Synthesis Of Nucleotide Sugars Research Articles

One-step synthesis of sugar nucleotides under optimized conditions

Sugar nucleotides are essential substrates used for synthesizing bioactive oligosaccharides in enzymatic reactions, however, their preparation via chemical and enzyme reactions is challenging. Therefore, we investigated the synthesis of sugar nucleotides under optimal conditions using a facile one-step reaction. Seven sugar nucleotides were synthesized via an optimized one-step reaction in 40–80% yield, exhibiting a preference for 1,2-trans glycoside. Moreover, a gram-scale synthesis of UDP-galactose resulted in a 46% (578 mg) yield.

Process Development for the Enzymatic Gram-Scale Production of the Unnatural Nucleotide Sugar UDP-6-Azido-GalNAc.

Azido sugars hold great promise as substrates in numerous click-chemistry applications. However, the synthesis of activated azido sugars is limited by cost and complexity. Conventional chemical activation methods are intricate and time-consuming. In response, we have developed a process for the large-scale production of UDP-6-azido-GalNAc through enzymatic nucleotide sugar synthesis on a gram scale. Our optimization strategies encompassed refining the process parameters of an enzyme cascade featuring NahK from Bifidobacterium longum and AGX1 from Homo sapiens. Using the repetitive-batch-mode technology, we synthesized up to 2.1 g of UDP-6-azido-GalNAc, achieving yields up to 97 % in five consecutive batch cycles using a single enzyme batch. The synthesis process demonstrated to have total turnover numbers (TTNs) between 4.4-4.8 g of product per gram of enzyme (gP/gE) and STYs ranging from 1.7-2.4 g per liter per hour (g*L-1*h-1). By purification of a product solution pool containing 2.6 g (4.1 mmol) UDP-6-azido-GalNAc, 2.1 g (2,122.1 mg) UDP-6-azido-GalNAc (sodium salt) with a purity of 99.96 % (HPLC) were obtained. The overall recovery after purification was 81 % (3.32 mmol). Our work establishes a robust production platform for the gram-scale synthesis of unnatural nucleotide sugars, opening new avenues for applications in glycan engineering.

Clinical and biochemical footprints of congenital disorders of glycosylation: Proposed nosology

We have identified 200 congenital disorders of glycosylation (CDG) caused by 189 different gene defects and have proposed a classification system for CDG based on the mode of action. This classification includes 8 categories: 1. Disorders of monosaccharide synthesis and interconversion, 2. Disorders of nucleotide sugar synthesis and transport, 3. Disorders of N-linked protein glycosylation, 4. Disorders of O-linked protein glycosylation, 5. Disorders of lipid glycosylation, 6. Disorders of vesicular trafficking, 7. Disorders of multiple glycosylation pathways and 8. Disorders of glycoprotein/glycan degradation. Additionally, using information from IEMbase, we have described the clinical involvement of 19 organs and systems, as well as essential laboratory investigations for each type of CDG. Neurological, dysmorphic, skeletal, and ocular manifestations were the most prevalent, occurring in 81%, 56%, 53%, and 46% of CDG, respectively. This was followed by digestive, cardiovascular, dermatological, endocrine, and hematological symptoms (17–34%). Immunological, genitourinary, respiratory, psychiatric, and renal symptoms were less frequently reported (8–12%), with hair and dental abnormalities present in only 4–7% of CDG. The information provided in this study, including our proposed classification system for CDG, may be beneficial for healthcare providers caring for individuals with metabolic conditions associated with CDG.

Isotopic Tracing of Nucleotide Sugar Metabolism in Human Pluripotent Stem Cells.

Metabolism not only produces energy necessary for the cell but is also a key regulator of several cellular functions, including pluripotency and self-renewal. Nucleotide sugars (NSs) are activated sugars that link glucose metabolism with cellular functions via protein N-glycosylation and O-GlcNAcylation. Thus, understanding how different metabolic pathways converge in the synthesis of NSs is critical to explore new opportunities for metabolic interference and modulation of stem cell functions. Tracer-based metabolomics is suited for this challenge, however chemically-defined, customizable media for stem cell culture in which nutrients can be replaced with isotopically labeled analogs are scarcely available. Here, we established a customizable flux-conditioned E8 (FC-E8) medium that enables stem cell culture with stable isotopes for metabolic tracing, and a dedicated liquid chromatography mass-spectrometry (LC-MS/MS) method targeting metabolic pathways converging in NS biosynthesis. By 13C6-glucose feeding, we successfully traced the time-course of carbon incorporation into NSs directly via glucose, and indirectly via other pathways, such as glycolysis and pentose phosphate pathways, in induced pluripotent stem cells (hiPSCs) and embryonic stem cells. Then, we applied these tools to investigate the NS biosynthesis in hiPSC lines from a patient affected by deficiency of phosphoglucomutase 1 (PGM1), an enzyme regulating the synthesis of the two most abundant NSs, UDP-glucose and UDP-galactose.

