4.2: Synthesis of di-saccharides and polysaccharides - Biology

4.2: Synthesis of di-saccharides and polysaccharides - Biology

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4.2: Synthesis of di-saccharides and polysaccharides

Difference Between Monosaccharides Disaccharides and Polysaccharides

Carbohydrates are the major components of all living organisms. All the carbohydrates are composed of Carbon (C), Hydrogen (H) and Oxygen (O) atoms in different combinations. Sugars are carbohydrates. The major types of sugars include Monosaccharides and disaccharides. Polysaccharides are complex carbohydrates. The main difference between Monosaccharides Disaccharides and Polysaccharides is that monosaccharides are monomers of sugars and disaccharides are composed of two monomers whereas polysaccharides are composed of a large number of monomers.

Key Areas Covered

Key Terms: Aldoses, Carbohydrates, Disaccharides, Fiber, Ketoses, Monosaccharides, Polysaccharides, Starch, Sugars

A novel synthesis of 1,2-cis-disaccharides

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Analysis of Glycans Polysaccharide Functional Properties Crystalline Conformations of Oligosaccharides The disaccharides

Important in their own right, disaccharides take great importance as the shortest components of the family of oligo- and polysaccharides and complex carbohydrates. As such a particular attention is given to their conformational properties because their molecular shapes are considered to be important determinants of their properties and those of their larger parents. The disaccharides result from the condensation of a reducing hydroxyl group (C-1 OH in aldose, C-2 OH in ketose) with another hydroxyl group. This linkage may be 1 → n, where n is 1–6, except 5. If two reducing groups are involved, the disaccharide is nonreducing as in sucrose and trehaloses.

For disaccharide moieties, the main conformational determinants are the ring shapes, the orientations of the hydroxyl groups, and the relative orientations of the monosaccharide units at the glycosidic linkage. 1 Ring shapes can be defined in terms of reference conformations (chair, C twist, T boat, B envelope, E skew, S) or by the so-called puckering parameters. 10 The exocyclic primary alcohol groups can adopt a number of low-energy conformations. They are in staggered arrangements that correspond to local minima. In the case of pyranoses, primary hydroxyl groups most frequently occupy two positions, avoiding interactions between O-4 and O-6. However, each of the secondary hydroxyl groups can rotate almost freely. The relative orientation of two consecutive monosaccharide units in a disaccharide is customarily described by the torsion angles Φ and Ψ around the glycosidic bonds. Φ represents the torsion angle about the C (anomeric)–O bond, whereasΨ The sign of the torsion angles is given in accordance with the IUPAC-IUBMB Joint Commission of Biochemical Nomenclature (1996). 15

The consideration of the axial/equatorial nature at the glycosidic linkage provides a useful framework for the classification of the disaccharide moieties, independently of the remaining and of the surrounding of the oligosaccharidic molecule ( Figure 1 ). As for six-membered ring-containing disaccharides, axial–axial, axial–equatorial, and equatorial–equatorial are found. The families involving axial-furanose and equatorial-furanose linkage to a hexopyranose are also indicated, along with the furanose–furanose cases. It is worth noting that there is no representative of the equatorial–axial class, even though such type of glycosidic linkage can be found in several carbohydrate-containing molecules and macromolecules.

Figure 1 . Schematic representation of the different types of glycosidic linkages observed in crystal structures of disaccharides and oligosaccharides.

Using such a classification, all the unsubstituted components of the linear oligosaccharide structures were reported and analyzed. 35 For each class, the nature of the glycosidic linkage, the magnitude of the angles ((Ω), Φ, Ψ, τ) at the glycosidic linkage, and the occurrence (if any) of inter-residual hydrogen bonds were reported. In order to have a comprehensive vision of the spatial occurrence of all the crystalline conformations as a function of their belonging to a given class, a schematic representation of their crystallographic conformations at the glycosidic linkage has been set with a superimposition onto the low-energy contours that have been computed for a prototypical motif using molecular mechanics calculations. All the crystalline conformations lie within the 5 kcal mol −1 energy contour of the corresponding potential energy surface. It is noteworthy to observe a limited dispersion about the glycosidic Φ angles compared to that observed about Ψ, as an expression of the influence of the exo-anomeric effect on the establishment of preferred conformation in crystalline oligosaccharides.

