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The collision reaction of H + O 2 = OH + O is a pivotal step in combustion. To investigate the influence of singlet oxygen on this reaction, we computed potential energy surfaces (PESs) for all six lowest states using high-level ab initio methods and coupled them with embedded atom neural network (EANN) fitting. By integrating quasi-classical trajectory (QCT) with trajectory surface hopping (TSH) based on the fitted PESs, we simulated the dynamics of both ground- and excited-states to derive the reaction rate constants for the forward and reverse processes. The results reveal that the forward reaction facilitates radical generation, promoting combustion reactions. Furthermore, calculations of reverse reaction rate constants indicate that all electronic states ultimately yield ground-state oxygen, leading to radical deactivation and exerting an inhibitory effect on combustion processes.

2-ketoGlc, which is the C(2)-carbon isostere of GlcNAc, is a novel GlcNAc analogue with a ketone group. The corresponding glycosyltransferase donor substrate, UDP-2-ketoGlc, is necessary for synthesizing 2-ketoGlc-containing molecules and is thus highly important for metabolic polysaccharide remodeling and engineering. We report here the first chemoenzymatic synthesis of UDP-2-ketoGlc using our two-enzyme (NahK and GlmU) system in vitro.

Isoglobotrihexosylceramide (iGb3) has been identified as an endogenous ligand recognized by NKT cells; however, it is a weak agonist compared to the exogenous alpha-galactosylceramide. Modification of the structure of iGb3 might improve its stimulatory activity. In this study, we assessed the stimulating activity of chemically-modified iGb3 analogues on murine hepatic NKT cells. We analyzed the percentage of IFN-gamma- or IL-4-producing cells in hepatic iNKT cell population and found that two chemically-modified iGb3 analogues, especially 4'''-dh-iGb3, induced significantly greater intracellular IFN-gamma+ NKT cells in liver by flow cytometry. In vivo experiments also showed that 4-HO-iGb3 and 4'''-dh-iGb3 are selectively strong inducer for rapid serum IFN-gamma production compared with unmodified iGb3. Comparing the structure of iGb3 and its two iGb3 analogues, 4-HO-iGb3 has an extra hydroxy group on C4, suggesting that the additional hydroxy group of phytosphingosine might augment the stability of the CD1d/glycoceramide complex forming and thereby possibly promote IFN-gamma producing. By further modifying the polysaccharide of glycolipid as did in 4'''-dh-iGb3, we found that 4'''-dh-iGb3 elicited more Th1-biased responses than iGb3 and 4-HO-iGb3. This modification might more strongly strengthen the affinity of the TCR/glycoceramide complex and ultimately polarize iNKT cells to release more Th1 cytokines. Our data suggests that a combination modification on both polysaccharide and sphingosine chain of iGb3 elicits preferential Th1-biased responses.

Invariant natural killer T (iNKT) cells are innate T lymphocytes that express T cell receptors binding to exogenous and endogenous glycosphingolpid antigens presented by a nonpolymorphic, non-MHC antigen presenting molecule, CD1d. The endogenous glycosphingolipid metabolite, isoglobotrihexosylceramide (iGb3), is the first known natural ligand for both human and mouse iNKT cells, whose activity has been confirmed in a variety of iNKT cell clones generated by different investigators, representing the majority of the iNKT cell population. The signaling pathway mediated by T cell receptor is largely influenced by the structural variation of glycosphingolpid antigens, leading to multiple and varied biological functions of iNKT cells. In order to investigate the structural requirements behind iGb3 triggered iNKT cell activation, the structure-activity relationship (SAR) of iGb3 needs to be characterized. In this study, iGb3 analogues containing 2' '', 3' '', 4' '' and 6' '' deoxy terminal galactose were synthesized for probing the SAR between iGb3 and TCR. The biological assays on the synthetic iGb3 analogues were performed with use of the murine iNKT cell hybridoma DN32.D3. The results showed that the 2' '' and 3' '' hydroxyl groups of terminal galactose play more important roles for the recognition of iGb3 by TCR; while 4' '' and 6' '' hydroxyl groups were not as crucial for this recognition. These studies might help to understand the general structural requirements for natural endogenous ligands recognized by iNKT cells.

Glycoceramides can activate NKT cells by binding with CD1d to produce IFN-gamma, IL-4, and other cytokines. An efficient synthetic pathway for alpha-galactosylceramide (KRN7000) was established by coupling a protected galactose donor to a properly protected ceramide. During the investigation, it was discovered that when the ceramide was protected with benzyl groups, only beta-galactosylceramide was produced from the glycosylation reaction. In contrast, the ceramide with benzoyl protecting groups produced alpha-galactosylceramide. Isoglobotrihexosylceramide (iGb3) was prepared by glycosylation of Galalpha1-3Galbeta1-4Glc donor with 2-azido-sphingosine in high yield. Biological assays on the synthetic KRN7000 and iGb3 were performed using human and murine iNKT cell clones or hybridomas.