pH and ROS Dual-Sensitive Nanocarriers for the Targeted Co-Delivery and On-Demand Sequential Release of Tofacitinib and Glucosamine for Synergistic Rheumatoid Arthritis Treatment.

Rheumatoid arthritis (RA) progression involves multiple cell types, and sequential drug action on target cells is necessary for RA treatment. Nanocarriers are widely used for RA treatment; however, the targeted delivery and on-demand release of multiple drugs remains challenging. Therefore, in this study, a dual-sensitive polymer is developed using chondroitin sulfate (CS) for the co-delivery of the cartilage repair agent, glucosamine (GlcN), and anti-inflammatory drug, tofacitinib (Tof). In the joint cavity, acidic pH facilitates the cleavage of GlcN from CS polymer to repair the cartilage damage. Subsequently, macrophage uptake via CS-CD44 binding and intracellular reactive oxygen species (ROS) mediate conversion of (methylsulfanyl)propylamine to a hydrophilic segment jointly triggered rapid Tof/GlcN release via micelle disassembly. The combined effects of Tof, GlcN, and ROS depletion promote the M1-to-M2 polarization shift to attenuate inflammation. The synergistic effects of these agents against RA are confirmed in vitro and in vivo. Overall, the dual pH/ROS-sensitive CS nanoplatform simultaneously delivers GlcN and Tof, providing a multifunctional approach for RA treatment with synergistic drug effects.

Synthesis of Novel 3-Deoxy-3-thio Derivatives of d-Glucosamine and Their Application as Ligands for the Enantioselective Addition of Diethylzinc to Benzaldehyde.

A series of novel thio-derivatives of d-glucosamine has been synthesized using double inversion procedures at the C3 atom. New compounds were applied as ligands for the diethylzinc addition to benzaldehyde and the products of the addition were obtained with a low to good enantiomeric ratio. The direction and the level of the asymmetric induction were highly dependent on the type of protecting groups on the nitrogen and sulfur atoms.

Optimization of enzymatic soy protein isolate-glucosamine conjugates to improve the freeze-thaw stability of emulsion.

BACKGROUND: Using transglutaminase (TGase) is a new method to improve protein properties in order to promote protein glycosylation. This article mainly studies soy protein isolate (SPI) and glucosamine to improve the freeze-thaw stability of emulsion under the action of TGase. The degree of glycosylation was studied by the content of free amino groups and the degree of conjugation. The optimal conditions for preparing soy protein isolate-glucosamine (SPI-G) conjugate were determined by a response surface optimization model based on single-factor experiments using the creaming index of the emulsion after the first freeze-thaw cycle as the response value. RESULTS: The results showed that the emulsion had the lowest creaming index when the conditions of protein concentration was 20 g L-1 , mass ratio of SPI-G was 5:3 (w/w), enzyme addition amount was 10 U g-1 , and reaction time was 2 h. The optimized modified product was measured for the creaming index after the first freeze-thaw cycle. It was found that the creaming index of the modified product SPI-G after the first freeze-thaw cycle was 9.02%, which was less than and close to the optimized model predicted value. The creaming index and optical microscopy results after three freeze-thaw cycles confirmed that the freeze-thaw stability of the SPI-G samples was significantly enhanced after optimization of the response surface model. CONCLUSION: It showed that glycosylation promoted by TGase could improve the freeze-thaw stability of SPI emulsion, thereby broadening the application of SPI in food. © 2022 Society of Chemical Industry.

Preparation, Anticoagulant and Antioxidant Properties of Glucosamine-Heparin Salt.

