鞘氨醇的化学

动植物细胞中鞘脂及其代谢产物鞘氨醇等具有多种生理作用，是生命体传递信息的化学信使。鞘氨醇等的合成在有机合成界形成了一个经久不衰的热潮，应用各种新技术的合成方法不断出现。本文主要综述了鞘氨醇等衍生脂质的化学研究，特别是它们的化学合成进展。     

Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues

The inositol pyrophosphate messengers (PP‐InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP‐InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non‐hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal‐coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP‐InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions.     

Functional Cellular Mimics for the Spatiotemporal Control of Multiple Enzymatic Cascade Reactions

Next‐generation therapeutic approaches are expected to rely on the engineering of biomimetic cellular systems that can mimic specific cellular functions. Herein, we demonstrate a highly effective route for constructing structural and functional eukaryotic cell mimics by loading pH‐sensitive polymersomes as membrane‐associated and free‐floating organelle mimics inside the multifunctional cell membrane. Metabolism mimicry has been validated by performing successive enzymatic cascade reactions spatially separated at specific sites of cell mimics in the presence and absence of extracellular organelle mimics. These enzymatic reactions take place in a highly controllable, reproducible, efficient, and successive manner. Our biomimetic approach to material design for establishing functional principles brings considerable enrichment to the fields of biomedicine, biocatalysis, biotechnology, and systems biology.     

Synthesis of Site‐Specifically Phosphate‐Caged siRNAs and Evaluation of Their RNAi Activity and Stability

A complete set of new photolabile nucleoside phosphoramidites were synthesized, then site‐specifically incorporated into sense or antisense strands of siRNA for phosphate caging. Single caging modification was made along siRNA strands and their photomodulation of gene silencing were examined by using the firefly luciferase reporter gene. Several key phosphate positions were then identified. Furthermore, multiple caging modifications at these key positions led to significantly enhanced photomodulation of gene silencing activity, suggesting a synergistic effect. The caging group on both the terminally phosphate‐caged siRNA and the single‐stranded caged RNA has comparatively high stability, whereas hydrolysis of the caged group from the internally caged siRNA was observed, irrespective of the presence of Mg²⁺. Molecular dynamic simulations demonstrated that enhanced hydrolysis of the caging group on internally phosphate‐caged siRNAs was due to easy fragmentation of the caging group upon formation of the pentavalent intermediate of the phosphotriester with attack by water. The caging group in the terminally phosphate‐caged siRNA or single‐stranded caged RNA prefers to form π–π stacks with nearby nucleobases. In addition to providing explanations for previous observations, this study sheds further light on the design of caged oligonucleotides and indicates the direction of future development of nucleic acid drugs with phosphate modifications.     

Comparative Bioinformatics Analyses and Profiling of Lysosome-Related Organelle Proteomes

Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for 7 lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.     

Dendrimer Conjugation Enables Multiphoton Chemical Neurophysiology Studies with an Extended π‐Electron Caging Chromophore

We have developed a caged neurotransmitter using an extended π‐electron chromophore for efficient multiphoton uncaging on living neurons. Widely studied in a chemical context, such chromophores are inherently bioincompatible due to their highly lipophilic character. Attachment of two polycarboxylate dendrimers, a method we call “cloaking”, to a bisstyrylthiophene (or BIST) core effectively transformed the chromophore into a water‐soluble optical probe, whilst maintaining the high two‐photon absorption of over 500 GM. Importantly, the cloaked caged compound was biologically inert at the high concentrations required for multiphoton chemical physiology. Thus, in contrast to non‐cloaked BIST compounds, the BIST‐caged neurotransmitter can be safely delivered onto neurons in acutely isolated brain slices, thereby enabling high‐resolution two‐photon uncaging without any side effects. We expect that our cloaking method will enable the development of new classes of cell‐compatible photolabile probes using a wide variety of extended π‐electron caging chromophores.     

