Direct Correlation between Donor-Acceptor Distance and Temperature Dependence of Kinetic Isotope Effects in Hydride-Tunneling Reactions of NADH/NAD+ Analogues.

Recent study of structural effects on primary kinetic isotope effects (1° KIEs) of H-transfer reactions in enzymes and solution revealed that a more rigid reaction system gave rise to a weaker temperature dependence of 1° KIEs, i.e., a smaller isotopic activation energy difference (ΔEa = EaD - EaH). This has been explained within the contemporary vibrationally assisted activated H-tunneling (VA-AHT) model in which rigidity is defined according to the density of donor-acceptor distance (DADTRS) populations at the tunneling ready state (TRS) sampled by heavy atom motions. To test the relationship between DADTRS and ΔEa in the model, we developed a computational method to obtain the TRS structures for H-transfer reactions. The method was applied to three hydride transfer reactions of NADH/NAD+ analogues for which the ΔEa's as well as secondary (2°) KIEs have been reported. The 2° KIEs computed from each TRS structure were fitted to the observed values to obtain the optimal TRSs/DADTRS's. It was found that a shorter DADTRS does correspond with a smaller ΔEa. This appears to support the VA-AHT model. Moreover, an analysis of hybridizations at the bent TRS structures shows that rehybridizations at the donor-acceptor centers are much more advanced than predicted from the classical mechanism. This implies that more orbital preparations are required for the nonclassical H-tunneling to take place.

A scalable synthesis of adjuvanting antigen depots based on metal-organic frameworks.

Vaccines have saved countless lives by preventing and even irradicating infectious diseases. Commonly used subunit vaccines comprising one or multiple recombinant proteins isolated from a pathogen demonstrate a better safety profile than live or attenuated vaccines. However, the immunogenicity of these vaccines is weak, and therefore, subunit vaccines require a series of doses to achieve sufficient immunity against the pathogen. Here, we show that the biomimetic mineralization of the inert model antigen, ovalbumin (OVA), in zeolitic imidazolate framework-8 (ZIF-8) significantly improves the humoral immune response over three bolus doses of OVA (OVA 3×). Encapsulation of OVA in ZIF-8 (OVA@ZIF) demonstrated higher serum antibody titers against OVA than OVA 3×. OVA@ZIF vaccinated mice displayed higher populations of germinal center (GC) B cells and IgG1+ GC B cells as opposed to OVA 3×, indicative of class-switching recombination. We show that the mechanism of this phenomenon is at least partly owed to the metalloimmunological effects of the zinc metal as well as the sustained release of OVA from the ZIF-8 composite. The system acts as an antigen reservoir for antigen-presenting cells to traffic into the draining lymph node, enhancing the humoral response. Lastly, our model system OVA@ZIF is produced quickly at the gram scale in a laboratory setting, sufficient for up to 20 000 vaccine doses.

TEMPO-conjugated tobacco mosaic virus as a magnetic resonance imaging contrast agent for detection of superoxide production in the inflamed liver.

Superoxide, an anionic dioxygen molecule, plays a crucial role in redox regulation within the body but is implicated in various pathological conditions when produced excessively. Efforts to develop superoxide detection strategies have led to the exploration of organic-based contrast agents for magnetic resonance imaging (MRI). This study compares the effectiveness of two such agents, nTMV-TEMPO and kTMV-TEMPO, for detecting superoxide in a mouse liver model with lipopolysaccharide (LPS)-induced inflammation. The study demonstrates that kTMV-TEMPO, with a strategically positioned lysine residue for TEMPO attachment, outperforms nTMV-TEMPO as an MRI contrast agent. The enhanced sensitivity of kTMV-TEMPO is attributed to its more exposed TEMPO attachment site, facilitating stronger interactions with water protons and superoxide radicals. EPR kinetics experiments confirm kTMV-TEMPO's faster oxidation and reduction rates, making it a promising sensor for superoxide in inflamed liver tissue. In vivo experiments using healthy and LPS-induced inflamed mice reveal that reduced kTMV-TEMPO remains MRI-inactive in healthy mice but becomes MRI-active in inflamed livers. The contrast enhancement in inflamed livers is substantial, validating the potential of kTMV-TEMPO for detecting superoxide in vivo. This research underscores the importance of optimizing contrast agents for in vivo imaging applications. The enhanced sensitivity and biocompatibility of kTMV-TEMPO make it a promising candidate for further studies in the realm of medical imaging, particularly in the context of monitoring oxidative stress-related diseases.

