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Stable tetrathiatriarylmethyl radicals have significantly contributed to the recent progress in biomedical electron paramagnetic resonance (EPR) due to their unmatched stability in biological media and long relaxation times. However, the lipophilic core of the most commonly used structure (Finland trityl) is responsible for its interaction with plasma biomacromolecules, such as albumin, and self-aggregation at high concentrations and/or low pH. While Finland trityl is generally considered inert toward many reactive radical species, we report that sulfite anion radical efficiently substitutes the three carboxyl moieties of Finland trityl with a high rate constant of 3.53 × 108 M–1 s–1, leading to a trisulfonated Finland trityl radical. This newly synthesized highly hydrophilic trityl radical shows an ultranarrow linewidth (ΔB pp = 24 mG), a lower affinity for albumin than Finland trityl, and a high aqueous solubility even at acidic pH. Therefore, this new tetrathiatriarylmethyl radical can be considered as a superior spin probe in comparison to the widely used Finland trityl. One of its potential applications was demonstrated by in vivo mapping oxygen in a mouse model of breast cancer. Moreover, we showed that one of the three sulfo groups can be easily substituted with S-, N-, and P-nucleophiles, opening access to various monofunctionalized sulfonated trityl radicals.

Tetrathiatriarylmethyl (TAM) radicals represent soluble paramagnetic probes for biomedical electron paramagnetic resonance (EPR)-based spectroscopy and imaging. There is an increasing demand in the development of multifunctional, biocompatible and targeted trityl probes hampered by the difficulties in derivatization of the TAM structure. We proposed a new straightforward synthetic strategy using click chemistry for the covalent conjugation of the TAM radical with a water-soluble biocompatible carrier exemplified here by dextran. A set of dextran-grafted probes varied in the degrees of Finland trityl radical loading and dextran modification by polyethelene glycol has been synthesized. The EPR spectrum of the optimized macromolecular probe exhibits a single narrow line with high sensitivity to oxygen and has advantages over the unbound Finland trityl of being insensitive to interactions with albumin. In vivo EPR imaging of tissue oxygenation performed in breast tumor-bearing mouse using dextran-grafted probe demonstrates its utility for preclinical oximetric applications. Synthesis of the first organic free radical, triphenylmethyl, was reported by Gomberg in 1900 1. By the late 1990s, these compounds with sterically protected trivalent carbon regained attention as the core structural fragment for the synthesis of stable organic radicals. Nycomed Innovation designed the sterically crowded trityl radicals, TAMs (tetrathiatriarylmethyl), in order to avoid hydrogen hyperfine coupling and enhance radical stability and water solubility 2-3. Currently TAMs represent one of the major classes of soluble paramagnetic probes characterized by extraordinary stability toward tissue redox processes, long relaxation time and narrow line width making them particularly attractive for electron paramagnetic resonance (EPR)-based spectroscopy and imaging applications 3-7. Figure 1 shows the most popular structures of TAM oximetric probes, Finland trityl (FTr) 8-9 and OX063 4 , their deuterated derivatives 5, 10-12 , and the recently synthesized multifunctional monophosphonated probe, HOPE 13-14

The advent of hybrid scanners, combining complementary modalities, has revolutionized the application of advanced imaging technology to clinical practice and biomedical research. In this project, we investigated the melding of two complementary, functional imaging methods: positron emission tomography (PET) and electron paramagnetic resonance imaging (EPRI). PET radiotracers can provide important information about cellular parameters, such as glucose metabolism. While EPR probes can provide assessment of tissue microenvironment, measuring oxygenation and pH, for example. Therefore, a combined PET/EPRI scanner promises to provide new insights not attainable with current imagers by simultaneous acquisition of multiple components of tissue microenvironments. To explore the simultaneous acquisition of PET and EPR images, a prototype system was created by combining two existing scanners. Specifically, a silicon photomultiplier (SiPM)-based PET scanner ring designed as a portable scanner was combined with an EPRI scanner designed for the imaging of small animals. The ability of the system to obtain simultaneous images was assessed with a small phantom consisting of four cylinders containing both a PET tracer and EPR spin probe. The resulting images demonstrated the ability to obtain contemporaneous PET and EPR images without cross-modality interference. Given the promising results from this initial investigation, the next step in this project is the construction of the next generation pre-clinical PET/EPRI scanner for multi-parametric assessment of physiologically-important parameters of tissue microenvironments.

Functional four-dimensional spectral-spatial electron paramagnetic imaging (EPRI) is routinely used in biomedical research. Positions and widths of EPR lines in the spectral dimension report oxygen partial pressure, pH, and other important parameters of the tissue microenvironment. Images are measured in the homogeneous external magnetic field. An application of EPRI is proposed in which the field is perturbed by a magnetized object. A proof-of-concept imaging experiment was conducted, which permitted visualization of the magnetic field created by this object. A single-line lithium octa-n-butoxynaphthalocyanine spin probe was used in the experiment. The spectral position of the EPR line directly measured the strength of the perturbation field with spatial resolution. A three-dimensional magnetic field map was reconstructed as a result. Several applications of this technology can be anticipated. First is EPRI/MPI co-registration, where MPI is an emerging magnetic particle imaging technique. Second, EPRI can be an alternative to magnetic field cameras that are used for the development of high-end permanent magnets and their assemblies, consumer electronics, and industrial sensors. Besides the high resolution of magnetic field readings, EPR probes can be placed in the internal areas of various assemblies that are not accessible by the standard sensors. Third, EPRI can be used to develop systems for magnetic manipulation of cell cultures.

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