Preparation and characterization of a homogeneous immunoassay for point-of-care testing (POCT) of procalcitonin (PCT).

Procalcitonin (PCT) has been recognized as a specific and early marker for microbial infection and sepsis. Sensitive measuring interaction-triggered luminescence experiment (SMILE), a homogeneous immunoassay method, was established for point-of-care testing (POCT) of PCT. SMILE is achieved through the principle of double antibody sandwich, where two antibodies immobilized on the surface of polystyrene microspheres (donor and acceptor beads) bind to the PCT antigen. The donor bead contains phthalocyanine dye (luminol chemiluminescent substance) and the acceptor bead contains dimethylthiophene derivatives and Eu chelates. Therefore, singlet oxygen can be transferred when the distance between donor and acceptor beads is within 200 nm, generating detectable luminescent signals. Scanning electron microscopy (SEM) was used to detect the diameter and polymer dispersity index (PDI) of microspheres before and after binding with antibodies to characterize the immobilization of antibodies. The reaction conditions for antibody immobilization including pH, mass ratio and reaction time have also been optimized. The limit of quantitation (LOQ) of the SMILE method (0.01 ng mL-1) was lower than that of the LFI method (0.1 ng mL-1), the working range (0.01-500 ng mL-1) was wider than that of the LFI method (0.1-50 ng mL-1), and the assay time (10 min) was shorter than that of the LFI method (15 min). So, SMILE is more suitable for POCT of PCT compared with lateral flow immunochromatography (LFI), which is the most used measuring method, due to its advantages of simple operation, saving time, convenience, wide detection range, and high sensitivity and accuracy.

Postmodification via Thiol-Click Chemistry Yields Hydrophilic Trityl-Nitroxide Biradicals for Biomolecular High-Field Dynamic Nuclear Polarization.

Dynamic nuclear polarization (DNP) is a powerful method to enhance nuclear magnetic resonance (NMR) signal intensities, enabling unprecedented applications in life and material science. An ultimate goal is to expand the use of DNP-enhanced solid-state NMR to ultrahigh magnetic fields where optimal spectral resolution and sensitivity are integrated. Trityl-nitroxide (TN) biradicals have attracted significant interest in high-field DNP, but their application to complex (bio)molecules has so far been limited. Here we report a novel postmodification strategy for synthesis of hydrophilic TN biradicals in order to improve their use in biomolecular applications. Initially, three TN biradicals (referred to as NATriPols 1-3) with amino-acid linkers were synthesized. EPR studies showed that the α-position of the amino-acid linkers is an ideal modification site for these biradicals since their electron-electron magnetic interactions are marginally affected by the substituents at this position. On the basis of this finding, we synthesized NATriPol-4 with pyridine disulfide appended at the α-position. Postmodification of NATriPol-4 via thiol-click chemistry resulted in various TN biradicals including hydrophilic NATriPol-5 in a quantitative manner. Interestingly, DNP enhancements at 18.8 T of NATriPols for 13C,15N-proline in a glycerol/water matrix are inversely correlated with their hydrophobicity. Importantly, applications of hydrophilic NATriPol-5 and NATriPol-3 to biomolecules including a globular soluble protein and a membrane targeting peptide reveal significantly improved performance compared to TEMTriPol-1 and AMUPol. Our work provides an efficient approach for one-step synthesis of new polarizing agents with tunable physicochemical properties, thus expediting optimization of new biradicals for biomolecular applications at ultrahigh magnetic fields.

Two radical-dependent mechanisms for anaerobic degradation of the globally abundant organosulfur compound dihydroxypropanesulfonate.

2(S)-dihydroxypropanesulfonate (DHPS) is a microbial degradation product of 6-deoxy-6-sulfo-d-glucopyranose (sulfoquinovose), a component of plant sulfolipid with an estimated annual production of 1010 tons. DHPS is also at millimolar levels in highly abundant marine phytoplankton. Its degradation and sulfur recycling by microbes, thus, play important roles in the biogeochemical sulfur cycle. However, DHPS degradative pathways in the anaerobic biosphere are not well understood. Here, we report the discovery and characterization of two O2-sensitive glycyl radical enzymes that use distinct mechanisms for DHPS degradation. DHPS-sulfolyase (HpsG) in sulfate- and sulfite-reducing bacteria catalyzes C-S cleavage to release sulfite for use as a terminal electron acceptor in respiration, producing H2S. DHPS-dehydratase (HpfG), in fermenting bacteria, catalyzes C-O cleavage to generate 3-sulfopropionaldehyde, subsequently reduced by the NADH-dependent sulfopropionaldehyde reductase (HpfD). Both enzymes are present in bacteria from diverse environments including human gut, suggesting the contribution of enzymatic radical chemistry to sulfur flux in various anaerobic niches.

Synthesis of Central Chirality-Containing Triarylmethanols and Triarylmethyl Radicals with Extraordinarily Stable Configurations.