Excessive inorganic ions in vivo may lead to electrolyte disorders and induce damage to the human body. Therefore, preparation of enhanced bioactivity compounds, composed of activated organic cations and organic anions, is of great interest among researchers. In this work, glucosamine-heparin salt (GHS) was primarily synthesized with positively charged glucosamine hydrochloride (GAH) and negatively charged heparin sodium (Heps) by ion exchange method. Then, the detailed structural information of the GHS was characterized by FTIR, 1H NMR spectroscopy and ICP-MS. In addition, its anticoagulant potency and antioxidant properties were evaluated, respectively. The results demonstrated that GHS salt achieved enhanced antioxidant activities, including 98.78% of O2•- radical scavenging activity, 91.23% of •OH radical scavenging rate and 66.49% of DPPH radical scavenging capacity at 1.6 mg/mL, severally. Meanwhile, anticoagulant potency (ATTP) of GHS strengthened from 153.10 ± 17.14 to 180.03 ± 6.02 at 0.75 µmol/L. Thus, introducing cationic glucosamine residues into GHS could improve its anticoagulant activity. The findings suggest that GHS product with a small amount of inorganic ions can greatly abate the prime cost of antioxidants and anticoagulants, and has significant economic benefits and practical significance.

Substrate Substitution in Kanosamine Biosynthesis Using Phosphonates and Phosphite Rescue.

Kanosamine is an antibiotic and antifungal compound synthesized from glucose 6-phosphate (G6P) in Bacillus subtilis by the action of three enzymes: NtdC, which catalyzes NAD-dependent oxidation of the C3-hydroxyl; NtdA, a PLP-dependent aminotransferase; and NtdB, a phosphatase. We previously demonstrated that NtdC can also oxidize substrates such as glucose and xylose, though at much lower rates, suggesting that the phosphoryloxymethylene moiety of the substrate is critical for effective catalysis. To probe this, we synthesized two phosphonate analogues of G6P in which the bridging oxygen is replaced by methylene and difluoromethylene groups. These analogues are substrates for NtdC, with second-order rate constants an order of magnitude lower than those for G6P. NtdA converts the resulting 3-keto products to the corresponding kanosamine 6-phosphonate analogues. We compared the rates to the rate of NtdC oxidation of glucose and xylose and showed that the low reactivity of xylose could be rescued 4-fold by the presence of phosphite, mimicking G6P in two pieces. These results allow the evaluation of the individual energetic contributions to catalysis of the bridging oxygen, the bridging C6 methylene, the phosphodianion, and the entropic gain of one substrate versus two substrate pieces. Phosphite also rescued the reversible formation 3-amino-3-deoxy-d-xylose by NtdA, demonstrating that truncated and nonhydrolyzable analogues of kanosamine 6-phosphate can be generated enzymatically.

Profiling protein expression in Klebsiella pneumoniae with a carbohydrate-based covalent probe.

We report the application of a covalent probe based on a d-glucosamine scaffold for the profiling of the bacterial pathogen Klebsiella pneumoniae. Incubation of K. pneumoniae lysates with the probe followed by electrophoretic separation and in-gel fluorescence detection allowed the generation of strain-specific signatures and the differentiation of a carbapenem-resistant strain. The labelling profile of the probe was independent of its anomeric configuration and included several low-abundance proteins not readily detectable by conventional protein staining. Initial target identification experiments by mass spectrometry suggest that target proteins include several carbohydrate-recognising proteins, which indicates that the sugar scaffold may have a role for target recognition.

Lignin/Carbohydrate Complex Isolated from Posidonia oceanica Sea Balls (Egagropili): Characterization and Antioxidant Reinforcement of Protein-Based Films.

A lignin fraction (LF) was extracted from the sea balls of Posidonia oceanica (egagropili) and extensively dialyzed and characterized by FT-IR and NMR analyses. LF resulted water soluble and exhibited a brownish-to-black color with the highest absorbance in the range of 250-400 nm, attributed to the chromophore functional groups present in the phenylpropane-based polymer. LF high-performance size exclusion chromatography analysis showed a highly represented (98.77%) species of 34.75 kDa molecular weight with a polydispersity index of 1.10 and an intrinsic viscosity of 0.15. Quantitative analysis of carbohydrates indicated that they represented 28.3% of the dry weight of the untreated egagropili fibers and 72.5% of that of LF. In particular, eight different monosaccharides were detected (fucose, arabinose, rhamnose, galactose, glucose, xylose, glucosamine and glucuronic acid), glucuronic acid (46.6%) and rhamnose (29.6%) being the most present monosaccharides in the LF. Almost all the phenol content of LF (113.85 ± 5.87 mg gallic acid eq/g of extract) was water soluble, whereas around 22% of it consisted of flavonoids and only 10% of the flavonoids consisted of anthocyanins. Therefore, LF isolated from egagropili lignocellulosic material could be defined as a water-soluble lignin/carbohydrate complex (LCC) formed by a phenol polymeric chain covalently bound to hemicellulose fragments. LCC exhibited a remarkable antioxidant activity that remained quite stable during 6 months and could be easily incorporated into a protein-based film and released from the latter overtime. These findings suggest egagropili LCC as a suitable candidate as an antioxidant additive for the reinforcement of packaging of foods with high susceptibility to be deteriorated in aerobic conditions.

