Facile Enzymatic Synthesis of Phosphorylated Ketopentoses

An efficient and convenient platform for the facile synthesis of phosphorylated ketoses is described. All eight phosphorylated ketopentoses were produced using this platform starting from two common and inexpensive aldoses (d-xylose and l-arabinose) in more than 84% isolated yield (gram scale). In this method, reversible conversions (isomerization or epimerization) were accurately controlled toward the formation of desired ketose phosphates by targeted phosphorylation reactions catalyzed by substrate-specific kinases. The byproducts were selectively removed by silver nitrate precipitation avoiding the tedious and time-consuming separation of sugar phosphate from adenosine phosphates (ATP and ADP). Moreover, the described strategy can be expanded for the synthesis of other sugar phosphates

Cysteine Sulfoxidation Increases the Photostability of Red Fluorescent Proteins

Photobleaching of fluorescent proteins (FPs) is a major limitation to their use in advanced microscopy, and improving photostability remains highly challenging due to limited understanding of its molecular mechanism. Here we discovered a new mechanism to increase FP photostability. Cysteine oxidation, implicated in only photobleaching before, was found to drastically enhance FP photostability to the contrary. We generated a far-red FP mStable by introducing a cysteine proximal to the chromophore. Upon illumination, this cysteine was oxidized to sulfinic and sulfonic acids, enabling mStable more photostable than its ancestor mKate2 by 12-fold and surpassing other far-red FPs. mStable outperformed in laser scanning confocal imaging and super-resolution structured illumination microscopy. Moreover, photosensitization to oxidize a cysteine similarly introduced in another FP mPlum also increased its photostability by 23-fold. This postfolding cysteine sulfoxidation cannot be simply substituted by the isosteric aspartic acid, representing a unique mechanism valuable for engineering better photostability into FPs

Efficient Enzymatic Synthesis of Guanosine 5′-Diphosphate-Sugars and Derivatives

An <i>N</i>-acetylhexosamine 1-kinase from <i>Bifidobacterium infantis</i> (NahK_15697), a guanosine 5′-diphosphate (GDP)-mannose pyrophosphorylase from <i>Pyrococcus furiosus</i> (PFManC), and an <i>Escherichia coli</i> inorganic pyrophosphatase (EcPpA) were used efficiently for a one-pot three-enzyme synthesis of GDP-mannose, GDP-glucose, their derivatives, and GDP-talose. This study represents the first facile and efficient enzymatic synthesis of GDP-sugars and derivatives starting from monosaccharides and derivatives

A One-Step Chemoenzymatic Labeling Strategy for Probing Sialylated Thomsen–Friedenreich Antigen

Abnormal expression of sialylated Thomsen–Friedenreich antigen (Neu5Acα2-3Galβ1-3GalNAcα-O-Ser/Thr, sialyl-T) has a strong relationship with various types of human cancers and many other diseases. However, the size and structural complexity, and relatively lower abundance of sialyl-T have posed a significant challenge to its detection. Therefore, details about the role of sialyl-T in a variety of physiological and pathological processes are still poorly understood. Here, a one-step chemoenzymatic labeling strategy to probe sialyl-T is described. This approach enables the sensitive, selective, and rapid detection of sialyl-T, and global profiling and identification of unknown sialyl-T-attached glycoproteins, which are potential therapeutic targets or biomarkers. The use of one-step labeling strategy not only has a higher sensitivity than a typical two-step reporter strategy but also avoids undergoing an additional chemical reaction step to introduce a reporter group after the labeling reaction, making it particularly useful for detecting low-abundance glycan epitopes on living cells

Glycosylated Platinum(IV) Complexes as Substrates for Glucose Transporters (GLUTs) and Organic Cation Transporters (OCTs) Exhibited Cancer Targeting and Human Serum Albumin Binding Properties for Drug Delivery

Glycosylated platinum­(IV) complexes were synthesized as substrates for GLUTs and OCTs for the first time, and the cytotoxicity and detailed mechanism were determined in vitro and in vivo. Galactoside Pt­(IV), glucoside Pt­(IV), and mannoside Pt­(IV) were highly cytotoxic and showed specific cancer-targeting properties in vitro and in vivo. Glycosylated platinum­(IV) complexes <b>5</b>, <b>6</b>, <b>7</b>, and <b>8</b> (IC₅₀ 0.24–3.97 μM) had better antitumor activity of nearly 166-fold higher than the positive controls cisplatin (<b>1a</b>), oxaliplatin (<b>3a</b>), and satraplatin (<b>5a</b>). The presence of a hexadecanoic chain allowed binding with human serum albumin (HSA) for drug delivery, which not only enhanced the stability of the inert platinum­(IV) prodrugs but also decreased their reduction by reductants present in human whole blood. Their preferential accumulation in cancer cells compared to noncancerous cells (293T and 3T3 cells) suggested that they were potentially safe for clinical therapeutic use

Identification of Target Proteins of Mangiferin in Mice with Acute Lung Injury Using Functionalized Magnetic Microspheres Based on Click Chemistry

