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1.
J Cell Physiol ; 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32162324

RESUMO

Noncollagenous proteins in the bone extracellular matrix, such as osteocalcin (OC) and osteopontin (OPN), inherent to evolution of bone as a skeletal tissue, are known to regulate bone formation and mineralization. However, the fundamental basis of this regulatory role remains unknown. Here, for the first time, we use mouse mesenchymal stem/stromal cells (MSC) lacking both OC and OPN to investigate the mechanistic roles of OC and OPN on the proliferation capacity and differentiation ability of MSC. We found that the loss of OC and OPN reduces stem cells self-renewal potential and multipotency, affects their differentiation into an osteogenic lineage, and impairs their angiogenic potential while maintaining chondrogenic and adipogenic lineages. Moreover, loss of OC and OPN compromises the extracellular matrix integrity and maturation, observed by an unexpected enhancement of glycosaminoglycans content that are associated with a more primitive skeletal connective tissue, and by a delay on the maturation of mineral species produced. Interestingly, exogenously supplemented OC and OPN were able to rescue MSC proliferative and osteogenic potential along with matrix integrity and mineral quality. Taken together, these results highlight the key contributions of OC and OPN in enhancing osteogenesis and angiogenesis over primitive connective tissue, and support a potential therapeutic approach based on their exogenous supplementation.

2.
Artigo em Inglês | MEDLINE | ID: mdl-32172323

RESUMO

Formaldehyde is a ubiquitous carcinogenic indoor pollutant. The treatment of formaldehyde has attracted increasing social attention. Over the past few decades, an increasing number of publications have reported approaches for removing indoor formaldehyde. These potential strategies include physical adsorption, chemical catalysis, and biodegradation. Although physical adsorption is widely used, it does not really remove pollution. Chemical catalysis is very efficient but adds the risk of introducing secondary pollutants. Biological removal strategies have attracted more research attention than the first two methods, because it is more efficient, clean, and economical. Plants and bacteria are the common organisms used in formaldehyde removal. However, both have limitations and shortcomings when used alone. This review discusses the mechanisms, applications, and improvements of existing biological methods for the removal of indoor gaseous formaldehyde. A combination strategy relying on plants, bacteria, and physical adsorbents exhibits best ability to remove formaldehyde efficiently, economically, and safely. When this combination system is integrated with a heating, ventilation, air conditioning, and cooling (HVAC) system, a practical combined system can be established in formaldehyde removal. Multivariate interactions of biological and non-biological factors are needed for the future development of indoor formaldehyde removal. KEY POINTS: • Indoor gaseous formaldehyde removal is necessary especially for new residence. • Biological removal strategies have attracted increasing research attentions. • Combined system of plants, bacteria, and physical adsorbents exhibits best efficiency. • Integrated device of biological and non-biological factors will be potential practical.

3.
Methods Cell Biol ; 156: 85-106, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32222228

RESUMO

Cell-derived extracellular matrices have emerged as promising scaffolds for tissue engineering (TE) strategies due to their ability to create a biomimetic microenvironment providing biochemical and physical cues to cells, without the limitations of availability and potential pathogen transmission associated with tissue-derived extracellular matrix (ECM) scaffolds. Glycosaminoglycans (GAGs) are important components of ECM with a crucial role in the maintenance of the mechanical properties of the tissue and as signaling regulators of several cellular processes, such as cell adhesion, growth and differentiation. However, despite their relevance to the field of TE, little information is available on the GAG composition of cell-derived ECM, mainly due to the lack of appropriate quantitative tools to determine different GAG and disaccharide subtypes in complex biological samples. In this chapter, we describe a highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to characterize decellularized cell-derived ECM generated in vitro in terms of their GAG and disaccharide composition.

