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The allene functional group in natural products isolated so far exists in a non-racemic form, but its axial chirality is difficult to elucidate. Allenes exhibit a characteristic antisymmetric C=C=C stretching mode at around 1950 cm(-1), and their VCD properties have not been studied in detail. This work, for the first time, applied VCD spectroscopy to allenic natural products and allenic molecules with other asymmetric centers focusing on the antisymmetric C=C=C stretching mode. This vibrational mode yielded a negligibly weak VCD signal for several molecules, but in the presence of electron-withdrawing and/or conjugating substituents, it generated a stronger one. Its sign was found to be influenced by the nature of substituents. These findings should deepen the understanding of the VCD properties of the allene functional group and should be useful for future studies of chiral allenes.

Oxidation of polyunsaturated fatty acids produces various oxidized lipids whose absolute configuration (AC) and conformations are difficult to analyze due to their flexibility. Through studies on hydroxy fatty acids, lipid hydroperoxides, and lipid epoxides, this work demonstrates the effectiveness of VCD spectroscopy to elucidate their AC and conformational preferences.

Background The protracted preclinical stage of Alzheimer's disease (AD) provides the opportunity for early intervention to prevent the disease; however, the lack of minimally invasive and easily detectable biomarkers and their measurement technologies remain unresolved. Extracellular vesicles (EVs) are nanosized membrane vesicles released from a variety of cells and play important roles in cell-cell communication. Neuron-derived and ganglioside-enriched EVs capture amyloid-ß protein, a major AD agent, and transport it into glial cells for degradation; this suggests that EVs influence Aß accumulation in the brain. EV heterogeneity, however, requires the use of a highly sensitive technique for measuring specific EVs in biofluid. In this study, immuno-digital invasive cleavage assay (idICA) was developed for quantitating target-intact EVs. Methods EVs were captured onto ganglioside GM1-specific cholera toxin B subunit (CTB)-conjugated magnetic beads and detected with a DNA oligonucleotide-labeled Aß antibody. Fluorescence signals for individual EVs were then counted using an invasive cleavage assay (ICA). This idICA examines the Aß-bound and GM1-containing EVs isolated from the culture supernatant of human APP-overexpressing N2a (APP-N2a) cells and APP transgenic mice sera. Results The idICA quantitatively detected Aß-bound and GM1-containing EVs isolated from culture supernatants of APP-N2a cells and sera of AD model mice. The idICA levels of Aß-associated EVs in blood gradually increased from 3- to 12-month-old mice, corresponding to the progression of Aß accumulations in the brain of AD model mice. Conclusions The present findings suggest that peripheral EVs harboring Aß and GM1 reflect Aß burden in mice. The idICA is a valuable tool for easy quantitative detection of EVs as an accessible biomarker for preclinical AD diagnosis.

The extracellular accumulation of aggregated amyloid-beta (A beta) in the brain leads to the early pathology of Alzheimer's disease (AD). The administration of exogenous plant-type ceramides into AD model mice can promote the release of neuronal exosomes, a subtype of extracellular vesicles, that can mediate A beta clearance. In vitro studies showed that the length of fatty acids in mammalian-type ceramides is crucial for promoting neuronal exosome release. Therefore, investigating the structures of plant ceramides is important for evaluating the potential in releasing exosomes to remove A beta. In this study, we assessed plant ceramide species with D-erythro-(4E,8Z)-sphingadienine and D-erythro-(8Z)-phytosphingenine as sphingoid bases that differ from mammalian-type species. Some plant ceramides were more effective than mammalian ceramides at stimulating exosome release. In addition, using deuterium chemistry-based lipidomics, most exogenous plant ceramides were confirmed to be derived from exosomes. These results suggest that the ceramide-dependent upregulation of exosome release may promote the release of exogenous ceramides from cells, and plant ceramides with long-chain fatty acids can effectively release neuronal exosomes and prevent AD pathology.