Synthesis and Bioinformatic Characterization of New Schiff Bases with Possible Applicability in Brain Disorders.

(1) Background: The research aims to find new treatments for neurodegenerative diseases, in particular, Alzheimer's disease. (2) Methods: This article presents a bioinformatics and pathology study of new Schiff bases, (EZ)-N'-benzylidene-(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanehydrazide derivatives, and aims to evaluate the drug-like, pharmacokinetic, pharmacodynamic and pharmacogenomic properties, as well as to predict the binding to therapeutic targets by applying bioinformatics, cheminformatics and computational pharmacological methods. (3) Results: We obtained these Schiff bases by condensing (2RS)-2-(6-chloro-9H-carbazol-2-yl)propanehydrazide with aromatic aldehydes, using the advantages of microwave irradiation. The newly synthesized compounds were characterized spectrally, using FT-IR and NMR spectroscopy, which confirmed their structure. Using bioinformatics tools, we noticed that all new compounds are drug-likeness features and may be proposed as potentially neuropsychiatric drugs (4) Conclusions: Using bioinformatics tools, we determined that the new compound 1e had a high potential to be used as a good candidate in neurodegenerative disorders treatment.

Effects of transglutaminase glycosylated soy protein isolate on its structure and interfacial properties.

BACKGROUNDS: The structural and interfacial properties of soybean protein isolate (SPI) after glycosylation by the transglutaminase method were studied. It is hoped that preliminary explorations will find a new food ingredient and broader application of SPI in the food industry. RESULTS: The contents of free amino proves that transglutaminase can insert glucosamine into SPI through its transamination, and realize the enzymatic glycosylated SPI. The results of structure properties showed that a decrease in the content of the α-helical structure indicates that the rigid structure of the protein is opened and the flexibility is increased. The blue shift of the maximum fluorescence intensity of soy protein isolate-glucosamine with transglutaminase (SPI-G) indicates the formation of a new substance; scanning electron microscopy shows that the SPI-G powder can be seen at a magnification of 2000×, and the protein structure becomes soft. The results of interfacial properties found that enzymatic protein glycosylation exposes the internal hydrophobic groups of SPI, resulting in increased surface hydrophobicity, increased emulsification and emulsification stability, and reduced surface tension. CONCLUSION: It shows that SPI-G effectively improves the interfacial properties of SPI, providing a theoretical basis for the application of enzymatic glycosylation of SPI in the food industry. © 2021 Society of Chemical Industry.

Biochemical and Structural Analysis of a Dehydrogenase, KanD2, and an Aminotransferase, KanS2, That Are Responsible for the Construction of the Kanosamine Moiety in Kanamycin Biosynthesis.

Kanosamine (3-amino-3-deoxy-d-glucose) is a characteristic sugar unit found in kanamycins, a group of aminoglycoside antibiotics. The kanosamine moiety originates from d-glucose in kanamycin biosynthesis. However, the timing of the replacement of the 3-OH group of the d-glucose-derived biosynthetic intermediate with the amino group is elusive. Comparison of biosynthetic gene clusters for related aminoglycoside antibiotics suggests that the nicotinamide adenine dinucleotide (NAD+)-dependent dehydrogenase KanD2 and the pyridoxal 5'-phosphate (PLP)-dependent aminotransferase KanS2 are responsible for the introduction of the amino group at the C3 position of kanosamine. In this study, we demonstrated that KanD2 and KanS2 convert kanamycin A, B, and C to the corresponding 3″-deamino-3″-hydroxykanamycins (3″-hks) in the presence of PLP, 2-oxoglutarate, and NADH via a reverse reaction in the pathway. Furthermore, we observed that all of the 3″-hks are oxidized by KanD2 with NAD+, but d-glucose, UDP-d-glucose, d-glucose 6-phosphate, and d-glucose 1-phosphate are not. Crystal structure analysis of KanD2 complexed with 3″-hkB and NADH illustrated the selective recognition of pseudotrisaccharides, especially the d-glucose moiety with 2-deoxystreptamine, by a combination of hydrogen bonds and CH-π interactions. Overall, it was clarified that the kanosamine moiety of kanamycins is constructed after the glucosylation of the pseudodisaccharide biosynthetic intermediates in kanamycin biosynthesis.