An optimized purification protocol for enzymatically synthesized S-adenosyl-L-methionine (SAM) for applications in solution state infrared spectroscopic studies.

S-adenosyl-L-methionine (SAM) is an abundant biomolecule used by methyltransferases to regulate a wide range of essential cellular processes such as gene expression, cell signaling, protein functions, and metabolism. Despite considerable effort, there remain many specificity challenges associated with designing small molecule inhibitors for methyltransferases, most of which exhibit off-target effects. Interestingly, NMR evidence suggests that SAM undergoes conformeric exchange between several states when free in solution. Infrared spectroscopy can detect different conformers of molecules if present in appreciable populations. When SAM is noncovalently bound within enzyme active sites, the nature and the number of different conformations of the molecule are likely to be altered from when it is free in solution. If there are unique structures or different numbers of conformers between different methyltransferase active sites, solution-state information may provide promising structural leads to increase inhibitor specificity for a particular methyltransferase. Toward this goal, frequencies measured in SAM's infrared spectra must be assigned to the motions of specific atoms via isotope incorporation at discrete positions. The incorporation of isotopes into SAM's structure can be accomplished via an established enzymatic synthesis using isotopically labeled precursors. However, published protocols produced an intense and highly variable IR signal which overlapped with many of the signals from SAM rendering comparison between isotopes challenging. We observed this intense absorption to be from co-purifying salts and the SAM counterion, producing a strong, broad signal at 1100 cm-1. Here, we report a revised SAM purification protocol that mitigates the contaminating salts and present the first IR spectra of isotopically labeled CD3-SAM. These results provide a foundation for isotopic labeling experiments of SAM that will define which atoms participate in individual molecular vibrations, as a means to detect specific molecular conformations.

In vivo biocompatibility of ZIF-8 for slow release via intranasal administration.

Zeolitic imidazolate framework-8 (ZIF-8) is becoming popular in research for its potential in antigen protection and for providing a thermally stable, slow-release platform. While papers applying this material for immunological applications are aplenty in the literature, studies that explore the biosafety of ZIF-8 in mammals-especially when administered intranasally-are not well represented. We checked the body clearance of uncoated and ZIF-8-coated liposomes and observed that the release slowed as ZIF-8 is easily degraded by mucosal fluid in the nasal cavity. We delivered varying doses of ZIF-8, checked its short- and long-term effects on diagnostic proteins found in blood serum, and found no noticeable differences from the saline control group. We also studied their lung diffusing capacity and tissue morphology; neither showed significant changes in morphology or function.

Rip it, stitch it, click it: A Chemist's guide to VLP manipulation.

Viruses are some of nature's most ubiquitous self-assembled molecular containers. Evolutionary pressures have created some incredibly robust, thermally, and enzymatically resistant carriers to transport delicate genetic information safely. Virus-like particles (VLPs) are human-engineered non-infectious systems that inherit the parent virus' ability to self-assemble under controlled conditions while being non-infectious. VLPs and plant-based viral nanoparticles are becoming increasingly popular in medicine as their self-assembly properties are exploitable for applications ranging from diagnostic tools to targeted drug delivery. Understanding the basic structure and principles underlying the assembly of higher-order structures has allowed researchers to disassemble (rip it), reassemble (stitch it), and functionalize (click it) these systems on demand. This review focuses on the current toolbox of strategies developed to manipulate these systems by ripping, stitching, and clicking to create new technologies in the biomedical space.

Substituent Effects on Temperature Dependence of Kinetic Isotope Effects in Hydride-Transfer Reactions of NADH/NAD+ Analogues in Solution: Reaction Center Rigidity Is the Key.

Substituent effects on the temperature dependence of primary kinetic isotope effects, characterized by ΔEa = EaD - EaH, for two series of the title reactions in acetonitrile were studied. The change from ΔEa ≈ 0 for a highly rigid system to ΔEa > 0 for systems with reduced rigidities was observed. The rigidities were controlled by the electronic and steric effects. This work replicates the observations in enzymes and opens a new research direction that studies structure-ΔEa relationship.