4.
Sci Transl Med ; 12(535)2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188725

RESUMO

Acetaminophen/paracetamol (APAP) overdose is the leading cause of drug-induced acute liver failure (ALF) in the United States and Europe. The progression of the disease is attributed to sterile inflammation induced by the release of high mobility group box 1 (HMGB1) and the interaction with receptor for advanced glycation end products (RAGE). A specific, effective, and safe approach to neutralize the proinflammatory activity of HMGB1 is highly desirable. Here, we found that a heparan sulfate (HS) octadecasaccharide (18-mer-HP or hepatoprotective 18-mer) displays potent hepatoprotection by targeting the HMGB1/RAGE axis. Endogenous HS proteoglycan, syndecan-1, is shed in response to APAP overdose in mice and humans. Furthermore, purified syndecan-1, but not syndecan-1 core protein, binds to HMGB1, suggesting that HMGB1 binds to HS polysaccharide side chains of syndecan-1. Last, we compared the protection effect between 18-mer-HP and N-acetyl cysteine, which is the standard of care to treat APAP overdose. We demonstrated that 18-mer-HP administered 3 hours after a lethal dose of APAP is fully protective; however, the treatment of N-acetyl cysteine loses protection. Therefore, 18-mer-HP may offer a potential therapeutic advantage over N-acetyl cysteine for late-presenting patients. Synthetic HS provides a potential approach for the treatment of APAP-induced ALF.

5.
Carbohydr Polym ; 235: 115904, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32122473

RESUMO

In vitro digestive conditions were simulated to investigate the digestibility of polysaccharides prepared from squash (SPS). A small amount of free glucose monosaccharide was released after salivary and intestinal digestion due to the breakdown of α-(1 → 4)-glucose linkages and may form SPS or a starch impurity. At the same time, there was no obvious change in molecular weight distribution and reducing sugar content throughout this digestion period, demonstrating that the main structure of SPS was relatively stable under the simulated digestive conditions. Thus, most SPS can be transported intact to the large intestine. In addition, SPS alleviated type 2 diabetes (T2D) in rats. Moreover, the content of short-chain fatty acids (SCFAs) in the colon significantly increased after treatment with SPS. The present research provides insight into the non-digestibility of SPS, and suggests its utility to alleviate T2D by promoting the production of SCFA in the colon.

6.
Matrix Biol ; 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32084457

RESUMO

Hyaluronan plays a key role in regulating inflammation and tumor angiogenesis. Of the three transmembrane hyaluronan synthases, HAS2 is the main pro-angiogenic enzyme responsible for excessive hyaluronan production. We discovered that HAS2 was degraded in vascular endothelial cells via autophagy evoked by nutrient deprivation, mTOR inhibition, or pro-autophagic proteoglycan fragments endorepellin and endostatin. Using live-cell and super-resolution confocal microscopy, we found that protracted autophagy evoked a dynamic interaction between HAS2 and ATG9A, a key transmembrane autophagic protein. This regulatory axis of HAS2 degradation occurred in various cell types and species and in vivo upon nutrient deprivation. Inhibiting in vivo autophagic flux via chloroquine showed increased levels of HAS2 in the heart and aorta. Functionally, autophagic induction via endorepellin or mTOR inhibition markedly suppressed extracellular hyaluronan production in vascular endothelial cells and inhibited ex vivo angiogenic sprouting. Thus, we propose autophagy as a novel catabolic mechanism regulating hyaluronan production in endothelial cells and demonstrate a new link between autophagy and angiogenesis that could lead to potential therapeutic modalities for angiogenesis.

7.
Int J Biol Macromol ; 149: 450-458, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32004605

RESUMO

Polysaccharide (HFSGF) was purified from Sargassum fusiforme. Autohydrolysis and gel column chromatography were performed to fractionate HFSGF into three components (HFSGF-S, HFSGF-L and HFSGF-H). Compositional analysis, mass spectrometry and nuclear magnetic resonance spectroscopy were used to elucidate the structural features of HFSGF. HFSGF-S was a mixture of sulfated galacto-fuco-oligomers, from the branches terminal ends; in HFSGF-L, the branches of HFSGF, was a sulfated galactofucan, containing a backbone of 1,3-linked α-L-fucan sulfated at C2/4 and/or C4 and interspersed with galactose (Gal); and in HFSGF-H, the backbone of HFSGF, was composed of alternating 1,2-linked α-D-mannose (Man) and 1,4-linked ß-D-glucuronic acid (GlcA), branched with sulfated galactofucan or sulfated fucan, 1,3-linked α-L-fucan sulfated at C2/4 and/or C4 and partly interspersed with Gal. Some fucose (Fuc) residues were also partially branched with xylose (Xyl). The anti-lung cancer activities of HFSGF-L and HFSGF-H against human lung cancer A549 cells in vitro and A549 xenograft tumor growth in vivo were determined. HFSGF-H had higher activity in vitro (IC50 ~12 mg/mL for 24 h) and in vivo (tumor inhibition ~51%.) than HFSGF-L, indicating that HFSGF-H might be a leading compound for a potential new therapeutics for the treatment of lung cancer.