An Intrinsically Disordered Peptide Facilitates Non‐Endosomal Cell Entry

Many cell‐penetrating peptides (CPPs) fold at cell surfaces, adopting α‐ or β‐structure that enable their intracellular transport. However, the same structural folds that facilitate cellular entry can also elicit potent membrane‐lytic activity, limiting their use in delivery applications. Further, a distinct CPP can enter cells through many mechanisms, often leading to endosomal entrapment. Herein, we describe an intrinsically disordered peptide (CLIP6) that exclusively employs non‐endosomal mechanisms to cross cellular membranes, while being remarkably biocompatible and serum‐stable. We show that a single anionic glutamate residue is responsible for maintaining the disordered bioactive state of the peptide, defines its mechanism of cellular entry, and is central to its biocompatibility. CLIP6 can deliver membrane‐impermeable cargo directly to the cytoplasm of cells, suggesting its broad utility for delivery of drug candidates limited by poor cell permeability and endosomal degradation.     

Discerning the Chemistry in Individual Organelles with Small‐Molecule Fluorescent Probes

Principle has it that even the most advanced super‐resolution microscope would be futile in providing biological insight into subcellular matrices without well‐designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small‐molecule fluorescent probes that not only allow cellular‐level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle‐anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology.     

Guiding Drugs to Target‐Harboring Organelles: Stretching Drug‐Delivery to a Higher Level of Resolution

The ratio between the dose of drug required for optimal efficacy and the dose that causes toxicity is referred to as the therapeutic window. This ratio can be increased by directing the drug to the diseased tissue or pathogenic cell. For drugs targeting fungi and malignant cells, the therapeutic window can be further improved by increasing the resolution of drug delivery to the specific organelle that harbors the drug's target. Organelle targeting is challenging and is, therefore, an under‐exploited strategy. Here we provide an overview of recent advances in control of the subcellular distribution of small molecules with the focus on chemical modifications. Highlighted are recent examples of active and passive organelle‐specific targeting by incorporation of organelle‐directing molecular determinants or by chemical modifications of the pharmacophore. The outstanding potential that lies in the development of organelle‐specific drugs is becoming increasingly apparent.     

“Stainomics”: Identification of mitotracker labeled proteins in mammalian cells

Organelle‐specific cell‐permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, mitotracker red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilization, denaturation, and electrophoresis. This enabled us to display MTR‐labeled proteins by 2DE. The MTR‐specific fluorescent signals on the gel revealed the spots that contained MTR‐conjugated proteins. These spots were analyzed by MS, resulting into the identification of ten proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock‐like responses. The identification of the molecular targets of biological dyes, or “stainomics,” can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes.     

Organelle‐Targeted BODIPY Photocages: Visible‐Light‐Mediated Subcellular Photorelease

Photocaging facilitates non‐invasive and precise spatio‐temporal control over the release of biologically relevant small‐ and macro‐molecules using light. However, sub‐cellular organelles are dispersed in cells in a manner that renders selective light‐irradiation of a complete organelle impractical. Organelle‐specific photocages could provide a powerful method for releasing bioactive molecules in sub‐cellular locations. Herein, we report a general post‐synthetic method for the chemical functionalization and further conjugation of meso‐methyl BODIPY photocages and the synthesis of endoplasmic reticulum (ER)‐, lysosome‐, and mitochondria‐targeted derivatives. We also demonstrate that 2,4‐dinitrophenol, a mitochondrial uncoupler, and puromycin, a protein biosynthesis inhibitor, can be selectively photoreleased in mitochondria and ER, respectively, in live cells by using visible light. Additionally, photocaging is shown to lead to higher efficacy of the released molecules, probably owing to a localized and abrupt release.     

Oligonucleotide loaded polypeptide‐peptide nanoparticles towards mitochondrial‐targeted delivery

Nanoparticles (NPs) consisting of biodegradable and biocompatible polymers may have the ability to deliver a cargo to specific tissue, cell type, and organelle. Various diseases, which are linked to mitochondrial genome (mtDNA) mutations and have no effective treatments, may be approached by gene therapy strategies. In this study, we adapted the recently developed mitochondria delivering polypeptide‐peptide nanoparticles (PoP‐NPs) system to carry an oligonucleotide cargo to the proximity of the mitochondria. PoP‐NPs are formulated by self‐assembly of the negatively charged polypeptide, poly gamma glutamic acid (γ‐PGA), with an amphiphilic and cationic β‐sheet peptide (PFK). Here, we show that PFK interacts favorably with oligonucleotides and thereby enables the formation of DNA‐loaded PoP‐NPs (DNA‐PoP‐NPs). DNA‐PoP‐NPs could be assembled with different peptide to oligonucleotide (N/P) ratios, and their targeting to the proximity of mitochondria in cell culture could be facilitated through NPs coating with PFK peptide.     

