ParaHydrogen Polarized Ethyl-[1-13C]pyruvate in Water,a Key Substrate for Fostering the PHIP-SAH Approach to Metabolic Imaging

An efficient synthesis of vinyl-[1-¹³C]pyruvate has been reported, from which ¹³C hyperpolarized (HP) ethyl-[1-¹³C]pyruvate has been obtained by means of ParaHydrogen Induced Polarization (PHIP). Due to the intrinsic lability of pyruvate, which leads quickly to degradation of the reaction mixture even under mild reaction conditions, the vinyl-ester has been synthesized through the intermediacy of a more stable ketal derivative. ¹³C and ¹H hyperpolarizations of ethyl-[1-¹³C]pyruvate, hydrogenated using ParaHydrogen, have been compared to those observed on the more widely used allyl-derivative. It has been demonstrated that the spin order transfer from ParaHydrogen protons to ¹³C, is more efficient on the ethyl than on the allyl-esterdue to the larger J-couplings involved. The main requirements needed for the biological application of this HP product have been met, i. e. an aqueous solution of the product at high concentration (40 mM) with a good ¹³C polarization level (4.8 %) has been obtained. The in vitro metabolic transformation of the HP ethyl-[1-¹³C]pyruvate, catalyzed by an esterase, has been observed. This substrate appears to be a good candidate for in vivo metabolic investigations using PHIP hyperpolarized probes.     

A Toolbox for Glutamine Use in Dissolution Dynamic Nuclear Polarization: from Enzymatic Reaction Monitoring to the Study of Cellular Metabolic Pathways and Imaging

Glutamine is under scrutiny regarding its metabolic deregulation linked to energetic reprogramming in cancer cells. Many analytical techniques have been used to better understand the impact of the metabolism of amino acids on biological processes, however only a few are suited to work with complex samples. Here, we report the use of a general dissolution dynamic nuclear polarization (D-DNP) formulation using an unexpensive radical as a multipurpose tool to study glutamine, with insights from enzymatic modelling to complex metabolic networks and fast imaging. First, hyperpolarized [5-¹³C] glutamine is used as molecular probe to study the kinetic action of two enzymes: L-asparaginase that has been used as an anti-metabolic treatment for cancer, and glutaminase. These results are also compared with those acquired with another hyperpolarized amino acid, [1,4-¹³C] asparagine. Second, we explored the use of hyperpolarized (HP) substrates to probe metabolic pathways by monitoring metabolic profiles arising from hyperpolarized glutamine in E. coli extracts. Finally, a highly concentrated sample formulation is proposed for the purpose of fast imaging applications. We think that this approach can be extended to formulate other amino acids as well as other metabolites and provide complementary insights into the analysis of metabolic networks.     

Order-Unity 13C Nuclear Polarization of [1-13C]Pyruvate in Seconds and the Interplay of Water and SABRE Enhancement

Signal Amplification By Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) is investigated to achieve rapid hyperpolarization of ¹³C₁ spins of [1-¹³C]pyruvate, using parahydrogen as the source of nuclear spin order. Pyruvate exchange with an iridium polarization transfer complex can be modulated via a sensitive interplay between temperature and co-ligation of DMSO and H₂O. Order-unity ¹³C (>50 %) polarization of catalyst-bound [1-¹³C]pyruvate is achieved in less than 30 s by restricting the chemical exchange of [1-¹³C]pyruvate at lower temperatures. On the catalyst bound pyruvate, 39 % polarization is measured using a 1.4 T NMR spectrometer, and extrapolated to >50 % at the end of build-up in situ. The highest measured polarization of a 30-mM pyruvate sample, including free and bound pyruvate is 13 % when using 20 mM DMSO and 0.5 M water in CD₃OD. Efficient ¹³C polarization is also enabled by favorable relaxation dynamics in sub-microtesla magnetic fields, as indicated by fast polarization buildup rates compared to the T₁ spin-relaxation rates (e. g., ∼0.2 s⁻¹ versus ∼0.1 s⁻¹, respectively, for a 6 mM catalyst-[1-¹³C]pyruvate sample). Finally, the catalyst-bound hyperpolarized [1-¹³C]pyruvate can be released rapidly by cycling the temperature and/or by optimizing the amount of water, paving the way to future biomedical applications of hyperpolarized [1-¹³C]pyruvate produced via comparatively fast and simple SABRE-SHEATH-based approaches.     

