34 research outputs found
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
platinumÂ(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 PtÂ(IV), glucoside
PtÂ(IV), and mannoside PtÂ(IV) were highly cytotoxic and showed specific
cancer-targeting properties in vitro and in vivo. Glycosylated platinumÂ(IV)
complexes <b>5</b>, <b>6</b>, <b>7</b>, and <b>8</b> (IC<sub>50</sub> 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 platinumÂ(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 1×10<sup>5</sup> 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