The exo-anomeric effect arises due to the particular bonding sequence C-5–O-5–C-1–O-g–C-x′ in glycopyranosides and disaccharides and influences the conformation about the glycosidic torsion angle Φ. In the case of an axial type of linkage (typically as in 4 C1 α- d -configuration), only one staggered conformation is preferred with Φ being in the vicinity of 60°. As for the equatorial type of linkage (typically as in 4 C1 β- d -configuration), two staggered conformations are preferred (Φ = 60° and −60°) of which that corresponding to Φ = –60° is favored due to further stabilization occurring from nonbonded interactions. The values of Φ generally vary between 40° and 120° for 1→ x axial linkages, and between –100° and –65° for 1→ x equatorial linkages. Obviously, the magnitude of the exo-anomeric effect is not strong enough to drive the Φ angle to one single conformation. It can be counterbalanced by the occurrence of inter-residue hydrogen bond or other favorable interatomic interactions. Not unexpectedly, the dispersion of the observed conformations about the torsion angle Ψ is much wider.

A somewhat similar set of observations can be made in the case of disaccharide segments belonging to the axial-furanose family, which encompasses most of the sucrosyl-containing molecules. In such cases, a double exo-anomeric effect, occurring from the C-5–O-5–C-1–O-g–C-x′–O-5′ sequence, could be even more influential in the establishment of glycosidic conformations. Whereas the dispersion in Φ angles spans from 60° to 100°, the Ψ torsion angles span more than 200°. Most of these observations have been rationalized throughout molecular modeling investigations. 14 The analogs (S, C, N, etc.)

The replacement of the interglycosidic oxygen atom in a disaccharide by S, C, N–H,…, generates a class of interesting analogs, namely thio-disaccharides, C-disaccharides, diglycosylamines, which all constitute potential nonhydrolyzable epitopes and substrate analogs. The crystal structures of those analogs were reported and analyzed. 35 For the analogs, accurate geometric and structural data are available providing some useful information about the influence of the chemical change upon the conformation at the glycosidic linkage, the influence (or lack) of the exo-anomeric effect and packing features. 42

Supplementary files

Synthesis of the β-linked GalNAc-Kdo disaccharide antigen of the capsular polysaccharide of Kingella kingae KK01

L. Zhuang, Y. Chen, Q. Lou and Y. Yang, Org. Biomol. Chem., 2019, 17, 1694 DOI: 10.1039/C8OB02340A

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2.2: Carbohydrates

All carbohydrates consist of carbon, hydrogen, and oxygen atoms and are polyhydroxy aldehydes or ketones or are compounds that can be broken down to form such compounds. Examples of carbohydrates include starch, fiber, the sweet-tasting compounds called sugars, and structural materials such as cellulose. The term carbohydrate had its origin in a misinterpretation of the molecular formulas of many of these substances. For example, because its formula is C6H12O6, glucose was once thought to be a &ldquocarbon hydrate&rdquo with the structure C6·6H2O.

Which compounds would be classified as carbohydrates?

  1. This is a carbohydrate because the molecule contains an aldehyde functional group with OH groups on the other two carbon atoms.
  2. This is not a carbohydrate because the molecule does not contain an aldehyde or a ketone functional group.
  3. This is a carbohydrate because the molecule contains a ketone functional group with OH groups on the other two carbon atoms.
  4. This is not a carbohydrate although it has a ketone functional group, one of the other carbons atoms does not have an OH group attached.

Which compounds would be classified as carbohydrates?