Stereochemical elucidation of molecules with multiple chiral centers is difficult. Even with VCD spectroscopy, excluding all but one diastereomeric structural candidates is challenging because stereochemical inversion of one chiral center among many centers does not always result in noticeable differences in their VCD spectra. This work demonstrates that introduction of a suitable VCD chromophore with absorption in the 2300-1900 cm-1 region can be used for extracting local stereochemical information and for stereochemical assignment of C-1 of various sugars as a case study. Through studies on a series of epimeric pairs of monosaccharides and their derivatives, we found that the introduction of one -OCD3 group to each C-1 position produced almost mirror-image VCD patterns in the 2300-1900 cm-1 region depending on the C-1 stereochemistry irrespective of the other molecular moiety. This work also shows that comparison of the observed VCD signals in this spectral region and calculated ones enables the stereochemical assignment of a chiral center in the vicinity of the chromophore. This study provides a proof of concept that the use of a VCD chromophore in the 2300-1900 cm-1 region enables the analysis of selected stereochemistry of suitable molecular systems. Further studies on this concept should lead to development of a method useful for the structural elucidation of other types of complex molecules.

The extracellular accumulation of aggregated amyloid-beta (A beta) in the brain leads to the early pathology of Alzheimer's disease (AD). The administration of exogenous plant-type ceramides into AD model mice can promote the release of neuronal exosomes, a subtype of extracellular vesicles, that can mediate A beta clearance. In vitro studies showed that the length of fatty acids in mammalian-type ceramides is crucial for promoting neuronal exosome release. Therefore, investigating the structures of plant ceramides is important for evaluating the potential in releasing exosomes to remove A beta. In this study, we assessed plant ceramide species with D-erythro-(4E,8Z)-sphingadienine and D-erythro-(8Z)-phytosphingenine as sphingoid bases that differ from mammalian-type species. Some plant ceramides were more effective than mammalian ceramides at stimulating exosome release. In addition, using deuterium chemistry-based lipidomics, most exogenous plant ceramides were confirmed to be derived from exosomes. These results suggest that the ceramide-dependent upregulation of exosome release may promote the release of exogenous ceramides from cells, and plant ceramides with long-chain fatty acids can effectively release neuronal exosomes and prevent AD pathology.

Oxidation of polyunsaturated fatty acids produces various oxidized lipids whose absolute configuration (AC) and conformations are difficult to analyze due to their flexibility. Through studies on hydroxy fatty acids, lipid hydroperoxides, and lipid epoxides, this work demonstrates the effectiveness of VCD spectroscopy to elucidate their AC and conformational preferences.

Interactions of two chromophores such as carbonyl groups yield a strong VCD couplet that reflects the molecular structures. The use of VCD couplets for biomacromolecular structural studies has been hampered by severe signal overlap caused by numerous functional groups that originally exist in biomacromolecules. Nitrile, isonitrile, alkyne, and azido groups show characteristic IR absorption in the 2300-2000 cm(-1) region, where biomolecules do not strongly absorb. We herein examined the usefulness of these functional groups as chromophores to observe a strong VCD couplet that can be readily interpreted using theoretical calculations. Studies on a chiral binaphthyl scaffold possessing two identical chromophores showed that nitrile and isonitrile groups generate moderately-strong but complex VCD signals due to anharmonic contributions. The nature of their anharmonic VCD patterns is discussed by comparison with the VCD spectrum of a mono-chromophoric molecule and by anharmonic DFT calculations. On the other hand, through studies on diazido binaphthyl and diazido monosaccharide, we demonstrated that the azido group is more promising for structural analysis of larger molecules due to its simple, strong VCD couplet whose spectral patterns are readily predicted by harmonic DFT calculations.

Interactions of two chromophores such as carbonyl groups yield a strong VCD couplet that reflects the molecular structures. The use of VCD couplets for biomacromolecular structural studies has been hampered by severe signal overlap caused by numerous functional groups that originally exist in biomacromolecules. Nitrile, isonitrile, alkyne, and azido groups show characteristic IR absorption in the 2300-2000 cm(-1) region, where biomolecules do not strongly absorb. We herein examined the usefulness of these functional groups as chromophores to observe a strong VCD couplet that can be readily interpreted using theoretical calculations. Studies on a chiral binaphthyl scaffold possessing two identical chromophores showed that nitrile and isonitrile groups generate moderately-strong but complex VCD signals due to anharmonic contributions. The nature of their anharmonic VCD patterns is discussed by comparison with the VCD spectrum of a mono-chromophoric molecule and by anharmonic DFT calculations. On the other hand, through studies on diazido binaphthyl and diazido monosaccharide, we demonstrated that the azido group is more promising for structural analysis of larger molecules due to its simple, strong VCD couplet whose spectral patterns are readily predicted by harmonic DFT calculations.