Metabolic Studies of Tumor Cells Using [1-13C] Pyruvate Hyperpolarized by Means of PHIP-Side Arm Hydrogenation

The kinetics of metabolic processes can be assessed, in real time by means of MR hyperpolarized (HP) metabolites. [1-¹³C]pyruvate, hyperpolarized by means of d-DNP, is, by far, the substrate most widely applied to the investigation of several pathologies characterized by deregulated glycolytic metabolic networks, including cancer. Hyperpolarization of [1-¹³C]pyruvate by means of the cost effective, fast and easy to handle PHIP-SAH (para-hydrogen induced polarization-side arm hydrogenation) method opens-up a pathway for the application of HP metabolites to a wide range of cancer-related studies. Herein, we report the first application of PHIP-SAH hyperpolarized [1-¹³C]pyruvate in the investigation of upregulated glycolysis in two murine breast cancer cell lines (168FARN and 4T1). The results obtained using HP pyruvate have been validated with a conventional biochemical assay and are coherent with previously-reported lactate dehydrogenase activity measured in those cells.     

Carbon-carbon coupling in [3-13C1]tryptophan

Carbon-carbon coupling constants have been observed in [90% 3-¹³C₁]tryptophan and used to assign the ¹³C resonances of the indole ring. The results support a reassignment of C-5 and C-6 recently suggested on the basis of the reassigment of the corresponding resonances in indole. Coupling constants are compared with theoretical values obtained using a finite perturbation theory Blizzard-Santry program, and excellent agreement is obtained for the ¹J(CC) values. The calculations predict that the Fermi contact contribution is dominant for all ⁿJ(CC) couplings to C-3.     

Synthesis and 15N NMR Signal Amplification by Reversible Exchange of [15N]Dalfampridine at Microtesla Magnetic Fields

Signal Amplification by Reversible Exchange (SABRE) technique enables nuclear spin hyperpolarization of wide range of compounds using parahydrogen. Here we present the synthetic approach to prepare ¹⁵N-labeled [¹⁵N]dalfampridine (4-amino[¹⁵N]pyridine) utilized as a drug to reduce the symptoms of multiple sclerosis. The synthesized compound was hyperpolarized using SABRE at microtesla magnetic fields (SABRE-SHEATH technique) with up to 2.0 % ¹⁵N polarization. The 7-hour-long activation of SABRE pre-catalyst [Ir(IMes)(COD)Cl] in the presence of [¹⁵N]dalfampridine can be remedied by the use of pyridine co-ligand for catalyst activation while retaining the ¹⁵N polarization levels of [¹⁵N]dalfampridine. The effects of experimental conditions such as polarization transfer magnetic field, temperature, concentration, parahydrogen flow rate and pressure on ¹⁵N polarization levels of free and equatorial catalyst-bound [¹⁵N]dalfampridine were investigated. Moreover, we studied ¹⁵N polarization build-up and decay at magnetic field of less than 0.04 μT as well as ¹⁵N polarization decay at the Earth's magnetic field and at 1.4 T.     

Biosynthesis of the antibiotic verrucarin E use of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]-acetates. Verrucarins and roridins, 37th communication [1]

The origin of the carbon skeleton of verrucarin E (1) from acetate as precursor is confirmed. Incorporation studies with [1,2-¹³C]-acetate have demonstrated that two acetoacetate units couple together as shown in pattern A (Scheme 2) and not as in B . Analysis of the deuterium distribution in both verrucarin E (1) isolated after the incorporation of [2-¹³C,2-²H₃]-acetate and in sodium acetate obtained after Kuhn-Roth oxidation of the metabolite demonstrated that C(7) is derived from the starter unit of one of the acetoacetate moieties. The deuterium exchange in verrucarin E (1) occurring during fermentation was investigated.     

Enzymic synthesis of 3-[3- 13C]dehydroquinic acid

The title compound has been prepared enzymically over four steps from commercially available D-[5- 13C]fructose.     

