kex ≤ Δω).[1] While the first reported CEST agents were diamagnetic molecules containing exchangeable NH and OH groups (Δω ≤ 5 ppm), it was later shown that the slow water exchange characteristics of certain paramagnetic Ln3+ complexes of DOTA-tetraamide ligands allows selective saturation of a hyperfine shifted Ln3+-bound water pool (Δω > 50 ppm) for creating CEST contrast.[2] Radio frequency (RF) saturation of highly shifted exchange resonances in paramagnetic systems offer significant advantages over diamagnetic CESTagents with small Δω values.[3]

Europium(III) DOTA-derivatives having ketone donor pendant arms display dramatically slower water exchange

A series of new 1,4,7,10-tetraazacyclododecane-derivatives having a combination of amide and ketone donor groups as side-arms were prepared, and their complexes with europium(III) studied in detail by high resolution NMR spectroscopy. The chemical shift of the Eu(3+)-bound water resonance, the chemical exchange saturation transfer (CEST) characteristics of the complexes, and the bound water residence lifetimes (τ(m)) were found to vary dramatically with the chemical structure of the side-arms. Substitution of ketone oxygen donor atoms for amide oxygen donor atoms resulted in an increase in residence water lifetimes (τ(m)) and a decrease in chemical shift of the Eu(3+)-bound water molecule (Δω). These experimental results along with density functional theory (DFT) calculations demonstrate that introduction of weakly donating oxygen atoms in these complexes results in a much weaker ligand field, more positive charge on the Eu(3+) ion, and an increased water residence lifetime as expected for a dissociative mechanism. These results provide new insights into the design of paramagnetic CEST agents with even slower water exchange kinetics that will make them more efficient for in vivo imaging applications.

Dispersion-free continuum two-dimensional electronic spectrometer

Electronic dynamics span broad energy scales with ultrafast time constants in the condensed phase. Two-dimensional (2D) electronic spectroscopy permits the study of these dynamics with simultaneous resolution in both frequency and time. In practice, this technique is sensitive to changes in nonlinear dispersion in the laser pulses as time delays are varied during the experiment. We have developed a 2D spectrometer that uses broadband continuum generated in argon as the light source. Using this visible light in phase-sensitive optical experiments presents new challenges in implementation. We demonstrate all-reflective interferometric delays using angled stages. Upon selecting an ~180  nm window of the available bandwidth at ~10  fs compression, we probe the nonlinear response of broadly absorbing CdSe quantum dots and electronic transitions of Chlorophyll a.

Communication: Coherences observed in vivo in photosynthetic bacteria using two-dimensional electronic spectroscopy

Energy transfer through large disordered antenna networks in photosynthetic organisms can occur with a quantum efficiency of nearly 100%. This energy transfer is facilitated by the electronic structure of the photosynthetic antennae as well as interactions between electronic states and the surrounding environment. Coherences in time-domain spectroscopy provide a fine probe of how a system interacts with its surroundings. In two-dimensional electronic spectroscopy, coherences can appear on both the ground and excited state surfaces revealing detailed information regarding electronic structure, system-bath coupling, energy transfer, and energetic coupling in complex chemical systems. Numerous studies have revealed coherences in isolated photosynthetic pigment-protein complexes, but these coherences have not been observed in vivo due to the small amplitude of these signals and the intense scatter from whole cells. Here, we present data acquired using ultrafast video-acquisition gradient-assisted photon echo spectroscopy to observe quantum beating signals from coherences in vivo. Experiments were conducted on isolated light harvesting complex II (LH2) from Rhodobacter sphaeroides, whole cells of R. sphaeroides, and whole cells of R. sphaeroides grown in 30% deuterated media. A vibronic coherence was observed following laser excitation at ambient temperature between the B850 and the B850(∗) states of LH2 in each of the 3 samples with a lifetime of ∼40-60 fs.

Improved synthesis of DOTA tetraamide ligands for lanthanide(III) ions: A tool for increasing the repertoire of potential PARACEST contrast agents for MRI and/or fluorescent sensors

The synthesis of new DOTA tetraamide (DOTAMR(4)) compounds is of great interest given their application in the formation of Ln(III) complexes as potential PARACEST contrast agents in MRI or fluorescent molecular probes. In this context amino acid and peptide DOTAMR(4) derivatives are particularly attractive since the amino-acid and/or peptide moiety can show responsive properties dependent on a given stimuli which might translate to changes in water exchange rates of the corresponding Ln(III) complex. Current synthesis of DOTAMR(4) derivatives is typically carried out by reacting haloacetamide intermediates with cyclen. However, this method fails to generate the tetra-substituted products when bulky substituents are present in the haloacetamide and in some cases this intermediate cannot be prepared by conventional acylation procedures limiting the number of DOTAMR(4) compounds available for study. As a solution to these limitations, an improved methodology for the synthesis of DOTAMR(4) by coupling DOTA to an appropriate amine containing reagent (i.e. protected amino-acids with the alpha-amino group free) is presented in this work. Several DOTAMR(4) derivatives which are difficult or impossible to prepare with the traditional methodologies were easily obtained starting with DOTA. A new protocol was derived using this methodology for the solution-phase synthesis of DOTA peptide derivatives. With this methodology, many other DOTAMR(4) peptide and non-peptide derivatives have been prepared in our laboratories with several of these new compounds showing interesting properties for molecular imaging.