Tracer metabolomics reveals the role of aldose reductase in glycosylation

Abnormal polyol metabolism is predominantly associated with diabetes, where excess glucose is converted to sorbitol by aldose reductase (AR). Recently, abnormal polyol metabolism has been implicated in phosphomannomutase 2 congenital disorder of glycosylation (PMM2-CDG) and an AR inhibitor, epalrestat, proposed as a potential therapy. Considering that the PMM2 enzyme is not directly involved in polyol metabolism, the increased polyol production and epalrestat's therapeutic mechanism in PMM2-CDG remained elusive. PMM2-CDG, caused by PMM2 deficiency, presents with depleted GDP-mannose and abnormal glycosylation. Here, we show that, apart from glycosylation abnormalities, PMM2 deficiency affects intracellular glucose flux, resulting in polyol increase. Targeting AR with epalrestat decreases polyols and increases GDP-mannose both in patient-derived fibroblasts and in pmm2 mutant zebrafish. Using tracer studies, we demonstrate that AR inhibition diverts glucose flux away from polyol production toward the synthesis of sugar nucleotides, and ultimately glycosylation. Finally, PMM2-CDG individuals treated with epalrestat show a clinical and biochemical improvement.

Enzyme cascades for the synthesis of nucleotide sugars: Updates to recent production strategies

Nucleotide sugars play an elementary role in nature as building blocks of glycans, polysaccharides, and glycoconjugates used in the pharmaceutical, cosmetics, and food industries. As substrates of Leloir-glycosyltransferases, nucleotide sugars are essential for chemoenzymatic in vitro syntheses. However, high costs and the limited availability of nucleotide sugars prevent applications of biocatalytic cascades on a large industrial scale. Therefore, the focus is increasingly on nucleotide sugar synthesis strategies to make significant application processes feasible. The chemical synthesis of nucleotide sugars and their derivatives is well established, but the yields of these processes are usually low. Enzyme catalysis offers a suitable alternative here, and in the last 30 years, many synthesis routes for nucleotide sugars have been discovered and used for production. However, many of the published procedures shy away from assessing the practicability of their processes. With this review, we give an insight into the development of the (chemo)enzymatic nucleotide sugar synthesis pathways of the last years and present an assessment of critical process parameters such as total turnover number (TTN), space-time yield (STY), and enzyme loading.

Engineering nucleotide sugar synthesis pathways for independent and simultaneous modulation of N-glycan galactosylation and fucosylation in CHO cells

Glycosylation of recombinant therapeutics like monoclonal antibodies (mAbs) is a critical quality attribute. N-glycans in mAbs are known to affect various effector functions, and thereby therapeutic use of such glycoproteins can depend on a particular glycoform profile to achieve desired efficacy. However, there are currently limited options for modulating the glycoform profile, which depend mainly on over-expression or knock-out of glycosyltransferase enzymes that can introduce or eliminate specific glycans but do not allow predictable glycoform modulation over a range of values. In this study, we demonstrate the ability to predictably modulate the glycoform profile of recombinant IgG. Using CRISPR/Cas9, we have engineered nucleotide sugar synthesis pathways in CHO cells expressing recombinant IgG for combinatorial modulation of galactosylation and fucosylation. Knocking out the enzymes UDP-galactose 4′-epimerase (Gale) and GDP-L-fucose synthase (Fx) resulted in ablation of de novo synthesis of UDP-Gal and GDP-Fuc. With Gale knock-out, the array of N-glycans on recombinantly expressed IgG is narrowed to agalactosylated glycans, mainly A2F glycan (89%). In the Gale and Fx double knock-out cell line, agalactosylated and afucosylated A2 glycan is predominant (88%). In the double knock-out cell line, galactosylation and fucosylation was entirely dependent on the salvage pathway, which allowed for modulation of UDP-Gal and GDP-Fuc synthesis and intracellular nucleotide sugar availability by controlling the availability of extracellular galactose and fucose. We demonstrate that the glycoform profile of recombinant IgG can be modulated from containing predominantly agalactosylated and afucosylated glycans to up to 42% and 96% galactosylation and fucosylation, respectively, by extracellular feeding of sugars in a dose-dependent manner. By simply varying the availability of extracellular galactose and/or fucose, galactosylation and fucosylation levels can be simultaneously and independently modulated. In addition to achieving the production of tailored glycoforms, this engineered CHO host platform can cater to the rapid synthesis of variably glycoengineered proteins for evaluation of biological activity.

