Rapidly Signal‐enhanced Metabolites for Atomic Scale Monitoring of Living Cells with Magnetic Resonance

Abstract

Nuclear magnetic resonance (NMR) is widely applied from analytics to biomedicine although it is an inherently insensitive phenomenon. Overcoming sensitivity challenges is key to further broaden the applicability of NMR and, for example, improve medical diagnostics. Here, we present a rapid strategy to enhance the signals of 13C‐labelled metabolites with para‐hydrogen and, in particular, 13C‐pyruvate, an important molecule for the energy metabolism. We succeeded to obtain an average of 27 % 13C polarization of 1‐13C‐pyruvate in water which allowed us to introduce two applications for studying cellular metabolism. Firstly, we demonstrate that the metabolism of 1‐13C‐pyruvate can serve as a biomarker in cellular models of Parkinson's disease and, secondly, we introduce the opportunity to combine real‐time metabolic analysis with protein structure determination in the same cells. Based on the here presented results, we envision the use of our approach for future biomedical studies to detect diseases.

A Field‐Independent Method for the Rapid Generation of Hyperpolarized [1‐13C]Pyruvate in Clean Water Solutions for Biomedical Applications

“…[20,21] In previous studies, we demonstrated that pulsed PHIP methods can lead to a high level of 13 C polarization ( � 60 %) on metabolite precursors in the organic phase within few seconds in weakly coupled spin systems at high magnetic fields. [22][23][24][25] Such results have demonstrated that para-H 2 -based methods can deliver 13 C polarization levels previously exclusive only to d-DNP.…”

“…Spin Order Transfer : after hydrogenation of the precursor, the para‐H 2 spin order is transferred on an intermediate heteronucleus in the sidearm employing the relayed MINERVA (Maximizing Insensitive Nuclei Enhancement Reached Via para‐H 2 Amplification) pulse sequence, [25] shown in figure 2…”

Parahydrogen-induced polarization and spin order transfer in ethyl pyruvate at high magnetic fields

“…When this manuscript was in preparation, remarkable polarization levels for pyruvate polarized at high magnetic elds were reported, although limited to low volumes in 5 mm NMR tubes [23] . As reported in this work, we repeated our experiments in acetone and observed spontaneous premature dissociation of the sidearm after hydrogenation (Figure S5, We measured the content of water in acetone to be about 30 mM which should be removed before the experiment.…”

“…The starting compound is an unsaturated pyruvate ester such as vinyl, propargyl, allyl, or another unsaturated ester, which is commonly labeled with 13 C in 1-position [22] . After hydrogenation with pH 2 and SOT, the reduced ester is cleaved yielding pyruvate that is hyperpolarized at the 1- 13 C nucleus with a relaxation time exceeding one minute [23,24] . Hence, all manipulations have to be performed rapidly to avoid loss of polarization.…”

A Field‐Independent Method for the Rapid Generation of Hyperpolarized [1‐13C]Pyruvate in Clean Water Solutions for Biomedical Applications

“…Hydrogenation: para-H 2 [1] is bubbled through an organic solvent solution (here acetone-d 6 ) containing a phenylpropynil pyruvate ester precursor (PPE) [2] and a homogeneous catalyst, here [Rh(dppb)COD]BF 4 ; 2. Spin Order Transfer: after hydrogenation of the precursor, the para-H 2 spin order is transferred on an intermediate heteronucleus in the sidearm employing the relayed MINERVA (Maximizing Insensitive Nuclei Enhancement Reached Via para-H 2 Amplification) pulse sequence, [25] shown in figure 2a), and in turn is transferred to the 13 The polarization transfer method presented here relies on the large J-couplings with a heteronucleus, to bring the Using the MINERVA pulse sequence, [25] the spin order is transferred to the carbonyl 13 C nucleus of the pyruvate moiety via the highlighted Jcouplings network ( J 12 ffi 11:6 Hz; J 13 ffi 0 Hz; J 23 ffi 160 Hz; J 34 ffi 2:3 Hz). Following the cleavage of the ester bond, the removal of the organic solvent and the filtration of the catalyst, HP [1-13 C]pyruvate [4] is left in a neat biocompatible water solution, see main text for details.…”