Photocaged morpholino oligomers for the light-regulation of gene function in zebrafish and Xenopus embryos

Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts EGFP production only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in multiple contexts.     

Influence of the Absolute Configuration of NPE‐Caged Cytosine on DNA Single Base Pair Stability

Photolabile protecting groups are a versatile tool to trigger reactions by light irradiation. In this study, we have investigated the influence of the absolute configuration of the 1‐(2‐nitrophenyl)ethyl (NPE) cage group on a 15‐base‐pair duplex DNA. Using UV melting, we determined the global stability of the unmodified and the selectively (S)‐ and (R)‐NPE‐modified DNA sequences, respectively. We observe a differently destabilizing effect for the two NPE stereoisomers on the global stability. Analysis of the temperature dependence of imino proton exchange rates measured by NMR spectroscopy reveals that this effect can be attributed to decreased base pair stabilities of the caged and the 3′‐neighbouring base pair, respectively. Furthermore, our NMR based structural models of the modified duplexes provide a structural basis for the distinct effect of the (S)‐ and the (R)‐NPE group.     

Quantitative sphingosine measurement as a surrogate for total ceramide concentration—preclinical and potential translational applications

Biomarkers are an increasingly important constituent of the drug development process, offering the potential of increased efficiency through reduced compound attrition and earlier proof of mechanism and/or efficacy. Assays developed for compound screening that can be directly translated for clinical trials are especially valuable, but their successful adoption requires a careful balance between assay performance and implementation costs. One such ‘fit‐for‐purpose’ biomarker assay, the indirect measurement of pharmacological modulation of sphingolipid biosynthesis and disposition, is presented here. Among spingolipids, numerous ceramide species are readily detectable in different lipoprotein fractions of mammalian plasma, but their parallel quantification can be prohibitively expensive and time consuming. Ceramides differ in their fatty acid moiety, which is readily removed by hydrolysis, yielding a common sphingosine derivative, the measurement of which serves as an indicator of total ceramide. When followed by liquid chromatography tandem mass spectrometry (LC/MS/MS) for detection, robust analyte quantification becomes relatively straightforward. The practical utility of a method developed to be fit for the purpose of rapidly and quantitatively measuring treatment‐induced variations in total ceramide from hamster plasma and individual lipoprotein fractions is described. With a linear calibration range from 0.003 to 33.4 μm sphingosine, precision and accuracy error in plasma‐based quality controls spiked with ceramides was less than 15%. The specificity of the assay for ceramides was also assessed. The simplicity of the method would allow for its potential translation to other preclinical species, as well as for clinical applications in later‐stage drug development. Copyright © 2009 John Wiley & Sons, Ltd.     

Cloaked Caged Compounds: Chemical Probes for Two‐Photon Optoneurobiology

Caged neurotransmitters, in combination with focused light beams, enable precise interrogation of neuronal function, even at the level of single synapses. However, most caged transmitters are, surprisingly, severe antagonists of ionotropic gamma ‐aminobutyric acid (GABA) receptors. By conjugation of a large, neutral dendrimer to a caged GABA probe we introduce a “cloaking” technology that effectively reduces such antagonism to very low levels. Such cloaked caged compounds will enable the study of the signaling of the inhibitory neurotransmitter GABA in its natural state using two‐photon uncaging microscopy for the first time.     

Practical Labeling Methodology for Choline‐Derived Lipids and Applications in Live Cell Fluorescence Imaging

Lipids of the plasma membrane participate in a variety of biological processes, and methods to probe their function and cellular location are essential to understanding biochemical mechanisms. Previous reports have established that phosphocholine‐containing lipids can be labeled by alkyne groups through metabolic incorporation. Herein, we have tested alkyne, azide and ketone‐containing derivatives of choline as metabolic labels of choline‐containing lipids in cells. We also show that 17‐octadecynoic acid can be used as a complementary metabolic label for lipid acyl chains. We provide methods for the synthesis of cyanine‐based dyes that are reactive with alkyne, azide and ketone metabolic labels. Using an improved method for fluorophore conjugation to azide or alkyne‐modified lipids by Cu(I)‐catalyzed azide‐alkyne cycloaddition (CuAAC), we apply this methodology in cells. Lipid‐labeled cell membranes were then interrogated using flow cytometry and fluorescence microscopy. Furthermore, we explored the utility of this labeling strategy for use in live cell experiments. We demonstrate measurements of lipid dynamics (lateral mobility) by fluorescence photobleaching recovery (FPR). In addition, we show that adhesion of cells to specific surfaces can be accomplished by chemically linking membrane lipids to a functionalized surface. The strategies described provide robust methods for introducing bioorthogonal labels into native lipids.     