The Crystal Structures of Bacillithiol Disulfide Reductase Bdr (YpdA) Provide Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductase.

Low G+C Gram-positive Firmicutes, such as the clinically important pathogens Staphylococcus aureus and Bacillus cereus, use the low-molecular weight thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. The protein YpdA has recently been shown to function as an essential NADPH-dependent reductase of oxidized bacillithiol disulfide (BSSB) resulting from stress responses and is crucial for maintaining the reduced pool of BSH and cellular redox balance. In this work, we present the first crystallographic structures of YpdAs, namely, those from S. aureus and B. cereus. Our analyses reveal a uniquely organized biological tetramer; however, the structure of the monomeric subunit is highly similar to those of other flavoprotein disulfide reductases. The absence of a redox active cysteine in the vicinity of the FAD isoalloxazine ring implies a new direct disulfide reduction mechanism, which is backed by the presence of a potentially gated channel, serving as a putative binding site for BSSB in the proximity of the FAD cofactor. We also report enzymatic activities for both YpdAs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria.

Chemoenzymatic Synthesis of Chito-oligosaccharides with Alternating N-d-Acetylglucosamine and d-Glucosamine.

Chito-oligosaccharides (CHOS) are homo- or hetero-oligomers of N-acetylglucosamine (GlcNAc, A) and d-glucosamine (GlcN, D). Production of well-defined CHOS-mixtures, or even pure CHOS, with specific lengths and sugar compositions, is of great interest since these oligosaccharides have interesting bioactivities. While direct chemical synthesis of CHOS is not straightforward, chemo-enzymatic approaches have shown some promise. We have used engineered glycoside hydrolases to catalyze oligomerization of activated DA building blocks through transglycosylation reactions. The building blocks were generated from readily available (GlcNAc)2-para-nitrophenol through deacetylation of the nonreducing end sugar with a recombinantly expressed deacetylase from Aspergillus niger (AnCDA9). This approach, using a previously described hyper-transglycosylating variant of ChiA from Serratia marcescens (SmChiA) and a newly generated transglycosylating variant of Chitinase D from Serratia proteamaculans (SpChiD), led to production of CHOS containing up to ten alternating D and A units [(DA)2, (DA)3, (DA)4, and (DA)5]. The most abundant compounds were purified and characterized. Finally, we demonstrate that (DA)3 generated in this study may serve as a specific inhibitor of the human chitotriosidase. Inhibition of this enzyme has been suggested as a therapeutic strategy against systemic sclerosis.

Classification, characterization and structural analysis of sugar nucleotidylyltransferase family of enzymes.

Sugar Nucleotidyl Transferases (SNTs) constitute a large family of enzymes that play important metabolic roles. Earlier, for one such SNT, termed N-acetylglucosamine-1-phosphate uridyltransferase- GlmU, we had established that two magnesium ions - Mg2+A and Mg2+B - catalyze the sugar-nucleotidyl transfer reaction. Despite a common structural framework that SNTs share, we recognized key differences around the active-site based on the analysis of available structures. Based on these differences, we had classified SNTs into two major groups, Group - I & II; and further, variation in 'Mg2+A-stabilizing motifs' led us to sub-classify them into five distinct sub-groups. Since group specific conservation of 'Mg2+A-stabilizing motifs' was based only for 45 available structures, here we validate this via an exhaustive analysis of 1,42,025 protein sequences. Previously, we had hypothesized that a metal-ion-catalyzed mechanism would be operative in all SNTs. Here, we validate it biochemically and establish that Mg2+ is a strict requirement for nucleotidyl transfer reactions in every group or sub-group and that a common metal ion dependent mechanism operates in SNTs. Further, mutating Mg2+A coordinating residue in each sub-group led to abolished catalysis, indicating an important role for both of these residues and suggest that SNTs employ variations over 'a conserved catalytic mechanism mediated by Mg2+ ion(s)', to bring about functional diversity. This would constitute a comprehensive study to establish the catalytic mechanism across the family of SNTs.