Cell‐free biocatalytic synthesis of nucleotide sugars

Cell‐free biocatalytic synthesis of nucleotide sugars

Insights into the role of external layers in cyanobacteria at varying temperatures and their regulatory mechanism

Abstract The dominance of cyanobacteria and their adaptability to changing climates might be attributed to their morphological plasticity related to external layers. The content of exopolysaccharides in cyanobacterial external layers has been shown to increase at elevated temperatures. However, the answers to the questions of why and how cyanobacteria adapt to varying temperatures by adjusting the physiological and biophysical properties of their external layers are still not clear. We cultivated three cyanobacterial strains, Nostoc sp. FACHB‐2009, Dolichospermum sp. FACHB‐82 and Microcystis sp. FACHB‐3296, which are embedded in three typical types of external layers—sheath, capsule and slime, respectively––and each grown at three different temperatures (10°C, 25°C and 35°C). The results showed that varying the temperature significantly changed the physicochemical properties of the external layers. High temperature conditions promoted increases in colony sizes compared to the control, with the external layers becoming thicker, looser and more adhesive, whereas cell sizes did not significantly change. Additionally, the expression patterns of related genes varied with temperature; it represents several temp dependent genes related to synthesis of nucleotide sugars were upregulated at high temperature but downregulated at low temperature. As the external layers are wrapped outside cells, we assumed a “spherical shell heat conduction model”. Through that, we inferred that the presence of external layers influenced heat transfer in the cyanobacteria, thereby playing a role in stabilising cell activity at various temperatures. Transcriptome tests showed that the use of intracellularly stored nucleotide sugars for external layer synthesis was the preferred process for cyanobacteria at low temperature, whereas de novo external layer synthesis was the preferred process for cyanobacteria at high temperature.

Mn-doped bimetallic synergistic catalysis boosts for enzymatic phosphorylation of N-Acetylglucosamine/ N-Acetylgalactosamine and their derivatives

Metal-organic frameworks (MOFs), as advanced enzyme immobilization platforms for improving biocatalysis and protein biophysics, are rarely investigated as solid supports in the enzymatic synthesis of carbohydrate and derivatives, which can be attributed to the complex biochemical reaction mechanisms and the adverse interactions between the high polarity of substrate sugars, glycoenzymes and traditional MOFs. Here, we introduced divalent metal ion Mn2+ into MOF to prepare bimetallic MOF microreactor that encapsulated N-acetylhexosamine 1-Kinase (NahK), a critical anomeric kinase involved in the enzymatic synthesis of sugar nucleotide. The introduced Mn ions not only adjusted the microstructure of MOFs, but also participated in the enzymatic catalysis as cofactor, thus facilitated the N-acetylglucosamine/ N-acetylgalactosamine (GlcNAc/GalNAc) phosphorylation. The Mn-doped NahK@Zn-metal organic material (MOM), integrated with high catalytic activity, high stability, and high recoverability, solved the issues of immobilization related to glucokinase activity. These features significantly improved the operability and reduced the processing cost, assuring industrial application prospects for sugar nucleotides synthesis.