8.
Carbohydr Polym ; 233: 115847, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32059898

RESUMO

Glycosaminoglycans (GAGs) are large, complex carbohydrate molecules that interact with a wide range of proteins involved in physiological and pathological processes. Several naturally derived GAGs have emerged as potentially useful therapeutics in clinical applications. Natural polysaccharides, however, generally have high molecular weights with a degree of polydispersity, making it difficult to investigate their structural properties. In this study, we establish a free-radical-mediated micro-reaction system and use hydrophilic interaction chromatography (HILIC)-Fourier transform mass spectrometry (FTMS) to profile the degraded products of various types of GAGs, heparin, chondroitin sulfate A, NS-heparosan, and oversulfated chondroitin sulfate (OSCS), to reveal the free-radical degradation mechanism of GAGs. The results show that the bulk fragments of GAGs generated by free-radical degradation can maintain their basic structural units and sulfate substituents. In addition, an abundance of oligomers modified with oxidation at their reducing ends or by dehydration also appeared. We discovered that these modifications were related in terms of the degree of sulfation and the α- or ß-linkage of HexNY (Y = SO3- or Ac), and especially that the different linkage of the disaccharide unit is the main factor in modification. In addition, the method based on micro-free-radical reaction and HILIC-FTMS is both effective and sensitive, thus suggesting its broad practical value for the structural characterization and in the biological structure-function studies of GAGs.

9.
Int J Biol Macromol ; 150: 765-774, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32035956

RESUMO

Ganoderma lucidum, commonly known as "Lingzhi" in Chinese, are well-known medicinal mushrooms. Lingzhi has been used in traditional Chinese herbal medicines for more than two thousand years. G. lucidum polysaccharides (GLPs) are present at high levels in G. lucidum cells and GLPs have molecular weights ranging from thousands to millions. GLPs have been widely studied for their various biological activities, such as antioxidant, antitumor, anti-inflammatory, antiviral, anti-diabetes, and immunomodulatory activities. The methods for GLPs extraction and characterization are mature, but the comprehensive research on the relationship between GLPs structure (i.e., molecular weight, tertiary structure, branching, substituents, and monosaccharide composition) and function is still quite limited. The aim of this review is to update and summarize the mechanisms of the various bioactive polysaccharides extracted from G. lucidum. The information presented on these bio-mechanisms should be valuable in the research and development of GLPs-derived therapeutics.

10.
Molecules ; 25(4)2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32093113

RESUMO

Polysaccharides are considered to be the most important active substances in Goji. However, the structure of polysaccharides varies according to the extraction methods applied, and the solution used to prepare Goji polysaccharides (LBPs) were limited. Thus, it is important to clarify the connection between extraction methods and structure of Goji polysaccharide. In view of the complex composition of cell wall polysaccharides and the various forms of interaction, different extraction methods will release different parts of the cell wall. The present study compared the effects of different extraction methods, which have been used to prepare different types of plant cell wall polysaccharides based on various sources, on the structure of cell-wall polysaccharides from Goji, by the single separate use of hot water, hydrochloric acid (0.4%) and sodium hydroxide (0.6%), at both high and low temperatures. Meanwhile, in order to explore the limitations of single extraction, sequential extraction methods were applied. Structural analysis including monosaccharide analysis, GPC-MALLS, AFM and 1H-NMR suggested the persistence of more extensively branched rhamnogalacturonan I (RG-I) domains in the procedures involving low-temperature-alkali, while procedures prepared by high-temperature-acid contains more homogalacturonan (HG) regions and results in the removal of a substantial part of the side chain, specifically the arabinan. A kind of acidic heteropolysaccharide was obtained by hot water extraction. SEC-MALLS and AFM confirmed large-size polymers with branched morphologies in alkali-extracted polysaccharides. Our results provide new insight into the extraction of Goji polysaccharides, which differ from the hot water extraction used by traditional Chinese medicine.

11.
Int J Biol Macromol ; 152: 199-206, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32088231

RESUMO

Hyaluronic acid (HA) is a major glycosaminoglycan, a family of structurally complex, linear, anionic hetero-co-polysaccharides. HA is important in various anatomical structures including the eyes, joints, heart and myriad intricate tissues, and is currently widely used in the therapeutics and cosmetics areas. The synthesis of HA of well-defined and uniform chain lengths is of major interest for the development of safer and more reliable drugs and to gain a better understanding of its structure-activity relationships. However, HA has received less attention from the synthetic carbohydrate community compared with other members of the glycosaminoglycan family. In this review, we examine the remarkable progress that has been made in the chemical and chemoenzymatic synthesis of HA, providing a broad spectrum of options to access HA of well controlled chain lengths.

