Investigation of quorum sensing in Candida albicans

The dimorphic fungus Candida albicans produces extracellular farnesol which acts as a quorum-sensing molecule (QSM) to suppress filamentation. Of four possible geometric isomers of farnesol, only the E,E isomer possesses QSM activity. We tested number of natural and synthetic analogs of farnesol for their activity in an N-acetylglucosamine-induced differentiation assay for germ tube formation (GTF). Modified structural features include the nature of the head group, chain length, presence or absence of the three double bonds, substitution of a backbone carbon by S, O, N, and Se heteroatoms, presence or absence of a 3-methyl branch, as well as the bulkiness and the hydrophobic tail. In order to elucidate localization a putative receptor we prepared ten polyenes typified by 3,7,11-trimethyl-2,4,6,8,10-dodecapentenaldehyde oxime. Four of the ten analogs display strong quorum-sensing activity in the human pathogen Candida albicans, also they are fluorescent. Therefore oxime anti-4 is demonstrated to be useful for confocal fluorescence microscopic imaging of fungal cells. Also, we have investigated of a series of farnesol analogs replacing the primary alcohol head group with several classes of heterocycles. High levels of quorum-sensing activity were associated with several types of heterocycles. Two non toxic analogs were tested in C. albicans inoculated immune-challenged mice. One of the two analogs has been shown to perfectly emulate farnesol in vivo behavior, which unfortunately includes an increase of C. Albicans virulence in the mice. In an attempt to isolate putative farnesol binding receptor we synthesized of affinity media designed to isolate a putative farnesol receptor or binding protein in C. albicans. The approach rests on a new class of farnesol analogs, which maintain the acidic head group and farnesyl-like backbone known from our earlier studies to be necessary for quorum sensing, but which also allow for covalent chemical linkage of the head group to an affinity support. In preliminary results, affinity chromatography of the total protein extract of C. albicans resulted in selective retention of a handful of proteins. Mass spectrometry of some of eluted proteins reveals two matches against known fungal proteins

Spin–Lattice Relaxation of Hyperpolarized Metronidazole in Signal Amplification by Reversible Exchange in Micro-Tesla Fields

Simultaneous reversible chemical exchange of <i>para</i>-hydrogen and to-be-hyperpolarized substrate on metal centers enables spontaneous transfer of spin order from <i>para</i>-hydrogen singlet to nuclear spins of the substrate. When performed at a sub-micro-tesla magnetic field, this technique of NMR signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH). SABRE-SHEATH has been shown to hyperpolarize nitrogen-15 sites of a wide range of biologically interesting molecules to a high polarization level (<i>P</i> > 20%) in 1 min. Here, we report on a systematic study of ¹H, ¹³C, and ¹⁵N spin–lattice relaxation (<i>T</i>₁) of metronidazole-¹³C₂-¹⁵N₂ in the SABRE-SHEATH hyperpolarization process. In the micro-tesla range, we find that all ¹H, ¹³C, and ¹⁵N spins studied share approximately the same <i>T</i>₁ values (ca. 4 s under the conditions studied) because of mixing of their Zeeman levels, which is consistent with the model of relayed SABRE-SHEATH effect. These <i>T</i>₁ values are significantly lower than those at a higher magnetic field (i.e. the Earth’s magnetic field and above), which exceed 3 min in some cases. Moreover, these relatively short <i>T</i>₁ values observed below 1 μT limit the polarization build-up process of SABRE-SHEATH, thereby limiting the maximum attainable ¹⁵N polarization. The relatively short <i>T</i>₁ values observed below 1 μT are primarily caused by intermolecular interactions with quadrupolar iridium centers or dihydride protons of the employed polarization transfer catalyst, whereas intramolecular spin–spin interactions with ¹⁴N quadrupolar centers have a significantly smaller contribution. The presented experimental results and their analysis will be beneficial for more rational design of SABRE-SHEATH (i) polarization transfer catalysts and (ii) hyperpolarized molecular probes in the context of biomedical imaging and other applications

Parahydrogen-Induced Polarization with a Rh-Based Monodentate Ligand in Water

Reported here is a water-soluble Rh­(I)-based catalyst for performing parahydrogen-induced polarization (PHIP). The [Rh­(I)­(norbornadiene)­(THP)₂]⁺[BF₄]⁻ catalyst utilizes the monodentate phosphine ligand tris­(hydroxymethyl)­phosphine (THP). The monodentate PHIP catalyst is less susceptible to oxygenation by air and the THP ligand is significantly less expensive than bidentate water-soluble PHIP ligands. In situ PHIP detection with this monodentate Rh­(I)-based catalyst in water yielded 12% ¹³C polarization for the parahydrogen addition product, 2-hydroxyethyl 1-¹³C-propionate-d_2,3,3 (HEP), with a ¹³C <i>T</i>₁ relaxation of 108 s at 0.0475 T. PHIP polarization yields were high, reflecting efficient hydrogenation even under conditions of high content of the oxidized phosphine form of the THP ligand

Efficient Synthesis of Nicotinamide-1-¹⁵N for Ultrafast NMR Hyperpolarization Using Parahydrogen