Effect of amino acids on free exopolysaccharide biosynthesis by Streptococcus thermophilus 937 in chemically defined medium

Free exopolysaccharide (f-EPS) produced by Streptococcus thermophilus improves the texture and functionality of fermented dairy foods. Our previous study showed a major improvement in f-EPS production of Strep. thermophilus 937 by increasing the concentrations of histidine, isoleucine, and glutamate to 15 mM in an optimized chemically defined medium. The aim of this study was to elucidate the effect of His, Ile, and Glu on the growth, f-EPS biosynthesis pathway, and carbohydrate metabolism profiles of Strep. thermophilus 937. The growth kinetics; transcript levels of key genes in the EPS biosynthesis pathway; enzyme activity involved in sugar nucleotide synthesis; concentrations of lactic acid, lactose, and galactose; and extracellular and intracellular pH were analyzed in chemically defined media with different initial histidine, isoleucine, and glutamate concentrations. The results showed that f-EPS production and viable cell counts of Strep. thermophilus 937 increased 2-fold after the concentrations of His, Ile, and Glu were increased. Additionally, increasing the concentrations of His, Ile, and Glu upregulated transcription of EPS biosynthesis genes and increased the activity of key enzymes in sugar nucleotide synthesis. Moreover, the consumption of lactose increased and secretion of galactose decreased, indicating that increasing the concentration of His, Ile, and Glu could enhance f-EPS production by maintaining viable cell counts, promoting sugar nucleotide synthesis, and increasing the transcript levels of the eps gene cluster. Our results provide a better understanding of the effect of AA on EPS biosynthesis in Strep. thermophilus.

Facile Synthesis of Sugar Nucleotides from Common Sugars by the Cascade Conversion Strategy.

Sugar nucleotides are essential glycosylation donors in the carbohydrate metabolism. Naturally, most sugar nucleotides are derived from a limited number of common sugar nucleotides by de novo biosynthetic pathways, undergoing single or multiple reactions such as dehydration, epimerization, isomerization, oxidation, reduction, amination, and acetylation reactions. However, it is widely believed that such complex bioconversions are not practical for synthetic use due to the high preparation cost and great difficulties in product isolation. Therefore, most of the discovered sugar nucleotides are not readily available. Here, based on de novo biosynthesis mainly, 13 difficult-to-access sugar nucleotides were successfully prepared from two common sugars D-Man and sucrose in high yields, at a multigram scale, and without the need for tedious purification manipulations. This work demonstrated that de novo biosynthesis, although undergoing complex reactions, is also practical and cost-effective for synthetic use by employing a cascade conversion strategy.

Pan-Genomic and Transcriptomic Analyses of Marine Pseudoalteromonas agarivorans Hao 2018 Revealed Its Genomic and Metabolic Features.

The genomic and carbohydrate metabolic features of Pseudoalteromonas agarivorans Hao 2018 (P. agarivorans Hao 2018) were investigated through pan-genomic and transcriptomic analyses, and key enzyme genes that may encode the process involved in its extracellular polysaccharide synthesis were screened. The pan-genome of the P. agarivorans strains consists of a core-genome containing 2331 genes, an accessory-genome containing 956 genes, and a unique-genome containing 1519 genes. Clusters of Orthologous Groups analyses showed that P. agarivorans harbors strain-specifically diverse metabolisms, probably representing high evolutionary genome changes. The Kyoto Encyclopedia of Genes and Genomes and reconstructed carbohydrate metabolic pathways displayed that P. agarivorans strains can utilize a variety of carbohydrates, such as d-glucose, d-fructose, and d-lactose. Analyses of differentially expressed genes showed that compared with the stationary phase (24 h), strain P. agarivorans Hao 2018 had upregulated expression of genes related to the synthesis of extracellular polysaccharides in the logarithmic growth phase (2 h), and that the expression of these genes affected extracellular polysaccharide transport, nucleotide sugar synthesis, and glycosyltransferase synthesis. This is the first investigation of the genomic and metabolic features of P. agarivorans through pan-genomic and transcriptomic analyses, and these intriguing discoveries provide the possibility to produce novel marine drug lead compounds with high biological activity.

Cofactor-Driven Cascade Reactions Enable the Efficient Preparation of Sugar Nucleotides.

Glycosylation is catalyzed by glycosyltransferases using sugar nucleotides or occasionally lipid-linked phosphosugars as donors. However, only very few common sugar nucleotides that occur in humans can be obtained readily, while the majority of sugar nucleotides that exist in bacteria, plants, archaea, or viruses cannot be synthesized in sufficient quantities by either enzymatic or chemical synthesis. The limited availability of such rare sugar nucleotides is one of the major obstacles that has greatly hampered progress in glycoscience. Herein we describe a general cofactor-driven cascade conversion strategy for the efficient synthesis of sugar nucleotides. The described strategy allows the large-scale preparation of rare sugar nucleotides from common sugars in high yields and without the need for tedious purification processes.