12.
Glycoconj J ; 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32086666

RESUMO

Glycosaminoglycans (GAGs) are major components of cartilage extracellular matrix (ECM), which play an important role in tissue homeostasis not only by providing mechanical load resistance, but also as signaling mediators of key cellular processes such as adhesion, migration, proliferation and differentiation. Specific GAG types as well as their disaccharide sulfation patterns can be predictive of the tissue maturation level but also of disease states such as osteoarthritis. In this work, we used a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to perform a comparative study in terms of temporal changes in GAG and disaccharide composition between tissues generated from human bone marrow- and synovial-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) after chondrogenic differentiation under normoxic (21% O2) and hypoxic (5% O2) micromass cultures. The chondrogenic differentiation of hBMSC/hSMSC cultured under different oxygen tensions was assessed through aggregate size measurement, chondrogenic gene expression analysis and histological/immunofluorescence staining in comparison to human chondrocytes. For all the studied conditions, the compositional analysis demonstrated a notable increase in the average relative percentage of chondroitin sulfate (CS), the main GAG in cartilage composition, throughout MSC chondrogenic differentiation. Additionally, hypoxic culture conditions resulted in significantly different average GAG and CS disaccharide percentage compositions compared to the normoxic ones. However, such effect was considerably more evident for hBMSC-derived chondrogenic aggregates. In summary, the GAG profiles described here may provide new insights for the prediction of cartilage tissue differentiation/disease states and to characterize the quality of MSC-generated chondrocytes obtained under different oxygen tension culture conditions.

13.
ACS Infect Dis ; 6(3): 503-514, 2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-31961652

RESUMO

Lyme disease (LD) is caused by the spirochete Borrelia burgdorferi sensu lato (Bbsl). After transmission to humans by ticks, Bbsl spreads to multiple organs, leading to arthritis, carditis, and neuroborreliosis. No effective prophylaxis against human LD prior to tick exposure is currently available. Thus, a pre-exposure prophylaxis (PrEP) against LD is needed. The establishment of LD bacteria at diverse sites is dictated partly by the binding of Bbsl to proteoglycans (PGs) and glycosaminoglycans (GAGs) in tissues. The drug heparin is structurally similar to these GAGs and inhibits Bbsl attachment to PGs, GAGs, cells, and tissues, suggesting its potential to prevent LD. However, the anticoagulant activity of heparin often results in hemorrhage, hampering the development of this compound as LD PrEP. We have previously synthesized a non-anticoagulant version of heparin (NACH), which was verified for safety in mice and humans. Here, we showed that NACH blocks Bbsl attachment to PGs, GAGs, and mammalian cells. We also found that treating mice with NACH prior to the exposure of ticks carrying Bbsl followed by continuous administration of this compound prevents tissue colonization by Bbsl. Furthermore, NACH-treated mice develop greater levels of IgG and IgM against Bbsl at early stages of infection, suggesting that the upregulation of antibody immune responses may be one of the mechanisms for NACH-mediated LD prevention. This is one of the first studies examining the ability of a heparin-based compound to prevent LD prior to tick exposure. The information presented might also be extended to prevent other infectious diseases agents.

14.
Int J Biol Macromol ; 149: 672-681, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31981665

RESUMO

To preserve bioactivity and achieve colon targeted release of phycocyanin (PC), the polysaccharides-based electrospun fiber mat (EFM) containing PC and prebiotics was prepared and characterized. In vitro release tests confirmed the colon targeting behavior of PC, in particular, faster release of PC was achieved due to the addition of prebiotics. Ritger-Peppas model confirmed that the release of PC in simulated colon fluids follows a mechanism of anomalous transport (non-Fickian). CCK-8 results showed that the combination of PC and prebiotics exerted a significant anti-proliferative effect on HCT116 cells with an IC50 values of 22.31, 17.12 and 11.63 mg/mL after 24, 48, and 72 h, respectively. Furthermore, the cell cycle and apoptosis analysis revealed that the inhibition activity on HCT116 cells was caused by arresting cell cycle at G0/G1 phase that is relevant to the inhibition of cyclin D1 and CDK4 and the up-regulation of p21 expression, and inducing cell apoptosis by mediating the mitochondrial pathway as well, in which the decrease of Bcl-2/Bax, activation of caspase 3 and release of cytochrome c were included. This study suggests that the PC-loaded EFM with GOS holds a great potential as an effective formulation for colon cancer prevention.