The potential application of gold-apoferritin nanocages conjugated with 2-amino-2-deoxy-glucose for imaging of breast cancer cells.

Development of biocompatible and multifunctional nanoprobes for tumor targeting, imaging, and therapy still remains a great challenge. Herein, gold nanoparticles (AuNPs) were synthesized in the cavity of horse spleen apoferritin protein (HoSAF) and protein surface was labeled with 2-amino-2-deoxy-glucose (2DG) as a cell surface glucose transport protein specific targeting probe to study the feasibility of its usage as a computer tomography (CT) contrast agent with tumor targeting capability through in vitro experiments. 2DG conjugated and gold-loaded apoferritin (Au-HoSAF-2DG) nanoparticles (NPs) showed selective targeting for human breast adenocarcinoma (MCF-7) cells when compared to normal breast (MCF-10A) cells. This AuNP-based imaging agent was found to be non-cytotoxic in a given concentration range with an apoptotic effect upon longer exposure times towards MCF-7 cells, while MCF-10A cells were affected less. This selective cell death would also be useful for further cancer treatments with the ability of X-ray attenuation in in vitro X-ray and computed tomography (CT) imaging.

Improving the analytical toolbox to investigate copurifying host cell proteins presence: N-(4)-(ß-acetylglucosaminyl)- l-asparaginase case study.

Levels of host cell proteins (HCPs) in purification intermediates and drug substances (DS) of monoclonal antibodies (mAbs) must be carefully monitored for the production of safe and efficacious biotherapeutics. During the development of mAb1, an immunoglobulin G1 product, unexpected results generated with HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit triggered an investigation which led to the identification of a copurifying HCP called N-(4)-(ß-acetylglucosaminyl)-l-asparaginase (AGA, EC3.5.1.26) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The risk assessment performed indicated a low immunogenicity risk for the copurifying HCP and an ad hoc stability study demonstrated no mAb glycan cleavage and thus no impact on product quality. Fractionation studies performed on polishing steps revealed that AGA was coeluted with the mAb. Very interestingly, the native digestion protocol implemented to go deeper in the MS-HCP profiling was found to be incompatible with correct AGA detection in last purification intermediate and DS, further suggesting a hitchhiking behavior of AGA. In silico surface characterization of AGA also supports this hypothesis. Finally, the combined support of HCP ELISA results and MS allowed process optimization and removal of this copurifying HCP.

N,O-Dialkyl deoxynojirimycin derivatives as CERT START domain ligands.

Dysregulation of the ceramide transport protein CERT is associated to diseases such as cancer. In search for new CERT START domain ligands, N-dodecyl-deoxynojirimycin (N-dodecyl-DNJ) iminosugar was found to display, as a ceramide mimic, significant protein recognition. To reinforce the lipophilic interactions and strengthen this protein binding, a docking study was carried out in order to select the optimal position on which to introduce an additional O-alkyl chain on N-dodecyl-DNJ. Analysis of the calculated poses for three different regioisomers indicated an optimal calculated interaction pattern for N,O3-didodecyl-DNJ. The two most promising regioisomers were prepared by a divergent route and their binding to the CERT START domain was evaluated with fluorescence intensity (FLINT) binding assay. N,O3-didodecyl-DNJ was confirmed to be a new binder prototype with level of protein recognition in the FLINT assay comparable to the best known ligands from the alkylated HPA-12 series. This work opens promising perspectives for the development of new inhibitors of CERT-mediated ceramide trafficking.

Synthesis, distribution analysis and mechanism studies of N-acyl glucosamine-bearing oleanolic saponins.

