Choline is a precursor of cellular phospholipid metabolism that provides Magnetic Resonance (MR) and Positron Emission Tomography (PET) biomarkers for cancer detection and response assessment. Employing Dynamic Nuclear Polarization we show that the MR signal of 15N in choline can be enhanced by at least 4 orders of magnitude with a relaxation time of ca. 4 min, providing a method to observe the action of choline kinase, an important target for novel cancer therapeutics.

Ups and downs of DNP: Polychlorinated trityl radicals (see structure: C black, Cl green, Na gray, O red) used for dynamic nuclear polarization (DNP) showed a new transfer mechanism involving quadrupolar chlorine nuclei. The observation of positive or negative enhancements, depending on the substrate, highlights the supramolecular character of the initial polarization‐transfer process.

Polychorinated trityl radicals bearing carboxilate substituents are water soluble persistent radicals that can be used for Dynamic Nuclear Polarization. In contrast to other trityl radicals, the polarization mechanism differs from the classical solid effect. DFT calculations performed to 10 rationalize this behaviour support the hypothesis that polarization is transferred from the unpaired electron to chlorine nuclei and from these to carbon by spin diffusion. The marked differences observed between neutral and anionic forms of the radical will be discussed.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene-directed enzyme prodrug therapy (GDEPT). Employing dynamic nuclear polarization (DNP) and natural abundance 13 C magnetic resonance spectroscopy (MRS), we observed the CPG2-mediated conversion of a novel hyperpolarized reporter probe 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu) to 3,5-difluorobenzoic acid (3,5-DFBA) and L-glutamic acid (L-Glu) in vitro. Isotopic labeling of the relevant nuclei with 13 C in 3,5-DFBGlu or related substrates will yield a further factor of 100 increase in the signal-to-noise. We discuss the feasibility of translating these experiments to generate metabolic images of CPG2 activity in vivo. New frontiers in metabolic imaging have recently been realized in vivo through a combination magnetic resonance spectroscopy (MRS) and novel hyperpolarization techniques employing dynamic nuclear polarization (DNP). The significant enhancement of the MR signal by more than a factor of 10,000 has transformed insensitive techniques such as 13 C and 15 N MRS into versatile strategies that afford a window on the dynamics of endogenous enzymatic processes by generating high spatial resolution and real-time maps of the metabolism of hyperpolarized substrates noninvasively (1,2). These techniques have primarily focused on hyperpolarized 13 C pyruvate as a substrate due to its favorable relaxation characteristics and central role in cellular energy metabolism (3,4). Other hyperpolarized imaging reporters have emerged in recent years to probe a range of key endogenous metabolic reactions (5-8), and further applications such as the measurement of pH in vivo (9).The bacterial enzyme carboxypeptidase G2 (CPG2, EC 3.4.17.11) and other exogenous enzymes have been utilized in promising targeted chemotherapeutic strategies to activate selectively nontoxic prodrugs into cytotoxic drugs in tumors (10). Several strategies have been developed to target the enzyme to a tumor, including the use of CPG2-antibody conjugates in antibody-directed enzyme prodrug therapy (ADEPT) (11), viral vectors that carry the gene encoding for CPG2 in gene-directed enzyme prodrug therapy (GDEPT) (12), and more recently the use of bacteria engineered to express CPG2 (13). These therapeutic strategies would benefit from robust imaging strategies that afford high spatial and temporal resolution images of the biodistribution of CPG2 activity.We have focused on developing MRI reporters for CPG2, a Zn 2ϩ -dependent exopeptidase that activates relatively nontoxic prodrugs into activated DNA alkylating agents by removing their glutamate moiety. 3,5-Difluorobenzoyl-Lglutamic acid (3,5-DFBGlu) is a reporter probe that can be used to detect CPG2 activity in vivo using 19 F MRS, utilizing a 1.4 ppm chemical shift difference associated with the CPG2-mediated conversion of 3,5-DFBGlu to 3,5-DFBA and L-glutamic acid (Fig. 1) (14). In this study we investigated the MR properties of the relevant 13 C nuclei (na...

Gadolinium contrast agents are known to affect the outer-sphere relaxivities of nuclei other than protons. We have investigated the heteronuclear relaxivities of four commercial gadolinium contrast agents (Magnevist, Omniscan, Dotarem and Gadovist) on the relaxation of (13)C in glycine, (13)C in pyruvate and (15)N in choline with long relaxation times. Marked differences in the relaxivities were found between different contrast agents and are attributed to electrostatic effects. This methodology may find applications in the field of hyperpolarized magnetic resonance and by way of example we show that injection of a bolus of contrast agent into an aqueous solution containing hyperpolarized (15)N labeled tetramethylammonium or (13)C labeled pyruvate leads to a predictable shortening of the lifetime of the hyperpolarized signal.

Choline metabolites are widely studied in cancer research as biomarkers of malignancy and as indicators of therapeutic response. However, endogenous phosphocholine levels are determined by a number of processes that confound the interpretation of these measurements, including membrane transport rates and a series of enzyme catalysed reactions in the Kennedy pathway. Employing a dynamic (31)P NMR assay that is specific to choline kinase (ChoK) we have measured the rates of this enzyme reaction in cell lysates of MDA-MB-231 breast, PC-3 prostate and HeLa cervical cancer cells and in solutions of purified human ChoK. The rates are sensitive to inhibition by hemicholinium-3 (HC-3), a competitive ChoK inhibitor, and to N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulphonamide (H-89), an agent commercialized as a specific cyclic-AMP-dependent protein kinase A (PKA) inhibitor.

Positiv oder negativ: Polychlorierte Tritylradikale (siehe Struktur: C schwarz, Cl grün, Na grau, O rot) zeigten beim Einsatz in der dynamischen Kernpolarisation (DNP) einen neuen Transfermechanismus unter Beteiligung der quadrupolaren Chlorkerne. Die Beobachtung positiver oder negativer Verstärkungen für verschiedene Substrate spricht für einen supramolekularen Charakter des anfänglichen Polarisationstransfers.

Pimonidazole is one of a series of nitroimidazole compounds that is widely used as a marker for qualitative and quantitative assessment of tumour hypoxia. We have observed a novel dynamic conformational exchange process in this molecule in aqueous solution. By a combination of 1H, 13C, two-dimensional 1H-1H EXchange SpectroscopY (EXSY) and spectral simulation, we unambiguously attribute the conformational exchange process to flipping of the six-membered heterocyclic ring.