15.
Diabetes ; 69(4): 760-770, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31974145

RESUMO

Long-term hyperglycemia in patients with diabetes leads to human serum albumin (HSA) glycation, which may impair HSA function as a transport protein and affect the therapeutic efficacy of anticoagulants in patients with diabetes. In this study, a novel mass spectrometry approach was developed to reveal the differences in the profiles of HSA glycation sites between patients with diabetes and healthy subjects. K199 was the glycation site most significantly changed in patients with diabetes, contributing to different interactions of glycated HSA and normal HSA with two types of anticoagulant drugs, heparin and warfarin. An in vitro experiment showed that the binding affinity to warfarin became stronger when HSA was glycated, while HSA binding to heparin was not significantly influenced by glycation. A pharmacokinetic study showed a decreased level of free warfarin in the plasma of diabetic rats. A preliminary retrospective clinical study also revealed that there was a statistically significant difference in the anticoagulant efficacy between patients with diabetes and patients without diabetes who had been treated with warfarin. Our work suggests that larger studies are needed to provide additional specific guidance for patients with diabetes when they are administered anticoagulant drugs or drugs for treating other chronic diseases.

16.
Artigo em Inglês | MEDLINE | ID: mdl-31916699

RESUMO

The clinical demand for tissue-engineered bone is growing due to the increase of non-union fractures and delayed healing in an aging population. Herein, we present a method combining additive manufacturing (AM) techniques with cell-derived extracellular matrix (ECM) to generate structurally well-defined bioactive scaffolds for bone tissue engineering (BTE). In this work, highly porous three-dimensional polycaprolactone (PCL) scaffolds with desired size and architecture were fabricated by fused deposition modeling and subsequently decorated with human mesenchymal stem/stromal cell (MSC)-derived ECM produced in situ. The successful deposition of MSC-derived ECM onto PCL scaffolds (PCL-MSC ECM) was confirmed after decellularization using scanning electron microscopy, elemental analysis, and immunofluorescence. The presence of cell-derived ECM within the PCL scaffolds significantly enhanced MSC attachment and proliferation, with and without osteogenic supplementation. Additionally, under osteogenic induction, PCL-MSC ECM scaffolds promoted significantly higher calcium deposition and elevated relative expression of bone-specific genes, particularly the gene encoding osteopontin, when compared to pristine scaffolds. Overall, our results demonstrated the favorable effects of combining MSC-derived ECM and AM-based scaffolds on the osteogenic differentiation of MSC, resulting from a closer mimicry of the native bone niche. This strategy is highly promising for the development of novel personalized BTE approaches enabling the fabrication of patient defect-tailored scaffolds with enhanced biological performance and osteoinductive properties.

17.
Carbohydr Polym ; 230: 115643, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31887910

RESUMO

Carbohydrates are often found linked to lipids or proteins, to form glycoconjugates such as glycolipids, glycoproteins and proteoglycans. These glycoconjugates play important biological roles, involving cell-cell recognition, inflammation, immune response, tumor metastasis and in viral/bacterial/parasitic infections. However, it is difficult to obtain these naturally occurring glycoconjugates as they are often heterogeneous compositions, which causes batch-to-batch variability of structure and activity often leading an incomplete understanding of their mechanism of action. Thus, the efficient preparation of synthetic glycoconjugates, possessing well-defined compositions and reproducible biological properties is highly desirable. In the present review, we summarize the advances in site-selective approaches for glycoconjugate synthesis and, in particular, the preparation of polysaccharide vaccines.

18.
J Pharm Sci ; 109(2): 975-980, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31705871

RESUMO

Heparin-induced thrombocytopenia (HIT) is an adverse immunological disorder caused by antibodies to platelet factor 4 (PF4)-heparin complexes. The analysis of HIT potential for different heparin and heparin-related products is important prior to their clinical application. Here, we report a rapid method for the evaluation of HIT potential of various heparin and heparin-related compounds using surface plasmon resonance (SPR). Solution competition between surface-immobilized heparin and soluble unfractionated heparin, low molecular weight heparin (LMWH), or ultra-LMWH binding to PF4 was performed using SPR to measure the half maximal inhibitory concentration (IC50) of different heparin products. The IC50 values of different unfractionated heparin active pharmaceutical ingredients (APIs) varied from 0.38 to 0.6 µg/mL and the IC50 values of different LMWH APIs ranged from 2.4 to 2.9 µg/mL. The IC50 of Arixtra® (a synthetic pentasaccharide ultra-LMWH) was not measurable even at very high concentrations. These differences in IC50 values for different heparin products suggest a quantitative means for evaluating the HIT potential of various heparins and heparin-related products.

