Eduard Y. Chekmenev

PASADENA Hyperpolarization of Succinic Acid for MRI and NMR Spectroscopy

We use the PASADENA (parahydrogen and synthesis allow dramatically enhanced nuclear alignment) method to achieve 13C polarization of approximately 20% in seconds in 1-13C-succinic-d2 acid. The high-field 13C multiplets are observed as a function of pH, and the line broadening of C1 is pronounced in the region of the pK values. The 2JCH, 3JCH, and 3JHH couplings needed for spin order transfer vary with pH and are best resolved at low pH leading to our use of pH approximately 3 for both the molecular addition of parahydrogen to 1-13C-fumaric acid-d2 and the subsequent transfer of spin order from the nascent protons to C1 of the succinic acid product. The methods described here may generalize to hyperpolarization of other carboxylic acids. The C1 spin-lattice relaxation time at neutral pH and 4.7 T is measured as 27 s in H2O and 56 s in D2O. Together with known rates of succinate uptake in kidneys, this allows an estimate of the prospects for the molecular spectroscopy of metabolism.

PASADENA hyperpolarization of 13C biomolecules: equipment design and installation

ObjectThe PASADENA method has achieved hyperpolarization of 16–20% (exceeding 40,000-fold signal enhancement at 4.7 T), in liquid samples of biological molecules relevant to in vivo MRI and MRS. However, there exists no commercial apparatus to perform this experiment conveniently and reproducibly on the routine basis necessary for translation of PASADENA to questions of biomedical importance. The present paper describes equipment designed for rapid production of six to eight liquid samples per hour with high reproducibility of hyperpolarization.Materials and methodsDrawing on an earlier, but unpublished, prototype, we provide diagrams of a delivery circuit, a laminar-flow reaction chamber within a low field NMR contained in a compact, movable housing. Assembly instructions are provided from which a computer driven, semi-automated PASADENA polarizer can be constructed.ResultsTogether with an available parahydrogen generator, the polarizer, which can be operated by a single investigator, completes one cycle of hyperpolarization each 52 s. Evidence of efficacy is presented. In contrast to competing, commercially available devices for dynamic nuclear polarization which characteristically require 90xa0min per cycle, PASADENA provides a low-cost alternative for high throughput.ConclusionsThis equipment is suited to investigators who have an established small animal NMR and wish to explore the potential of heteronuclear (13C and 15N) MRI, MRS, which harnesses the enormous sensitivity gain offered by hyperpolarization.

Hyperpolarized 1H NMR Employing Low γ Nucleus for Spin Polarization Storage

Here, we demonstrate the utility of low gamma nuclei for spin storage of hyperpolarization followed by proton detection, which theoretically can provide up to approximately (gamma[1H]/gamma[X])(2) gain in sensitivity in hyperpolarized biomedical MR. This is exemplified by hyperpolarized 1-(13)C sites of 2,2,3,3-tetrafluoropropyl 1-(13)C-propionate-d(3) (TFPP), (13)C T(1) = 67 s in D(2)O, and 1-(13)C-succinate-d(2), (13)C T(1) = 105 s in D(2)O, pH 11, using PASADENA. In a representative example, the spin polarization was stored on (13)C for 24 and 70 s, respectively, while the samples were transferred from a low magnetic field polarizer operating at 1.76 mT to a 4.7 T animal MR scanner. Following sample delivery, the refocused INEPT pulse sequence was used to transfer spin polarization from (13)C to protons with an efficiency of 50% for TFPP and 41% for 1-(13)C-succinate-d(2) increasing the overall NMR sensitivity by a factor of 7.9 and 6.5, respectively. The low gamma nuclei exemplified here by (13)C with a T(1) of tens of seconds acts as an efficient spin polarization storage, while J-coupled protons are better for NMR detection.

Quality assurance of PASADENA hyperpolarization for 13C biomolecules.

ObjectDefine MR quality assurance procedures for maximal PASADENA hyperpolarization of a biological 13C molecular imaging reagent.Materials and methodsAn automated PASADENA polarizer and a parahydrogen generator were installed. 13C enriched hydroxyethyl acrylate, 1-13C, 2,3,3-d3 (HEA), was converted to hyperpolarized hydroxyethyl propionate, 1-13C, 2,3,3-d3 (HEP) and fumaric acid, 1-13C, 2,3-d2 (FUM) to hyperpolarized succinic acid, 1-13C, 2,3-d2 (SUC), by reaction with parahydrogen and norbornadiene rhodium catalyst. Incremental optimization of successive steps in PASADENA was implemented. MR spectra and in vivo images of hyperpolarized 13C imaging agents were acquired at 1.5 and 4.7 T.ResultsApplication of quality assurance (QA) criteria resulted in incremental optimization of the individual steps in PASADENA implementation. Optimal hyperpolarization of HEP of Pxa0=xa020% was achieved by calibration of the NMR unit of the polarizer (B0 field strengthxa0±xa00.002 mT). Mean hyperpolarization of SUC, Pxa0=xa0[15.3xa0±xa01.9]% (Nxa0=xa016) in D2O, and Pxa0=xa0[12.8xa0±xa03.1]% (Nxa0=xa012) in H2O, was achieved every 5–8xa0min (range 13–20%). An in vivo 13C succinate image of a rat was produced.ConclusionPASADENA spin hyperpolarization of SUC to 15.3% in average was demonstrated (37,400 fold signal enhancement at 4.7 T). The biological fate of 13C succinate, a normally occurring cellular intermediate, might be monitored with enhanced sensitivity.