Development of Dissolution Dynamic Nuclear Polarization of [15N3]Metronidazole: A Clinically Approved Antibiotic

We report dissolution Dynamic Nuclear Polarization (d-DNP) of [¹⁵N₃]metronidazole ([¹⁵N₃]MNZ) for the first time. Metronidazole is a clinically approved antibiotic, which can be potentially employed as a hypoxia-sensing molecular probe using ¹⁵N hyperpolarized (HP) nucleus. The DNP process is very efficient for [¹⁵N₃]MNZ with an exponential build-up constant of 13.8 min using trityl radical. After dissolution and sample transfer to a nearby 4.7 T Magnetic Resonance Imaging scanner, HP [¹⁵N₃]MNZ lasted remarkably long with T₁ values up to 343 s and ¹⁵N polarizations up to 6.4 %. A time series of HP [¹⁵N₃]MNZ images was acquired in vitro using a steady state free precession sequence on the ¹⁵NO₂ peak. The signal lasted over 13 min with notably long T₂ of 20.5 s. HP [¹⁵N₃]MNZ was injected in the tail vein of a healthy rat, and dynamic spectroscopy was performed over the rat brain. The in vivo HP ¹⁵N signals persisted over 70 s, demonstrating an unprecedented opportunity for in vivo studies.     

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.     

13C tracer recovery in human stools after digestion of a fat-rich meal labelled with [1,1,1-13C3]tripalmitin and [1,1,1-13C3]triolein

Lipid metabolism studies focus mainly on oxidation and storage but rarely on faecal elimination, which is needed to assess total lipid distribution during the postprandial period. The purpose of the present work was to set up and validate the analysis of lipid tracers in stools, with an aim of later using this methodology in studies of postprandial lipid tracer metabolism. Eight subjects received a mixture of [1,1,1-(13)C3]tripalmitin and [1,1,1-(13)C3]triolein with a fat-rich meal. The nature and amounts of (13)C lipids excreted in stools during 3 days post-dose were determined by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis of fatty acid methyl esters (FAMEs) from total fatty acid (TFA), free fatty acid (FFA) and triacylglycerol (TAG) fractions. The results were expressed as the Cumulative Tracer Recovery of the administered dose (CTR%). The quantities and labelling of FAMEs were higher in FFA than in TAG, indicating that label loss was not due to a lack of digestive lipase activity. The labelling was higher for C16:0 than for C18:1. The CTRs were 7.03 ± 0.77% and 6.87 ± 0.91%, respectively, in TFA and FFA for [1-(13)C] C16:0, while they were 0.60 ± 0.15% and 0.51 ± 0.11% for [1-(13)C] C18:1 (mean ± sem). By studying the kinetics of lipid excretion from subjects, two groups emerged. The first one showed rapid excretion in stool #1, whereas the second showed slower excretion in stools #2-#3. A significant difference was found in the FFA in stool #1 for C16:0 (p < 0.01) and C18:1 (p < 0.05). Individual excretion kinetics showed marked variability. Nevertheless, the CTR over the 3-day study period was substantial and homogenous for all subjects. These results confirm that the assessment of faecal elimination is of great importance when establishing total lipid distribution during the postprandial period and validate the analysis of cumulative tracer loss during 72 h post-tracer ingestion.     

Synthesis of [1, 2-13C2]-S-Lysine Hydrochloride

Cobalt catalyzed hydroformylation-amidocarbonylation of 3-cyanopropene by carbon-13 monoxide and acetamide produced a [1,2-¹³C₂]-labelled mixture of 5-cyano-2-acetamidopentanoic acid and 4-cyano-3-methyl-2-acetamidobutanoic acid (n to b ratio of 7 to 3. 55% yield). Acylase I resolution of this mixture gave the free S-amino acids which could be separated by crystallization. Catalytic reduction of the cyano group of the straight chain acid furnished [1,2-¹³C₂] -S-lysine. HCl.     

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.     

Synthesis of dl-[2-13C]leucine and it use in the preparation of [3-dl-[2-13C]leucine]oxytocin and [8-dl-[2-13C]leucine]oxytocin: Preparative separation of diastereoisomeric peptides by partition chromatography and high pressure liquid chromatography

dl-[2-¹³C]Leucine was prepared by condensing the sodium salt of ethyl acetamido-[2-¹³C]cyanoacetate with isobutylbromide in hexamethylphosphoroustriamide followed by acid hydrolysis. N-Boc-dl-[2-¹³C]Leucine was prepared and incorporated into [8-dl-[2-¹³C]leucine]oxytocin by total synthesis. The ¹³C-labeled hormone derivative [8-[2-¹³C]leucine]oxytocin was separated from its 8-position diastereoisomer by partition chromatography. The specifically ¹³C-labeled peptide hormone diastereoisomeric analog [3-dl-[2-¹³C]leucine]oxytocin also was prepared by solid phase peptide synthesis. No suitable solvent system for partition chromatography separation of the latter diastereoisomeric peptide mixture could be found. However an excellent preparative separation of the diastereoisomers could be obtained by reverse phase high pressure liquid chromatography on a partisil 10 M9 ODS column using the solvent system 0.05 M ammonium acetate (pH 4.0), acetonitrile (81:19, $\text{v}\text{v}$) to give pure [3-(2-¹³C]leucine]oxytocin and [3-D-(2-¹³C]leucine]oxytocin. An excellent separation of [8[2-¹³C]leucine]oxytocin and the corresponding 8-D-leucine diastereoisomer derivative could also be accomplished by high pressure liquid chromatography.     