Green plants are capable of synthesizing glucose (C6H12O6) from carbon dioxide (CO2) and water (H2O) by using solar energy in the process known as photosynthesis :

(The 686 kcal come from solar energy.) Plants can use the glucose for energy or convert it to larger carbohydrates, such as starch or cellulose. Starch provides energy for later use, perhaps as nourishment for a plant&rsquos seeds, while cellulose is the structural material of plants. We can gather and eat the parts of a plant that store energy&mdashseeds, roots, tubers, and fruits&mdashand use some of that energy ourselves. Carbohydrates are also needed for the synthesis of nucleic acids and many proteins and lipids.

Animals, including humans, cannot synthesize carbohydrates from carbon dioxide and water and are therefore dependent on the plant kingdom to provide these vital compounds. We use carbohydrates not only for food (about 60%&ndash65% by mass of the average diet) but also for clothing (cotton, linen, rayon), shelter (wood), fuel (wood), and paper (wood).

The simplest carbohydrates&mdashthose that cannot be hydrolyzed to produce even smaller carbohydrates&mdashare called monosaccharides . Two or more monosaccharides can link together to form chains that contain from two to several hundred or thousand monosaccharide units. Prefixes are used to indicate the number of such units in the chains. Disaccharide molecules have two monosaccharide units, trisaccharide molecules have three units, and so on. Chains with many monosaccharide units joined together are called polysaccharides . All these so-called higher saccharides can be hydrolyzed back to their constituent monosaccharides.

Compounds that cannot be hydrolyzed will not react with water to form two or more smaller compounds.

Metabolic engineering for GAGs and their analogues

Metabolic engineering is the process of optimizing genetic and regulatory process that results in the production of a desired product accompanied by the suppression of competing pathways, through the transfer of product-specific enzymes or complete metabolic pathways from the intractable host organism into a more easily manipulated and readily available engineered microorganism [15, 46,47,48,49,50].

The potential shortage and safety concerns about commercial GAGs have pushed a new direction from traditional animal-sourced methods of GAG production to biomanufacturing. Synthetic biology techniques are becoming increasingly important tools for pathway optimization and metabolic engineering [15]. Some GAGs including chondroitin and hyaluronic acid have been prepared using metabolic engineering [45, 51,52,53,54,55,56,57]. Currently, commercial scale production of hyaluronan relies on bacterial expression systems in Streptococci and endotoxin-free microorganisms such as Bacilli.

In general, there are three issues regarding the biomanufacturing of GAGs, productivity, efficiency and cost. Even though much work has been carried out on the biomanufacturing of non-sulfated heparin-precursor, heparosan and the chondroitin backbone of chondroitin sulfate by fermentation, modifications involving epimerization and specific sulfations are still required to produce the desired final GAGs products [15, 58].

Heparin is biosynthesized as a proteoglycan in the Golgi of eukaryotic mast cells [8, 59]. Heparin might 1 day be biosynthesized in eukaryotic systems, such as insect cells, yeast and Chinese hamster ovary (CHO) cells, having a Golgi [15, 18, 60]. However, the engineering of bacterial expression systems for GAG production is much more complex.

For the biomanufacturing of other GAGs and their analogues, there are currently two systems under evaluation, eukaryotic systems (including mammalian cells) and prokaryotic cells. Eukaryotic systems and mammalian cells can be engineered to produce the heparan sulfate, and 1 day might be capable of producing heparin [18, 61, 62]. Yeast, the simplest eukaryote, is capable of generating some of the essential glycosylation patterns found in mammals unfortunately, yeast does not produce heparan sulfate [18]. However, the use of yeast for the expression of sulfotransferases, including N-sulfotransferases (NST), 3-O-sulfotransferases (3-OST), 2-O-sulfotransferases (2-OST) and 6-O-sulfotransferases (6-OST), and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in yeast had been reported [63]. CHO cells are mammalian cell lines that are capable of producing heparan sulfate. However, CHO cells only express 2/4 NST, 1/3 6-OST but none of 3-OST-1, and do not have granules [64,65,66]. Genetic engineering of CHO cells can result in the production of heparan sulfate and heparin capable of antithrombin binding, heparin cofactor binding, and herpes simplex virus entry [67, 68]. Unfortunately, the production levels of engineering CHO cells are still relatively low in comparison with the high levels in mammalian mast cells [65]. While further increases in yield and activity could be improved by changes in the fermentation condition, feeding strategies, media composition and through genetic engineering, it is doubtful that a eukaryotic expression system could ever produce the 100 metric ton quantities needed to fill the worldwide market [69, 70].