Tritopic Triazatruxene Ligands for Multicomponent Metal‐Organic Frameworks

Multicomponent metal‐organic frameworks (MOFs) are built up from multiple ligands that are geometrically distinct. These ligands occupy specific positions in the MOF lattice. Installing different functionalities at precise locations in the framework is an important step in making MOFs for specific applications. This can be achieved by designing functionalized ligands for multicomponent MOFs. Here, we report a simple synthetic procedure for a new tritopic triazatruxene based tricarboxylic acid, H₃tat. We show that this ligand can be symmetrically derivatized with various substituents on its nitrogen centres. We report a new isoreticular series of well‐ordered quaternary MOFs based on these new triazatruxene ligands together with two linear carboxylate ligands and Zn₄O clusters. These MOFs are isostructural to the previously reported MUF‐77 series and show similar high surface areas and large pore volumes. Furthermore, H‐bonding between the NH sites of the incorporated triazatruxene ligands and guest molecules is employed to modify their fluorescence behavior.     

Synthesis and Anti‐tumor Evaluation of B‐ring Modified Caged Xanthone Analogues of Gambogic Acid

Gambogic acid (GA, 1 ), the most prominent member of Garcinia natural products, has been reported to be a promising anti‐tumor agent. Previous studies have suggested that the planar B ring and the unique 4‐oxa‐tricyclo[4.3.1.0^3,7]dec‐2‐one caged motif were essential for anti‐tumor activity. To further explore the structure‐activity relationship (SAR) of caged Garcinia xanthones, two new series of B‐ring modified caged GA analogues 13a – 13e and 15a – 15e were synthesized utilizing a Claisen/Diel‐Alder cascade reaction. Subsequently, these compounds were evaluated for their in vitro anti‐tumor activities against A549, MCF‐7, SMMC‐7721 and BGC‐823 cancer cell lines by MTT assay. Among them, 13b – 13e exhibited micromolar inhibition against several cancer cell lines, being approximately 2–4 fold less potent in comparison to GA. SAR analysis revealed that the peripheral gem‐dimethyl groups are essential for maintaining anti‐tumor activity and substituent group on C1 position of B‐ring has a significant effect on potency, while modifications at C‐2, C‐3 and C‐4 positions are relatively tolerated. These findings will enhance our understanding of the SAR of Garcinia xanthones and lead to the development of simplified analogues as potential anti‐tumor agents.     

Urinary metabolomics analysis reveals the effect of volatile oil from Angelica sinensis on LPS‐induced inflammation rats

Lipopolysaccharide (LPS)‐induced inflammation occurs commonly and volatile oil from Angelica sinensis (VOAS) can be used as an anti‐inflammatory agent. The molecular mechanisms that allow the anti‐inflammatory factors to be expressed are still unknown. In this paper, we applied gas chromatography–mass spectrometry (GC–MS) and high‐performance liquid chromatography–time‐of‐flight mass spectrometry (LC‐Q/TOF–MS) based on a metabolomics platform coupled with a network approach to analyze urine samples in three groups of rats: one with LPS‐induced inflammation (MI); one with intervention with VOAS; and normal controls (NC). Our study found definite metabolic footprints of inflammation and showed that all three groups of rats, MI, intervention with VOAS and NC have distinct metabolic profiles in urine. The concentrations of 48 metabolites differed significantly among the three groups. The metabolites in urine were screened by the GC–MS and LC‐Q/TOF–MS methods. The significantly changed metabolites (p < 0.05, variable importance in projection > 1.5) between MI, NC and VOAS were included in the metabolic networks. Finally, hub metabolites were screened, including glycine, arachidonic acid, l ‐glutamate, pyruvate and succinate, which have high values of degree (k). the Results suggest that disorders of glycine, arachidonic acid, l ‐glutamate, pyruvate and succinate metabolism might play an important part in the predisposition and development of LPS‐induced inflammation. By applying metabolomics with network methods, the mechanisms of diseases are clearly elucidated.