19.
Nat Chem ; 12(1): 26-35, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31767992

RESUMO

DNA, when folded into nanostructures with a specific shape, is capable of spacing and arranging binding sites into a complex geometric pattern with nanometre precision. Here we demonstrate a designer DNA nanostructure that can act as a template to display multiple binding motifs with precise spatial pattern-recognition properties, and that this approach can confer exceptional sensing and potent viral inhibitory capabilities. A star-shaped DNA architecture, carrying five molecular beacon-like motifs, was constructed to display ten dengue envelope protein domain III (ED3)-targeting aptamers into a two-dimensional pattern precisely matching the spatial arrangement of ED3 clusters on the dengue (DENV) viral surface. The resulting multivalent interactions provide high DENV-binding avidity. We show that this structure is a potent viral inhibitor and that it can act as a sensor by including a fluorescent output to report binding. Our molecular-platform design strategy could be adapted to detect and combat other disease-causing pathogens by generating the requisite ligand patterns on customized DNA nanoarchitectures.

20.
Acc Chem Res ; 53(2): 335-346, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-31714740

RESUMO

Glycosaminoglycans (GAGs) are a family of structurally complex heteropolysaccharides composed of alternating hexosamine and uronic acid or galatose residue that include hyaluronan, chondroitin sulfate and dermatan sulfate, heparin and heparan sulfate, and keratan sulfate. GAGs display a range of critical biological functions, including regulating cell-cell interactions and cell proliferation, inhibiting enzymes, and activating growth factor receptors during various metabolic processes. Indeed, heparin is a widely used GAG-based anticoagulant drug. Unfortunately, naturally derived GAGs are highly heterogeneous, limiting studies of their structure-activity relationships and even resulting in safety concerns. For example, the heparin contamination crisis in 2007 reportedly killed more than a hundred people in the United States. Unfortunately, the chemical synthesis of GAGs, or their oligosaccharides, based on repetitive steps of protection, activation, coupling, and deprotection, is incredibly challenging. Recent advances in chemoenzymatic synthesis integrate the flexibility of chemical derivatization with enzyme-catalyzed reactions, mimicking the biosynthetic pathway of GAGs, and represent a promising strategy to solve many of these synthetic challenges. In this critical Account, we examine the recent progress made, in our laboratory and by others, in the chemoenzymatic synthesis of GAGs, focusing on heparan sulfate and heparin, a class of GAGs with profound physiological and pharmacological importance. A major challenge for the penetration of the heparin market by homogeneous heparin products is their cost-effective large-scale synthesis. In the past decade, we and our collaborators have systematically explored the key factors that impact this process, including better enzyme expression, improved biocatalysts using protein engineering and immobilization, low cost production of enzyme cofactors, optimization of the order of enzymatic transformations, as well as development of efficient technologies, such as using ultraviolet absorbing or fluorous tags, to detect and purify synthetic intermediates. These improvements have successfully resulted in multigram-scale synthesis of low-molecular-weight heparins (LMWHs), with some showing excellent anticoagulant activity and even resulting in more effective protamine reversal than commercial, animal-sourced LMWH drugs. Sophisticated structural analysis is another challenge for marketing heparins, since impurities and contaminants can be present that are difficult to distinguish from heparin drug products. The availability of the diverse library of structurally defined heparin oligosaccharides has facilitated the systematic analytical studies undertaken by our group, resulting in important information for characterizing diverse heparin products, safeguarding their quality. Recently, a series of chemically modified nucleotide sugars have been investigated in our laboratory and have been accepted by synthases to obtain novel GAGs and GAG oligosaccharides. These include fluoride and azido regioselectively functionalized sugars and stable isotope-enriched GAGs and GAG oligosaccharides, critical for better understanding the biological roles of these important biopolymers. We speculate that the repertoire of unnatural acceptors and nucleotide sugar donors will soon be expanded to afford many new GAG analogues with new biological and pharmacological properties including improved specificity and metabolic stability.

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