A general chemical shift decomposition method for hyperpolarized 13C metabolite magnetic resonance imaging

Metabolic imaging with hyperpolarized carbon‐13 allows sequential steps of metabolism to be detected in vivo. Potential applications in cancer, brain, muscular, myocardial, and hepatic metabolism suggest that clinical applications could be readily developed. A primary concern in imaging hyperpolarized nuclei is the irreversible decay of the enhanced magnetization back to thermal equilibrium. Multiple methods for rapid imaging of hyperpolarized substrates and their products have been proposed with a multi‐point Dixon method distinguishing itself as a robust protocol for imaging [1‐¹³C]pyruvate. We describe here a generalized chemical shift decomposition method that incorporates a single‐shot spiral imaging sequence plus a spectroscopic sequence to retain as much spin polarization as possible while allowing detection of metabolites that have a wide range of chemical shift values. The new method is demonstrated for hyperpolarized [1‐¹³C]pyruvate, [1‐¹³C]acetoacetate, and [2‐¹³C]dihydroxyacetone. Copyright © 2016 John Wiley & Sons, Ltd.     

Direct Monitoring of γ‐Glutamyl Transpeptidase Activity In Vivo Using a Hyperpolarized 13C‐Labeled Molecular Probe

The γ‐glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ‐Glu‐[1‐¹³C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) ¹³C magnetic resonance (MR). The properties of γ‐Glu‐[1‐¹³C]Gly are suitable for in vivo HP ¹³C metabolic analysis since the chemical shift between γ‐Glu‐[1‐¹³C]Gly and its metabolic product, [1‐¹³C]Gly, is large (4.3 ppm) and the T₁ of both compounds is relatively long (30 s and 45 s, respectively, in H₂O at 9.4 T). We also demonstrate that γ‐Glu‐[1‐¹³C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin.     

Hyperpolarized 13C Magnetic Resonance Imaging of Fumarate Metabolism by Parahydrogen-induced Polarization: A Proof-of-Concept in vivo Study

Hyperpolarized [1-¹³C]fumarate is a promising magnetic resonance imaging (MRI) biomarker for cellular necrosis, which plays an important role in various disease and cancerous pathological processes. To demonstrate the feasibility of MRI of [1-¹³C]fumarate metabolism using parahydrogen-induced polarization (PHIP), a low-cost alternative to dissolution dynamic nuclear polarization (dDNP), a cost-effective and high-yield synthetic pathway of hydrogenation precursor [1-¹³C]acetylenedicarboxylate (ADC) was developed. The trans-selectivity of the hydrogenation reaction of ADC using a ruthenium-based catalyst was elucidated employing density functional theory (DFT) simulations. A simple PHIP set-up was used to generate hyperpolarized [1-¹³C]fumarate at sufficient ¹³C polarization for ex vivo detection of hyperpolarized ¹³C malate metabolized from fumarate in murine liver tissue homogenates, and in vivo ¹³C MR spectroscopy and imaging in a murine model of acetaminophen-induced hepatitis.     

Amino Acid-Derived Sensors for Specific Zn2+ Detection Using Hyperpolarized 13C Magnetic Resonance Spectroscopy

Alterations in Zn²⁺ concentration are seen in normal tissues and in disease states, and for this reason imaging of Zn²⁺ is an area of active investigation. Herein, enriched [1-¹³C]cysteine and [1-¹³C₂]iminodiacetic acid were developed as Zn²⁺-specific imaging probes using hyperpolarized ¹³C magnetic resonance spectroscopy. [1-¹³C]cysteine was used to accurately quantify Zn²⁺ in complex biological mixtures. These sensors can be employed to detect Zn²⁺ via imaging mechanisms including changes in ¹³C chemical shift, resonance linewidth, or T₁.     

