& NMR Spectroscopy Filterable Agents for Hyperpolarization of Water, Metabolites, and Proteins Basile Vuichoud,* [a] AurØlien Bornet, [a] Florian de Nanteuil, [a] Jonas Milani, [a] Estel Canet, [a, b, c] Xiao Ji, [a, b, c] Pascal MiØville, [a] Emmanuelle Weber, [b] Dennis Kurzbach, [b] Andrea Flamm, [d] Robert Konrat, [d] Alvar D. Gossert, [e] Sami Jannin,* [a] and Geoffrey Bodenhausen [a, b, c] Abstract: Hyperpolarization is generated by dissolution dy- namic nuclear polarization (d-DNP) using a polymer-based polarizing agent dubbed FLAP (filterable labeled agents for polarization). It consists of a thermo-responsive poly(N-iso- propylacrylamide), also known as pNiPAM-COOH, labeled with nitroxide radicals. The polymer powder is impregnated with an arbitrary solution of interest and frozen as is. Disso- lution is followed by a simple filtration, leading to hyperpo- larized solutions free from any contaminants. We demon- strated the use of FLAP to hyperpolarize partially deuterated water up to P( 1 H) = 6 % with a long relaxation T 1 > 36 s char- acteristic of high purity. Water hyperpolarization can be transferred to drugs, metabolites, or proteins that are wait- ing in an NMR spectrometer, either by exchange of labile protons or through intermolecular Overhauser effects. We also show that FLAPs are suitable polarizing agents for 13 C- labeled metabolites such as pyruvate, acetate, and alanine. Introduction In the last decade, many applications of solution-state NMR and MRI have greatly benefited from dissolution dynamic nu- clear polarization (d-DNP). [1] At low temperatures and moder- ate magnetic fields (typically 1.2 < T < 4.2 K and 3.35 < B 0 < 7 T) the polarization of protons can be boosted by four to five orders of magnitude by saturating the ESR transitions of polar- izing agents such as TEMPOL (4-hydroxy-2,2,6,6-tetramethylpi- peridin-1-oxyl). Unfortunately, such polarizing agents lead to rapid nuclear spin relaxation during dissolution and transfer to the NMR or MRI systems. Protons are more sensitive to para- magnetic relaxation than low-gamma nuclei such as carbon-13 or nitrogen-15. Hyperpolarizing H 2 O or HDO represents a partic- ular challenge, because even low concentrations of free radi- cals cause the protons to relax significantly after dissolution, during transfer and in the NMR or MRI systems. Hyperpolarized H 2 O or HDO can be used in DNP-enhanced Water-LOGSY (Water-Ligand Observed via Gradient SpectroscopY) experi- ments [2] to detect ligand binding to macromolecules, or as a contrast agent for angiography. [3] Unfortunately, such applica- tions are hampered by the short T 1 ( 1 H) of HDO arising from the presence of free radicals. Following methods used in a pre- vious DNP-enhanced Water-LOGSY study, [2] we determined a disappointingly short T 1 ( 1 H) = 9.8 s in HDO after dissolution at 500 MHz, whereas T 1 ( 1 H) = 37 s in degassed and pure HDO. [3] Strategies have been proposed for neutralizing the polariz- ing agents via precipitation and filtration, [4] solvent extrac- tion, [5] or scavenging, [6] which typically require a few seconds during which relaxation occurs. We have recently introduced a new class of polarizing agents called HYPSO (HYbrid Polariz- ing SOlids) [7] consisting of mesoporous silica matrices with free radicals covalently anchored at the surface of the porous net- work. Though these polarizing agents turned out to be partic- ularly suitable for 13 C hyperpolarization, we found that proton polarization suffers considerable losses during the dissolution process, presumably because before being expelled from the porous network, protons are subjected to intense paramagnet- ic relaxation. Water hyperpolarization has been demonstrated recently with thermo-responsive hydrogels [8] via Overhauser DNP by Dollmann et al. [9] Modest enhancements of e( 1 H) = 27 were obtained at 0.345 T. Most recently, such hydrogels were used with dissolution-DNP to generate 13 C polarization of P( 13 C) = 2.1 % in tert-butanol. [10] [a] B. Vuichoud, Dr. A. Bornet, Dr. F. de Nanteuil, J. Milani, E. Canet, X. Ji, Dr. P. MiØville, Dr. S. Jannin, Prof. G. Bodenhausen Institut des Sciences et IngØnierie Chimiques Ecole Polytechnique FØdØrale de Lausanne 1015 Lausanne (Switzerland) E-mail : basile.vuichoud@epfl.ch sami.jannin@epfl.ch [b] E. Canet, X. Ji, E. Weber, Dr. D. Kurzbach, Prof. G. Bodenhausen DØpartement de Chimie Ecole Normale SupØrieure-PSL Research University 24 rue Lhomond, 75005 Paris (France) [c] E. Canet, X. Ji, Prof. G. Bodenhausen Sorbonne UniversitØs, UPMC Univ Paris 06 Ecole Normale SupØrieure, CNRS LBM 75005 Paris (France) [d] A. Flamm, Prof. R. Konrat Institute of Biomolecular Structural Chemistry University of Vienna, 1030 Vienna (Austria) [e] Dr. A. D. Gossert Institutes for BioMedical Research, Novartis 4002 Basel (Switzerland) Chem. Eur. J. 2016, 22, 14696 – 14700  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 14696 Full Paper DOI: 10.1002/chem.201602506