Fluorine-19 NMR Chemical Shift Probes Molecular Binding to Lipid Membranes

The binding of amphiphilic molecules to lipid bilayers is followed by 19F NMR using chemical shift and line shape differences between the solution and membrane-tethered states of -CF 3 and -CHF 2 groups. A chemical shift separation of 1.6 ppm combined with a high natural abundance and high sensitivity of 19F nuclei offers an advantage of using 19F NMR spectroscopy as an efficient tool for rapid time-resolved screening of pharmaceuticals for membrane binding. We illustrate the approach with molecules containing both fluorinated tails and an acrylate moiety, resolving the signals of molecules in solution from those bound to synthetic dimyristoylphosphatidylcholine bilayers both with and without magic angle sample spinning. The potential in vitro and in vivo biomedical applications are outlined. The presented method is applicable with the conventional NMR equipment, magnetic fields of several Tesla, stationary samples, and natural abundance isotopes.

Anisotropie Chemical Shift Perturbation Induced by Ions in Conducting Channels

Anisotropic chemical shifts observed from solid-state NMR spectroscopy of uniformly aligned samples can be influenced by three primary factors: a change in orientation of the nuclear site, a change in dynamics, or a change in the chemical shift tensor element magnitudes or orientation to the molecular frame. These features are particularly attractive for characterizing the influence of ions in ion conducting channels. Cation binding results in far more subtle effects than had previously been imagined. Prior to the analysis of the first solid-state NMR characterizations of ion binding [1,2] the experimental data were primarily in the form of a few water-soluble protein structures in the Protein Data Bank to which monovalent ions were bound [e.g. 3,4]. Such binding sites showed optimized solvation for the ions associated with strong binding. Computational modeling efforts on ion channels, for the most part, also showed substantial structural deformation upon ion binding [5–7]. We now realize that much better models for how ions interact with channels can be realized from the characterization of substrate binding to enzymes, for which we have a great deal of information represented in every biochemistry textbook. A delicate balance of molecular interactions and thermodynamic parameters has evolved for enzymes, so that substrates are attracted to the active sites of proteins while not compromising the primary function of these proteins to conduct chemistry on the substrates and to release the products efficiently. Similarly, ions must be attracted to the channel and yet the primary function of these proteins is to facilitate the transfer of ions from one side of the membrane to the other. Here, we describe how this can be done through a model system, the monovalent cation channel gramicidin A, produced by Bacillus brevis, to lyse cells in its environment and from the lysate the bacillus harvests amino acids following the additional export of proteases. This unique polypeptide has an alternating sequence of d and l amino acid residues that forms a β-strand with all of its side chains on one side of the strand resulting in a helical conformation with an aqueous pore approximately 4.5 Å in diameter. The high-resolution structure has been fully characterized using solid-state NMR orientational restraints from uniformly aligned samples in lipid bilayers (PDB # 1 mag) [8,9]. Analysis of the refined structure illustrates the unique high-resolution detail of this time averaged structure [10]. This polypeptide spans the lipid bilayer as a symmetric dimer [11], the amino-termini of which are formylated at the bilayer center. The approximate location of two symmetric ion binding sites in the vicinity of the monolayer interfacial region was initially characterized by X-ray diffraction [12]. Gramicidin shares a number of important features with the more recently characterized K+ channels [13,14]. In particular, it is the polypeptide backbone and the carbonyl oxygens that provide much of the solvation environment following ion dehydration in the ion binding site of gramicidin A and in the selectivity filter of the KcsA channel. However, in KcsA the ion binding sites are considerably closer together (approximately 7 Å for KcsA [15] and approximately 20 Å for gramicidin A) permitting stronger ion binding and a much higher degree of ion selectivity. Nevertheless, the principles gleaned from studies of gramicidin appear to have very general applicability. Solid-state NMR of uniformly aligned samples leads to high-resolution spectra. The frequencies from chemical shift, dipolar and quadrupolar interactions can be used as structural restraints by interpreting the frequencies within the context of the appropriate motionally averaged spin interaction tensor [16–18]. Here, we primarily describe the use of 15N NMR spectroscopy of the amide nitrogen sites in the polypeptide backbone of gramicidin A in hydrated liquid crystalline preparations. These tensor element magnitudes in both static and liquid crystalline environments have been characterized for each of the amide sites [8,19] and many of the tensor orientations to the molecular frame have also been characterized [19,20]. These 15N anisotropic chemical shifts represented some of the data used for solving the 3D structure in the absence of ions. The anisotropic shifts in the cation binding region change upon the introduction of ions [21,22] (Figure 1). The influence of the ions is surprisingly small compared to the 40–50 ppm anisotropic shifts calculated [23] from the structural changes predicted in the first molecular dynamics study of gramicidin ion binding [5]. Consequently, it