Heterogeneous 1H and 13C Parahydrogen-Induced Polarization of Acetate and Pyruvate Esters

Magnetic resonance imaging of [1-¹³C]hyperpolarized carboxylates (most notably, [1-¹³C]pyruvate) allows one to visualize abnormal metabolism in tumors and other pathologies. Herein, we investigate the efficiency of ¹H and ¹³C hyperpolarization of acetate and pyruvate esters with ethyl, propyl and allyl alcoholic moieties using heterogeneous hydrogenation of corresponding vinyl, allyl and propargyl precursors in isotopically unlabeled and 1-¹³C-enriched forms with parahydrogen over Rh/TiO₂ catalysts in methanol-d₄ and in D₂O. The maximum obtained ¹H polarization was 0.6±0.2 % (for propyl acetate in CD₃OD), while the highest ¹³C polarization was 0.10±0.03 % (for ethyl acetate in CD₃OD). Hyperpolarization of acetate esters surpassed that of pyruvates, while esters with a triple carbon-carbon bond in unsaturated alcoholic moiety were less efficient as parahydrogen-induced polarization precursors than esters with a double bond. Among the compounds studied, the maximum ¹H and ¹³C NMR signal intensities were observed for propyl acetate. Ethyl acetate yielded slightly less intense NMR signals which were dramatically greater than those of other esters under study.     

Inside Cover: Parahydrogen-Induced Carbon-13 Radiofrequency Amplification by Stimulated Emission of Radiation (Angew. Chem. Int. Ed. 5/2023)

The feasibility of Carbon-13 Radiofrequency (RF) Amplification by Stimulated Emission of Radiation (C-13 RASER) is demonstrated on a bolus of liquid hyperpolarized ethyl [1-¹³C]acetate. Hyperpolarized ethyl [1-¹³C]acetate was prepared via pairwise addition of parahydrogen to vinyl [1-¹³C]acetate and polarization transfer from nascent parahydrogen-derived protons to the carbon-13 nucleus via magnetic field cycling yielding C-13 nuclear spin polarization of approximately 6 %. RASER signals were detected from samples with concentration ranging from 0.12 to 1 M concentration using a non-cryogenic 1.4T NMR spectrometer equipped with a radio-frequency detection coil with a quality factor (Q) of 32 without any modifications. C-13 RASER signals were observed for several minutes on a single bolus of hyperpolarized substrate to achieve 21 mHz NMR linewidths. The feasibility of creating long-lasting C-13 RASER on biomolecular carriers opens a wide range of new opportunities for the rapidly expanding field of C-13 magnetic resonance hyperpolarization.     

A Strategy to Design Hyperpolarized 13C Magnetic Resonance Probes Using [1‐13C]α‐Amino Acid as a Scaffold Structure

Hyperpolarization is an emerging method that dramatically enhances NMR signal intensity. As a result of their increased sensitivity, hyperpolarized (HP) NMR molecular probes can be used to perform time‐resolved spectroscopy and imaging in vitro and in vivo. It is, however, challenging to design such probes de novo. Herein, the [1‐¹³C]α‐amino acid is reported as a scaffold structure to design HP ¹³C NMR molecular probes. The [1‐¹³C]α‐amino acid can be converted to various HP ¹³C chemical probes that show sufficient chemical shift change by altering the chemical state of the α nitrogen upon interaction with the target. Several previously reported HP probes could be explained by this design principle. To demonstrate the versatility of this approach, two α‐amino‐acid‐based HP ¹³C chemical probes, sensitive to pH and Ca²⁺ ion, were developed and used to detect targets.     

Biosynthesis of Mikrolin

Mikrolin (8) and dechloromikrolin (9) have been shown to exist as tautomeric mixtures in solution. The structures of mono-O-trifluoroacetyl Mikrolin (10) and di-O-acetyl Mikrolin (11) have been elucidated. The products 15 to 23 from reduction of the metabolites 8 9 with Pd/C and Zn in aqueous acetic acid have been identified. The ¹³C-NMR. spectra of Mikrolin (8) and dechloromikrolin (9) and their derivatives have been completely assigned Based on the results of incorporation experiments with sodium [1-¹³C]-, [2-¹³C]- and [1, 2-¹³C]-acetate, a biosynthetic pathway is proposed for Mikrolin (8) .     

Biosynthesis of cytochalasans. Part 4. The mode of incorporation of common naturally-occurring carboxylic acids into cytochalasin D1.