Cofactor‐Driven Cascade Reactions Enable the Efficient Preparation of Sugar Nucleotides

Glycosylation is catalyzed by glycosyltransferases using sugar nucleotides or occasionally lipid-linked phosphosugars as donors. However, only very few common sugar nucleotides that occur in humans can be obtained readily, while the majority of sugar nucleotides that exist in bacteria, plants, archaea, or viruses cannot be synthesized in sufficient quantities by either enzymatic or chemical synthesis. The limited availability of such rare sugar nucleotides is one of the major obstacles that has greatly hampered progress in glycoscience. Herein we describe a general cofactor-driven cascade conversion strategy for the efficient synthesis of sugar nucleotides. The described strategy allows the large-scale preparation of rare sugar nucleotides from common sugars in high yields and without the need for tedious purification processes.

The Trypanosome UDP-Glucose Pyrophosphorylase Is Imported by Piggybacking into Glycosomes, Where Unconventional Sugar Nucleotide Synthesis Takes Place.

ABSTRACTGlycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nucleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knockdown UGP mutant cell line (RNAiUGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle (RNAiUGP/EXPrUGP-GPDH, where GPDH is glycerol-3-phosphate dehydrogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography–high-resolution mass spectrometry [IC-HRMS]) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones.

Gram-scale production of sugar nucleotides and their derivatives

Herein, we reported a practical approach for multi-gram scale synthesis of natural and unnatural sugar nucleotides.

Synthesis of functional oligosaccharides and their derivatives through cocultivation and cellular NTP regeneration.

Carbohydrates play an important role in the life cycle. Among them, functional oligosaccharides show a complex and diverse structures with unique physiological activities and biological functions. However, different preparation methods directly affect the structure, molecular weight, and other functions of oligosaccharides, as well as their application fields and manufacturing costs. In the preparation of β-1,3-glucan oligosaccharides (OBGs), water insolubility of β-1,3-glucans hampers the hydrolysis efficiency. The synthesis of some functional oligosaccharides requires the consumption of energy substrates, such as ATP, CTP, and uridine triphosphate, for sugar nucleotide synthesis, leading to increased capital costs. A more economical solution to solve energy supply is to adopt microbial cocultivation or cellular nucleoside triphosphate regeneration. This review focused on the sources, preparation methods, biological activities of OBG, and the cultivation methods and applications of microbial cocultivation and fermentation. We also reviewed the preparation methods of other functional oligosaccharides, such as sialylated oligosaccharides, β-nicotinamide mononucleotide, and α-galacto-oligosaccharides.

Nucleotide Sugars in Chemistry and Biology.

Nucleotide sugars have essential roles in every living creature. They are the building blocks of the biosynthesis of carbohydrates and their conjugates. They are involved in processes that are targets for drug development, and their analogs are potential inhibitors of these processes. Drug development requires efficient methods for the synthesis of oligosaccharides and nucleotide sugar building blocks as well as of modified structures as potential inhibitors. It requires also understanding the details of biological and chemical processes as well as the reactivity and reactions under different conditions. This article addresses all these issues by giving a broad overview on nucleotide sugars in biological and chemical reactions. As the background for the topic, glycosylation reactions in mammalian and bacterial cells are briefly discussed. In the following sections, structures and biosynthetic routes for nucleotide sugars, as well as the mechanisms of action of nucleotide sugar-utilizing enzymes, are discussed. Chemical topics include the reactivity and chemical synthesis methods. Finally, the enzymatic in vitro synthesis of nucleotide sugars and the utilization of enzyme cascades in the synthesis of nucleotide sugars and oligosaccharides are briefly discussed.

One-Step Synthesis of Sugar Nucleotides.

The chemical synthesis of sugar nucleotides requires a multistep procedure to ensure a selective reaction. Herein, sugar nucleotides were synthesized in one step using 2-chloro-1,3-dimethylimidazolinium chloride as the condensation reagent. The products were obtained in yields of 12-30%, and the yields were increased to 35-47% by the addition of a tuning reagent. NMR identification of the sugar nucleotides showed that mainly 1,2-trans-glycosides were present. The reported method represents a one-step route to sugar nucleotides from commercially available materials.