Nicotinamide (a vitamin B₃ amide) is one of the key vitamins as well as a drug for treatment of M. tuberculosis, HIV, cancer, and other diseases. Here, an improved Zincke reaction methodology is presented allowing for straightforward and scalable synthesis of nicotinamide-1-¹⁵N with an excellent isotopic purity (98%) and good yield (55%). ¹⁵N nuclear spin label in nicotinamide-1-¹⁵N can be NMR hyperpolarized in seconds using parahydrogen gas. NMR hyperpolarization using the process of temporary conjugation between parahydrogen and to-be-hyperpolarized biomolecule on hexacoordinate iridium complex via the Signal Amplification By Reversible Exchange (SABRE) method significantly increases detection sensitivity (e.g., >20 000-fold for nicotinamide-1-¹⁵N at 9.4 T) as has been shown by Theis T. et al. (<i>J. Am. Chem. Soc.</i> <b>2015</b>, <i>137</i>, 1404), and hyperpolarized in this fashion, nicotinamide-1-¹⁵N can be potentially used to probe metabolic processes in vivo in future studies. Moreover, the presented synthetic methodology utilizes mild reaction conditions, and therefore can also be potentially applied to synthesis of a wide range of ¹⁵N-enriched N-heterocycles that can be used as hyperpolarized contrast agents for future in vivo molecular imaging studies

Parahydrogen Induced Polarization of 1-¹³C‑Phospholactate‑<i>d</i>₂ for Biomedical Imaging with >30,000,000-fold NMR Signal Enhancement in Water

The synthetic protocol for preparation of 1-¹³C-phosphoenolpyruvate-<i>d</i>₂, precursor for parahydrogen-induced polarization (PHIP) of 1-¹³C-phospholactate-<i>d</i>₂, is reported. ¹³C nuclear spin polarization of 1-¹³C-phospholactate-<i>d</i>₂ was increased by >30,000,000-fold (5.75 mT) in water. The reported ¹³C polarization level approaching unity (>15.6%), long lifetime of ¹³C hyperpolarized 1-¹³C-phospholactate-<i>d</i>₂ (58 ± 4 s versus 36 ± 2 s for nondeuterated form at 47.5 mT), and large production quantities (52 μmoles in 3 mL) in aqueous medium make this compound useful as a potential contrast agent for the molecular imaging of metabolism and other applications

Synthesis of Unsaturated Precursors for Parahydrogen-Induced Polarization and Molecular Imaging of 1-¹³C‑Acetates and 1-¹³C‑Pyruvates via Side Arm Hydrogenation

Hyperpolarized forms of 1-¹³C-acetates and 1-¹³C-pyruvates are used as diagnostic contrast agents for molecular imaging of many diseases and disorders. Here, we report the synthetic preparation of 1-¹³C isotopically enriched and pure from solvent acetates and pyruvates derivatized with unsaturated ester moiety. The reported unsaturated precursors can be employed for NMR hyperpolarization of 1-¹³C-acetates and 1-¹³C-pyruvates via parahydrogen-induced polarization (PHIP). In this PHIP variant, Side arm hydrogenation (SAH) of unsaturated ester moiety is followed by the polarization transfer from nascent parahydrogen protons to ¹³C nucleus via magnetic field cycling procedure to achieve hyperpolarization of ¹³C nuclear spins. This work reports the synthesis of PHIP-SAH precursors: vinyl 1-¹³C-acetate (55% yield), allyl 1-¹³C-acetate (70% yield), propargyl 1-¹³C-acetate (45% yield), allyl 1-¹³C-pyruvate (60% yield), and propargyl 1-¹³C-pyruvate (35% yield). Feasibility of PHIP-SAH ¹³C hyperpolarization was verified by ¹³C NMR spectroscopy: hyperpolarized allyl 1-¹³C-pyruvate was produced from propargyl 1-¹³C-pyruvate with ¹³C polarization of ∼3.2% in CD₃OD and ∼0.7% in D₂O. ¹³C magnetic resonance imaging is demonstrated with hyperpolarized 1-¹³C-pyruvate in aqueous medium

Parahydrogen-induced polarization of 14N nuclei

Hyperpolarization techniques provide a dramatic increase in sensitivity of nuclear magnetic resonance spectroscopy and imaging. In spite of the outstanding progress in solution-state hyperpolarization of spin-1/2 nuclei, hyperpolarization of quadrupolar nuclei remains challenging. Here, hyperpolarization of quadrupolar 14N nuclei with natural isotopic abundance of >99% is demonstrated. This is achieved via pairwise addition of parahydrogen to tetraalkylammonium salts with vinyl or allyl unsaturated moieties followed by a subsequent polarization transfer from 1H to 14N nuclei at high magnetic field using PH-INEPT or PH-INEPT+ radiofrequency pulse sequence. Catalyst screening identified water-soluble rhodium complex [Rh(P(m-C6H4SO3Na)3)3Cl] as the most efficient catalyst for hyperpolarization of the substrates under study, providing up to 1.3% and up to 6.6% 1H polarization in the cases of vinyl and allyl precursors, respectively. The performance of PH-INEPT and PH-INEPT+ pulse sequences was optimized with respect to interpulse delays, and the resultant experimental dependences were in good agreement with simulations. As a result, 14N NMR signal enhancement of up to 760-fold at 7.05 T (corresponding to 0.15% 14N polarization) was obtained