Utilization of sodium [1-¹⁴C]-, [2–¹⁴C]-, and [1,2-¹³C]-acetates, [1-¹⁴C]-, [1-¹³C]-, or [2-¹⁴C]-propionates, [1-¹⁴C]-or [2-¹⁴C]-malonates, of [1-¹⁴C]- or of [1-¹⁴C]-myristic acid, or of [1-¹⁴C]- and [1-¹⁴C]-palmitic acid in the biosynthesis of cytochalasin D ( 1 ) by Zygosporium masonii was determined by degradation studies or by carbon magnetic resonance spectroscopy. The precursors were incorporated primarily via the acetate-malonate pathway to generate 1 from nine intact acetate units, eight of which are coupled in a head to tail fashion to form the C₁₆-polyketide moiety.     

An asymmetric synthesis of l-[3-C]phenylalanine and l-[3-C]tyrosine from [C]carbon monoxide

-[3-¹³C]Phenylalanine and -[3-¹³C]tyrosine were synthesized. [α-¹³C]Benzyl bromides were prepared from [¹³C]carbon monoxide via the palladium-catalyzed carboalkoxylation of aryl halides. The asymmetric carbon corresponding to the 2-position in phenylalanine was introduced by the diastereoselective alkylation of Dellaria's oxazinone with [α-¹³C]benzyl bromides. Finally, ethanolysis, deprotection, hydrogenolysis and acid hydrolysis of the resulting alkylated oxazinones gave -[3-¹³C]phenylalanine and -[3-¹³C]tyrosine in high optical purity.     

Probing lactate dehydrogenase activity in tumors by measuring hydrogen/deuterium exchange in hyperpolarized l-[1-(13)C,U-(2)H]lactate

(13)C magnetic resonance spectroscopy and spectroscopic imaging measurements of hyperpolarized (13)C label exchange between exogenously administered [1-(13)C]pyruvate and endogenous lactate, catalyzed by lactate dehydrogenase (LDH), has proved to be a powerful approach for probing tissue metabolism in vivo. This experiment has clinical potential, particularly in oncology, where it could be used to assess tumor grade and response to treatment. A limitation of the method is that pyruvate must be administered in vivo at supra-physiological concentrations. This problem can be avoided by using hyperpolarized [1-(13)C]lactate, which can be used at physiological concentrations. However, sensitivity is limited in this case by the relatively small pyruvate pool size, which would result in only low levels of labeled pyruvate being observed even if there was complete label equilibration between the lactate and pyruvate pools. We demonstrate here a more sensitive method in which a doubly labeled lactate species can be used to measure LDH-catalyzed exchange in vivo. In this experiment exchange of the C2 deuterium label between injected hyperpolarized l-[1-(13)C,U-(2)H]lactate and endogenous unlabeled lactate is observed indirectly by monitoring phase modulation of the spin-coupled hyperpolarized (13)C signal in a heteronuclear (1)H/(13)C spin-echo experiment.     

Quantitative evaluation of the biosynthetic pathways leading to δ-aminolevulinic acid from the Shemin precursor glycine via the C5 pathway in Arthrobacter hyalinus by analysis of 13C-labeled coproporphyrinogen III biosynthesized from [2-13C]glycine, [1-13C]acetate, and [2-13C]acetate using 13C NMR spectroscopy

The biosynthetic pathways leading to δ-aminolevulinic acid (ALA) from the Shemin precursor glycine via the C5 pathway in Arthrobacter hyalinus were quantitatively evaluated by means of feeding experiments with [2-¹³C]glycine, sodium [1-¹³C]acetate, and sodium [2-¹³C]acetate, followed by analysis of the labeling patterns of coproporphyrinogen III (Copro’gen III) (biosynthesized from ALA) using ¹³C NMR spectroscopy. Two biosynthetic pathways leading to ALA from glycine via the C5 pathway were identified: i.e., transformation of glycine to l-serine catalyzed by glycine hydroxymethyltransferase, and glycine synthase-catalyzed catabolism of glycine to N ⁵,N ¹⁰-methylene-tetrahydrofolic acid (THF), which reacts with another molecule of glycine to afford l-serine. l-Serine is transformed to acetyl-CoA via pyruvic acid. Acetyl-CoA enters the tricarboxylic acid cycle, affording 2-oxoglutaric acid, which in turn is transformed to l-glutamic acid. The l-glutamic acid enters the C5 pathway, affording ALA in A. hyalinus. A ¹³C NMR spectroscopic comparison of the labeling patterns of Copro’gen III obtained after feeding of [2-¹³C]glycine, sodium [1-¹³C]acetate, and sodium [2-¹³C]acetate showed that [2-¹³C]glycine transformation and [2-¹³C]glycine catabolism in A. hyalinus proceed in the ratio of 52 and 48 %. The reaction of [2-¹³C]glycine and N ⁵,N ¹⁰-methylene-THF, that of glycine and N ⁵,N ¹⁰-[methylene-¹³C]methylene-THF generated from the [2-¹³C]glycine catabolism, and that of [2-¹³C]glycine and N ⁵,N ¹⁰-[methylene-¹³C]methylene-THF transformed the fed [2-¹³C]glycine to [1-¹³C]acetyl-CoA, [2-¹³C]acetyl-CoA, and [1,2-¹³C₂]acetyl-CoA in the ratios of 42, 37, and 21 %, respectively. These labeled acetyl-CoAs were then incorporated into ALA. Our results provide a quantitative picture of the pathways of biosynthetic transformation to ALA from glycine in A. hyalinus.     