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.     

Rational Design of [13C,D14]Tert‐butylbenzene as a Scaffold Structure for Designing Long‐lived Hyperpolarized 13C Probes

Dynamic nuclear polarization (DNP) is a technique to polarize the nuclear spin population. As a result of the hyperpolarization, the NMR sensitivity of the nuclei in molecules can be dramatically enhanced. Recent application of the hyperpolarization technique has led to advances in biochemical and molecular studies. A major problem is the short lifetime of the polarized nuclear spin state. Generally, in solution, the polarized nuclear spin state decays to a thermal spin equilibrium, resulting in loss of the enhanced NMR signal. This decay is correlated directly with the spin‐lattice relaxation time T₁. Here we report [¹³C,D₁₄]tert‐butylbenzene as a new scaffold structure for designing hyperpolarized ¹³C probes. Thanks to the minimized spin‐lattice relaxation (T₁) pathways, its water‐soluble derivative showed a remarkably long ¹³C T₁ value and long retention of the hyperpolarized spin state.     

13C NMR spectra of benzo[b]thiophene and 1-(X-benzo[b]thienyl)ethyl acetate derivatives

Carbon-13 chemical shift assignments are reported for benzo[b]thiophene and 1-(X-benzo[b]thienyl)ethyl acetate derivatives, where X=? CH(OAc)CH₃ substituted at positions 2-7. Substituent chemical shift (SCS) effects for the ethyl acetate group are additive at all positions. A substantial upfield shift was observed at C-3, arising from the peri interaction of H-3 and the 4-ethyl acetate substituent. Carbon-13 relaxation times (T₁) and nuclear Overhauser enhancements (η) have been measured for benzo[b]thiophene and its derivatives, and the contributions of dipolar, T^DD₁, and spin rotation, T^SR₁, relaxation have been determined. Intramolecular dipole–dipole interactions are found to provide by far the most important spin-lattice relaxation mechanism whenever protons are bound directly to the carbons under investigation. Nonprotonated ring carbons are relaxed by both DD and SR mechanisms. Anisotropic motion has an easily observable effect on the DD contribution to T₁, and can form the basis for spectral assignments, as in 1-phenylethyl acetate. Long-range ¹³C? ¹H coupling constants were observed both between ring carbons and between ring carbons with ring side-chain hydrogens. These results have been used for the structure determination of the title compounds.     

Furopyridines. XI. 13C NMR Spectra of 2- or 3-Substituted Furo[2,3-b]-, Furo[3,2-b]-, Furo[2,3-c]- and Furo[3,2-c]pyridine

The ¹³C nmr spectra of 2- or 3-monosubstituted furo[2,3-b]- 1a-1j , furo[3,2-b]- 2a-2j , furo[2,3-c]- 3a-3j and furo[3,2-c]pyridine derivatives 4a-4j are reported. Effects by change in annelation and substituent effects on ¹³C chemical shifts and carbon-proton coupling constants are discussed. The spectra of benzo[b]furan derivatives 5a-5j having the corresponding substituent are also reported for comparison.     

1H and 13C NMR data for C-6 substituted 3-azabicyclo[3.3.1]nonane-1-carboxylates

The 1H and 13C NMR spectra of 3-azabicyclo[3.3.1]nonanes with various oxygenated substituents at C-6 were assigned using 1D (DEPT) and 2D (COSY, HSQC, HMBC, NOESY) experiments. Close examination of this NMR data details the effects of substitution and stereochemistry at C-6 in these compounds.     

Synthesis and Crystal Structure of 2,2-Dimethyl-4-[1H-（ 1,2,4-triazolyl）]-4- [ 1H-benzotriazolyl]-3- butanone[t-BuCOCH（C2H2N3）（C6H4N3）]

1 INTRODUCTION Triazole nuclei appear frequently in the structures of various natural products and biologically active compounds, notably thiamine (vitamin B), peni- cillins, antibiotics such as micrococcin[1], and many metabolic products of fungi and p…