A series ofN-acyl glucosamine-bearingtriterpenoidsaponins has been synthesized with cytotoxic activities evaluated against HL-60, PC-3, HCT-116, and CT-26 tumor cells. Saponins incorporated anoleanolic acid (OA) triterpenoidal core exhibited the highest cytotoxic activity. To study the influence of the lengths of acyl-carbon chain onN-position of glucosamine, cells were treated with28-propargylamides and then reacted with an azido-fluorogenic probe under CuAACclickreactions to visualize the intact distributions of these compounds by confocal microscopy and flow cytometry; it was found that cytotoxic-active compounds (30-32) located in the cytosol and inactivecompounds bearing longer carbon chains (33-35) were impenetrable across cell membranes.Our study demonstrated the defined lipophilic acyl-carbon chain length can precisely regulate thecytotoxic activityof saponins, which is useful for the future development of cytotoxic agents.Furthermore, using quantitative proteomics and immunolabeling,the mechanism ofcytotoxicity induced by the synthetic saponin after membrane penetration could be a result of activation of death receptor pathway and inhibition of PI3K/Akt/mTOR pathway.

Qualitative analysis of enzymatic and chemical depolymerized low molecular weight heparins by UHPLC coupled with electrospray ionization quadrupole time-of-flight-mass spectrometry.

Complete heparin digestion with heparin lyase I and II results in a mixture of hexasaccharides and tetrasaccharides with 3-O-sulfo group-containing glucosamine residues at their reducing ends. Because these tetrasaccharides are derived from antithrombin III-binding sites of heparin, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. Therefore, this paper presents a new low molecular weight heparin sample preparation method-chemical depolymerization. Qualitative analysis of the studied compounds and a comparison of their composition are an important contribution to the structural analysis of low molecular weight heparins, which has not been fully conducted so far. Qualitative on-line liquid chromatography-mass spectrometric analysis of these resistant oligosaccharides is also described in this paper.

Preparation and evaluation of inhalable dry powder containing glucosamine-conjugated gefitinib SLNs for lung cancer therapy.

Gefitinib as an epidermal growth factor receptor tyrosine kinase inhibitor has strong potential in lung cancer therapy. However, a major challenge of using gefitinib is its toxicities. In the present study, we developed a dry powder inhaler dosage form containing gefitinib loaded glucosamine targeted solid lipid nanopaticles (Gef-G-SLNs) to locally transfer anticancer agent to the lung tumor. The Gef-G-SLNs were prepared by emulsion-solvent diffusion and evaporation method and optimized with irregular factorial design. The optimized nanoformulation was tested for action against A549 cells. Mannitol or lactose based dry powders were obtained from Gef-G-SLNs after spray drying and characterized using Anderson Cascade Impactor. The optimized formulation had drug loading of 33.29%, encapsulation efficiency of 97.31 ± 0.23%, zeta potential of -15.53 ± 0.47 mV, particle size of 187.23 ± 14.08 nm, polydispersity index of 0.28 ± 0.02 and release efficiency of 35.46 ± 2.25%. The Gef-G-SLNs showed superior anticancer effect compared to free gefitinib. The increased cellular uptake of G-SLNs in A549 cells was demonstrated compared with non-targeted SLNs using flow cytometry and fluorescence microscopy. The produced mannitol based microparticles showed suitable aerodynamic properties with an acceptable mass median aerodynamic diameter of 4.48 µm and fine particle fraction of 44.41%. Therefore, it can be concluded that this formulation represents promising drug delivery to treatment of lung cancer.

Tandem Electrospray Mass Spectrometry of Cyclic N-Substituted Oligo-ß-(1→6)-D-glucosamines.

High-resolution electrospray mass spectra (MS and MS/MS CID) of positive ions of a series of protonated, ammoniated, and metallated molecules of cyclic N-substituted oligo-ß-(1→6)-D-glucosamines differing in cycle size and N-acyl substituents were registered and interpreted. It was shown that the main type of fragmentation is a cleavage of glycosidic bonds of a cycle, and in some cases fragmentation of amide side chains is possible. If labile fragments in substituents (e.g., carbohydrate chains) are present, a decay of the cycle and an elimination of labile fragments are of comparable possibility. It was found that in some cases rearrangements with loss of an internal carbohydrate residue (IRL), or an internal part of a side chain, are feasible.