With respect to prokaryotic expression systems, several microorganisms have been reported for the biosynthesis of GAGs and their analogues. The capsular polysaccharide (CPS) of Escherichia coli K4 consists of a repeating disaccharide unit [→ 4)-β- d -GlcA-(1 → 3)-β- d -GalNAc-(1 →] branched with β-linked fructose at C3 of GlcA [71, 72]. After th ekfoE gene was knocked out, non-fructosylated chondroitin was obtained, which is the polysaccharide backbone of chondroitin sulfate [56]. Chondroitin production can reach maximum levels of 2.4 g/L in a modified rich defined medium using oxygen-stat fed batch bioreactor after re-arranging the gene sequence in the order of kfoC, kfoA and kfoF in a pETM6_PCAF construct and expressing in the non-pathogenic E. coli BL21 Star (DE3) strain [15, 56]. Similarly, heparosan, the backbone of heparin and heparan sulfate is the CPS of E. coli K5, which act as the principal protection of these enteric bacteria against intrinsic host defense within the cell surface [15, 56]. Many microorganisms, such as E. coli K5, K-12, BL21 and Pasteurella multocida are capable of producing heparosan. In the case of E. coli K5, the chain size of heparosan produced is considerably larger than that of heparan sulfate or heparin. Studies show that the region 2 genes of the K5 gene cluster control heparosan biosynthesis [73]. By optimizing heparosan production through genetic engineering, fermentation and metabolic engineering by the expression of a lyase gene and the overexpression of two glycosyltransferases genes (kfiA and kfiC) heparosan yields can be increased [74]. For example, E. coli K5 produces 15 g/L heparosan in a defined medium using exponential fed-batch glucose supplement with oxygen enrichment [75]. A recombinant bacterial strain E. coli K-12 that contains the cloned genes kfiABCD can express heparosan of different molecular weights [15]. Non-pathogenic E. coli BL21 can be engineered to produce both chondroitin and heparosan although the yields are lower than obtained from E. coli K4 and K5. A competitive relationship between E. coli K-12 cell growth and heparosan production has been observed in a mineral culture medium under fed-batch cultivation [76]. This competitive relationship was also found in the biosynthesis of hyaluronic acid [77]. Bacillus subtilis and B. magaterium can also be prepared to express heparosan, chondroitin and hyaluronic acid. It was reported that heparosan synthase encoding genes from E. coli K5 (kfiA and kfiC), the chondroitin pathway genes (kfoA and kfoC) and the Streptococcus hasA gene were cloned and inserted into Bacillus to express the corresponding GAG analogues [78,79,80]. For example, Streptococcus hasA gene had been expressed in B. subtilis A1645 with a mineral salts medium, resulting in the production of hyaluronic acid [78].


One contribution of 12 to a theme issue ‘Synthetic glycobiology’.

Published by the Royal Society. All rights reserved.


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Synthesis and medical applications of oligosaccharides

Our understanding of the different glycoconjugates present on cells, proteins and entire organisms is lagging far behind advances in genomics and proteomics. Carbohydrate sequencing and the synthesis of defined oligosaccharides are two key technologies that have contributed to progress in glycomics research. Synthetic tools and high-throughput experiments such as carbohydrate arrays are beginning to affect biological research. These techniques are now being applied to the development of carbohydrate-based diagnostics, vaccines and therapeutics.