Chemoenzymatic synthesis of [3,9-C]-labeled NeuAc and KDN

The chemoenzymatic synthesis of ¹³C-labeled sialic acid (NeuAc) and 3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (KDN) as useful molecular probes for studying the conformation of sialyl or KDN oligosaccharides attached to proteins was performed by using [6-¹³C]-ManNAc, [6-¹³C]-Man and [3-¹³C]-pyruvic acid sodium salt. In the synthesis of the compounds, 5,6-anhydro intermediates were found to easily provide not only 6-¹³C-labeled but also 5-, and 6-modified NeuAc and KDN analogs. Furthermore, it was demonstrated that identical results are obtained by NMR for both [3,9-¹³C]-NeuAc and 1:1 mixtures of [3-¹³C]- and [9-¹³C]-NeuAc.     

Biosynthesis of highly modified meroterpenoids in aspergillus variecolor. Incorporation of 13C-labelled acetates and methionine into anditomin and andilesin C

Incorporations of [1-¹³C]?, [2-¹³C]?, [1,2-¹³C₂]-acetates and [¹³C]-methionine into anditomin, a metabolite of $\text{Aspergillus variecolor}$, indicate its formation by a mixed polyketide-terpenoid biosynthetic pathway similar to that elucidated for andibenin; observations are made on the possible biosynthetic relationship of the $\text{A. variecolor}$ metabolites with austin and terretonin, mycotoxins recently isolated from $\text{A. ustus}$ and $\text{A. terreus}$ respectively.     

Studies on the Biosynthesis of Spirostaphylotrichin A

Incorporation of ¹⁴C-labelled acetate and amino acids as well as of [1-¹³C]-, [2-¹³C]-, and [1,2-¹³C₂] acetate, L -[methyl¹³C] methionine, [2,3-¹³C₂] succinate, and L -[2,3-¹³C₂] aspartate into spirostaphylotrichin A ( 1 ) by Staphylotrichum coccosporum demonstrates that the building blocks of 1 are 5 units of acetate/malonate, 1 unit of methionine, and a C₄-dicarboxylic acid. The latter is most likely aspartate and derived from the citric-acid cycle. Using [2-¹³C, 2-²H₃] acetate as a precursor, the starter unit of the polyketide chain was identified.     

Practical synthesis of d-[1-C]mannose, l-[1-C] and l-[6-C]fucose

The chemical synthesis of ¹³C-labeled mannose and fucose is important for the preparation of molecular probes used in the conformational study of the oligosaccharide portions of glycoproteins. A new method for the synthesis of the title [1-¹³C]-labeled compounds via the corresponding olefin compounds, which are in turn derived from d-mannitol or l-arabinose by efficient introduction of ¹³C, by the Wittig reaction using Ph₃P¹³CH₃I and n-BuLi, is described. The introduction of ¹³CH₃I to produce the [1-¹³C]- and [6-¹³C]-labeled compounds was accomplished in 62%, 56%, and 71% yields, respectively. All mannose and fucose protons, from H-1 to H-6, were observed by the HMQC-TOCSY technique using 1:1 mixtures of [1-¹³C]- and [6-¹³C]-labeled compounds.