15 N-Permethylated amino acids as efficient probes for MRI-DNP applications Enrico Chiavazza a , Alessandra Viale a , Magnus Karlsson b and Silvio Aime a * The synthesis, NMR properties and preliminary polarization tests on protonated and perdeuterated forms of a-trimethylglutamine (NMe 3 Gln), a-trimethylglutamate (NMe 3 Glu) and «-trimethyllysine (NMe 3 Lys) are reported. The 15 N-permethylated, perdeuterated amino acids display very long 15 N-T 1 values, ranging between 190 and 330 s, are well polarized by the dynamic nuclear polarization (DNP) procedure, yielding good polarization levels (10%), and appear to be well tolerated by cells and mice. The obtained results make perdeuterated amino acids excellent candidates for innovative DNP 15 N-MRI applications such as perfusion or targeting studies. Copyright © 2013 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this paper. Keywords: DNP; hyperpolarized probe; 15 N; perfusion agent; spin–lattice relaxation time 1. INTRODUCTION There are a number of molecular imaging methodologies available; however, MRI with hyperpolarized substances has come under more intense scrutiny than others because it allows the intrinsic low sensitivity that is usually associated with MRI probes to be overcome. Over the last decade, the highly demanding requirements of this approach have driven impressive advances in hardware, pulse sequences and an increase in the number of hyperpolarized agents that are tailored for specific experiments (1). Three methods are commonly used to produce hyperpolarized systems: the optical pumping and spin exchange of noble gases, para-hydrogen-induced polarization and dynamic nuclear polarization (DNP) (2). DNP is the most versatile approach as, in principle, it allows every nucleus in every molecule to be polarized. Furthermore, fast dissolution methods have been developed that allow polarization maintenance in the liquid state (3). For these reasons DNP has become the method of choice for enhancing the sensitivity of nuclei in a number of metabolites and other molecules for potential MR applications, such as 13 C in urea, pyruvic acid, bicarbonate, sodium acetate (4,5), fumarate (6) and glycine (7), 1 H in alanine–glycine (8,9), 15 N in acetylcholine and choline chloride (10), 89 Y in yttrium chloride and its complexes with DOTAM and similar ligands (11–13) and 6 Li in lithium chloride (14). 13 C pyruvate is currently in the clinical phase of trials as a probe for the visualization of prostate tumors (NCT01229618) (15). The possibility of using hyperpolarized resonances in MR spectroscopy or in MR imaging is strictly dependent on the relaxation time. In fact, polarization decays as a function of the longitudinal relaxation time T 1 . Great effort has therefore been devoted to identifying species that display long T 1 values. Low g nuclei that do not present any directly bound protons and are confined to highly symmetrical environments are characterized by long T 1 values. Relaxation time can be further elongated in perdeuterated molecules because the dipolar contribution to the relaxation arising from non-directly bound protons is markedly reduced in these systems. For example, it has been reported that the 15 N-T 1 in choline is much longer upon the perdeuteration of the methyl groups than in the parent [390  110s (10) vs 202  12s (16)]. In spite of the outstanding enhancement, hyperpolarized substrates are used at relatively high concentrations in order to yield detectable responses in the corresponding MR images. This is because they naturally become more diluted after injection, and relaxation is faster in blood. These factors may constitute an issue when dealing with metabolic substrates (17,18). An alternative approach to the use of endogenous substances for hyperpolarized assays is the use of non-naturally occurring substrates that do not enter metabolic cycles (17). Of course, these non-naturally occurring substrates have to be well tolerated by living systems (18). The use of the long T 1 hyperpolarized resonances of non-naturally occurring molecules may be particu- larly useful in diagnostic applications in the field of quantitative perfusion studies (as an alternative to nuclear medicine and Gd-based MRI approaches) and/or of targeting applications. On this basis, we deemed that a new class of long relaxation time substrates may be found in 15 N-permethylated amino acids that contain a long-T 1 moiety analogous to that of choline. Methylation is a post-translational process found in vivo for many proteins and, upon enzymatic hydrolysis, the released methylated amino acids appear to be well tolerated by living systems. We herein report the synthesis, NMR properties and preliminary polarization tests on both protonated and perdeuterated forms of a-trimethylglutamine (NMe 3 Gln), a-trimethylglutamate (NMe 3 Glu) and e-trimethyllysine (NMe 3 Lys) as representative examples of a * Correspondence to: A. Silvio, Department of Chemistry and Center for Molecular ImagingUniversity of TurinVia Nizza 52, 10125 Torino, Italy. E-mail: silvio.aime@unito.it a E. Chiavazza, A. Viale, S. Aime Department of Chemistry and Center for Molecular Imaging, University of Turin, Via Nizza 52, 10125 Torino, Italy b M. Karlsson Albeda Research, Gamle Carlsberg, Vej 10, 1799 Copenhagen, Denmark Short Communication Received: 14 September 2012, Revised: 15 January 2013, Accepted: 3 February 2013, Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cmmi.1538 Contrast Media Mol. Imaging 2013, 8 417–421 Copyright © 2013 John Wiley & Sons, Ltd. 417