Prevention of the occurrence and development of inflammation is a vital therapeutic strategy for treating acute lung injury (ALI). Increasing evidence has shown that a wealth of ingredients from natural foods and plants have potential anti-inflammatory activity. In the present study, mangiferin, a natural <i>C</i>-glucosyl xanthone that is primarily obtained from the peels and kernels of mango fruits and the bark of the Mangifera indica L. tree, alleviated the inflammatory responses in lipopolysaccharide (LPS)-induced ALI mice. Mangiferin-modified magnetic microspheres (MMs) were developed on the basis of click chemistry to capture the target proteins of mangiferin. Mass spectrometry and molecular docking identified 70 kDa heat-shock protein 5 (Hspa5) and tyrosine 3-monooxygenase (Ywhae) as mangiferin-binding proteins. Furthermore, an enzyme-linked immunosorbent assay (ELISA) indicated that mangiferin exerted its anti-inflammatory effect by binding Hspa5 and Ywhae to suppress downstream mitogen-activated protein kinase (MAPK) signaling pathways. Thoroughly revealing the mechanism and function of mangiferin will contribute to the development and utilization of agricultural resources from M. indica L

l‑Rhamnose Enhances the Immunogenicity of Melanoma-Associated Antigen A3 for Stimulating Antitumor Immune Responses

Vaccines based on melanoma-associated antigens (MAGEs) present a promising strategy for tumor immunotherapy, albeit with weak immunogenicity. In this study, the xenoantigen l-rhamnose (Rha) was chemically conjugated with truncated MAGE-A3 (tMAGE-A3) to generate Rha-tMAGE-A3. The product showed good antigenicity with anti-Rha antibodies purified from human serum. FITC-labeled Rha-tMAGE-A3 was detected in THP-1 human macrophage cells via the anti-Rha antibody-dependent antigen uptake process. Furthermore, peripheral blood mononuclear cells (PBMCs) stimulated with Rha-tMAGE-A3 in the presence of anti-Rha antibodies showed better cytotoxicity toward A375 human melanoma cells surfaced by MAGE-A3 antigen compared to PBMCs stimulated with tMAGE-A3. All data reveal that linking of Rha epitopes to MAGE enhances the immunogenicity of MAGE by harnessing the immune effector functions of human naturally existing anti-Rha antibodies. Rha epitopes could become immunogenicity enhancers of tumor associated antigens in the development of tumor immunotherapies

Successfully Engineering a Bacterial Sialyltransferase for Regioselective α2,6-sialylation

A β-galactoside α2,6-sialyltransferase from <i>Photobacterium damselae</i> (Pd2,6ST) that is capable of sialylating both terminal and internal galactose and <i>N</i>-acetylgalactosamine was herein redesigned for regioselectively producing terminal α2,6-sialosides. Guided by a recently developed bump-hole strategy, a series of mutations at Ala200 and Ser232 sites were created for reshaping the acceptor binding pocket. Finally, a Pd2,6ST double mutant A200Y/S232Y with an altered L-shaped acceptor binding pocket was identified to be a superior α2,6-sialyltransferase which can efficiently catalyze the regioselective α2,6-sialylation of galactose or <i>N</i>-acetylgalactosamine at the nonreducing end of a series of glycans. Meanwhile, A200Y/S232Y remains flexible donor substrate specificity and is able to transfer Neu5Ac, Neu5Gc, and KDN

Chemoenzymatic Synthesis of Unnatural Nucleotide Sugars for Enzymatic Bioorthogonal Labeling

In recent years, the development of the enzymatic bioorthogonal labeling strategy has offered exciting possibilities in the probing of structure-defined glycan epitopes. This strategy takes advantage of relaxed donor specificity and strict acceptor specificity of glycosyltransferases to label target glycan epitopes with bioorthogonal reactive groups carried by unnatural nucleotide sugars in vitro. The subsequent covalent conjugation by bioorthogonal chemical reactions with either fluorescent or affinity tags allows further visualization, quantification, or enrichment of target glycan epitopes. However, the application and development of the enzymatic labeling strategy have been hindered due to the limited availability of unnatural nucleotide sugars. Herein, a platform that combines chemical synthesis and enzymatic synthesis for the facile preparation of unnatural nucleotide sugars modified with diverse bioorthogonal reactive groups is described. By this platform, a total of 25 UDP-GlcNAc and UDP-GalNAc derivatives, including the most well explored bioorthogonal functional groups, were successfully synthesized. Furthermore, the potential application of these compounds for use in enzymatic bioorthogonal labeling reactions was also evaluated

Cytokines determined by ELISPOT.

<p>(<b>A</b>), IFN-γ; (<b>B</b>), IL-4. The spots for cytokine-producing lymphocytes after stimulation with MBP or O-Ag-MBP were counted and expressed based on 1×10⁵ cells. Results are expressed as the arithmetic mean ±SD indicated by error bars. Differences of three groups stimulated at the same dose of MBP are indicated with symbols (*: P<0.05; **: P<0.001).</p