Triarylmethanol adopts a propeller-shaped conformation with either right-handed (P) or left-handed (M) configuration. Herein, new triarylmethanols with two chiral centers were obtained via introduction of two cis-hydroxyl groups on the side chains, affording four stereoisomers. These four stereoisomers were easily separated by silica gel column chromatography into two pairs of propeller-shaped enantiomers, as shown by NMR and X-ray crystallographic studies. High-performance liquid chromatography (HPLC) studies showed that the configurations of the hydroxyl-bearing triarylmethanols are much more stable than those of the bulky tert-butyldimethylsilyl-protected precursors, inconsistent with the general strategy in which the steric repulsion is largely responsible for the configurational stability. Similarly, two hydroxyl-bearing tetrathiatriarylmethyl (TAM) radicals also exhibit excellent configurational stability and are thus separable by CS-HPLC into four stereoisomers. Interestingly, both helical chirality from triaryl group (M or P) and central chirality (R and S) on the side chain have little effect on their electron paramagnetic resonance properties. Our present study provides a new strategy to construct configurationally stable triaryl compounds and demonstrates that the side chain on TAM radicals is a new site for their structural modifications.

A gene cluster for taurine sulfur assimilation in an anaerobic human gut bacterium.

Aminoethylsulfonate (taurine) is widespread in the environment and highly abundant in the human body. Taurine and other aliphatic sulfonates serve as sulfur sources for diverse aerobic bacteria, which carry out cleavage of the inert sulfonate C-S bond through various O2-dependent mechanisms. Taurine also serves as a sulfur source for certain strict anaerobic fermenting bacteria. However, the mechanism of C-S cleavage by these bacteria has long been a mystery. Here we report the biochemical characterization of an anaerobic pathway for taurine sulfur assimilation in a strain of Clostridium butyricum from the human gut. In this pathway, taurine is first converted to hydroxyethylsulfonate (isethionate), followed by C-S cleavage by the O2-sensitive isethionate sulfo-lyase IseG, recently identified in sulfate- and sulfite-reducing bacteria. Homologs of the enzymes described in this study have a sporadic distribution in diverse strict and facultative anaerobic bacteria, from both the environment and the taurine-rich human gut, and may enable sulfonate sulfur acquisition in certain nutrient limiting conditions.

Synthesis and Characterization of Hydrophilic Trityl Radical TFO for Biomedical and Biophysical Applications.

Tetrathiatriarylmethyl (TAM, trityl) radicals have found wide applications as spin probes/labels for EPR spectroscopy and imaging, and as polarizing agents for dynamic nuclear polarization. The high hydrophilicity of TAM radicals is essential for their biomedical applications. However, the synthesis of hydrophilic TAM radicals (e.g., OX063) is extremely challenging and has only been reported in the patent literature, to date. Herein, an efficient synthesis of a highly water-soluble TAM radical bis(8-carboxyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d']bis([1,3]dithiol-4-yl)-mono-(8-carboxyl-2,2,6,6-tetrakis(2-hydroxyethyl)benzo[1,2-d:4,5-d']bis([1,3]dithiol-4-yl)methyl (TFO), which contains four additional hydroxylethyl groups, relative to the Finland trityl radical CT-03, is reported. Similar to OX063, TFO exhibits excellent properties, including high water solubility in phosphate buffer, low log P, low pKa , long relaxation times, and negligible binding with bovine serum albumin. On the other hand, TFO has a sharper EPR line and higher O2 sensitivity than those of OX063. Therefore, in combination with its facile synthesis, TFO should find wide applications in magnetic resonance related fields and this synthetic approach would shed new light on the synthesis of other hydrophilic TAM radicals.

Diastereoisomers of l-proline-linked trityl-nitroxide biradicals: synthesis and effect of chiral configurations on exchange interactions.

The exchange (J) interaction of organic biradicals is a crucial factor controlling their physiochemical properties and potential applications and can be modulated by changing the nature of the linker. In the present work, we for the first time demonstrate the effect of chiral configurations of radical parts on the J values of trityl-nitroxide (TN) biradicals. Four diastereoisomers (TNT1, TNT2, TNL1 and TNL2) of TN biradicals were synthesized and purified by the conjugation of a racemic (R/S) nitroxide with the racemic (M/P) trityl radical vial-proline. The absolute configurations of these diastereoisomers were assigned by comparing experimental and calculated electronic circular dichroism (ECD) spectra as (M, S, S) for TNT1, (P, S, S) for TNT2, (M, S, R) for TNL1 and (P, S, R) for TNL2. Electron paramagnetic resonance (EPR) results showed that the configuration of the nitroxide part instead of the trityl part is dominant in controlling the exchange interactions and the order of the J values at room temperature is TNT1 (252 G) > TNT2 (127 G) ≫ TNL2 (33 G) > TNL1 (14 G). Moreover, the J values of TNL1/TNL2 with the S configuration in the nitroxide part vary with temperature and the polarity of solvents due to their flexible linker, whereas the J values of TNT1/TNT2 are almost insensitive to these two factors due to the rigidity of their linkers. The distinct exchange interactions between TNT1,2 and TNL1,2 in the frozen state led to strongly different high-field dynamic nuclear polarization (DNP) enhancements with ε = 7 for TNT1,2 and 40 for TNL1,2 under 800 MHz DNP conditions.