Glycomics is the study of the structural and functional aspects of the various glycoconjugates present on proteins, cells and, in some cases, entire organisms. Compared with its counterparts, genomics 1 and proteomics 2 — which deal with nucleic acids and proteins, respectively — the field of glycomics is much less developed. The types of bio-oligomers and biopolymers covered by the term glycoconjugate are diverse. Different natural products such as glycoproteins, glycolipids, glycosaminoglycans and glycosylphosphatidylinositol anchors are summarily known as sugars 3 . Even when only the oligosaccharide chains are considered, studies of processes involving carbohydrates are complicated because, unlike peptides and oligonucleotides (which are usually linear), many oligosaccharides have branched structures. Because they are not under direct genetic control, glycoconjugates are typically heterogeneous. Amplification methods — such as the polymerase chain reaction (PCR) for nucleic acids or bacterial expression systems for protein production — do not exist for glycoconjugates. Consequently, until recently, isolation of carbohydrates was the only way to procure these natural products 3 . Overall progress in glycobiology has suffered from a lack of tools such as those that are readily available for studying nucleic acids and proteins, including automated sequencing 4,5 , automated synthesis 6,7 , high-throughput microarray screening, and detailed structure elucidation, including X-ray analyses. Carbohydrate synthesis 8 methods are time-consuming and practised mostly by specialized laboratories 9,10,11 . The products of such syntheses have aided the development of modern sequencing methods 12,13 .

Improved synthetic protocols for solution-phase oligosaccharide synthesis 9,10,11 , as well as the use of automated carbohydrate assembly, have provided more straightforward access to usable quantities of pure oligosaccharides. Synthetic carbohydrates have allowed the development of chemical approaches to glycomics that provide a molecular picture of biological processes involving carbohydrates. Synthetic sugars are beginning to be used in the development of diagnostic tests, vaccines and carbohydrate therapeutics. Some of these exciting advances in this rapidly growing field are highlighted in this review.

The usual first step when investigating a biological signal-transduction event is to establish which molecule is responsible for the activity. If the biomolecule is a nucleic acid or a protein, an answer can be obtained quickly because there are reliable, automated sequencing techniques. If, however, a carbohydrate is involved, sequencing is less straightforward. Carbohydrate analysis has improved tremendously in the past two decades 12,13,14,15,16,17,18 , but there is still no single method to determine the composition of all glycoconjugates. Given the structural diversity of glycoconjugates, different analytical approaches may persist for the analysis of different classes of sugar.

Synthesis of carbohydrates

Once a particular oligosaccharide (or a set of oligosaccharides) has been identified as being responsible for a biological effect, it often has to be synthesized in order to establish or confirm its structure assignment. In addition, defined oligosaccharides and their analogues are key tools for biochemical, biophysical and biological studies. The synthesis of carbohydrates has been pursued for more than a century, and many oligosaccharides can now be synthesized, albeit with considerable effort 6,9,10,11 . Specialized laboratories synthesize oligosaccharides using processes that may take months to years owing to the structural complexity of carbohydrates. This situation is reminiscent of the solution-phase total syntheses of peptides and DNA that were practised before the advent of automated solid-phase synthesis. A host of improvements has accelerated solution-phase oligosaccharide syntheses 19 .

The one-pot synthesis strategy aims to automate the planning of oligosaccharide synthesis 20 . On the basis of the relative reactivity of hundreds of monosaccharide 'building blocks', a computer program known as Optimer selects the appropriate building blocks, as well as the order in which they should be added to the reaction vessel during oligosaccharide assembly (Fig. 1a). The Optimer method works well for oligosaccharides of up to six units, but requires an extensive set of building blocks. An automated instrument known as the 'Golgi apparatus' 21 has been used for enzymatic syntheses 22 , building on the superb regiospecificity and stereospecificity of glycosyltransferases that avoid the need for protective groups to assemble oligosaccharides 23 (see page 1008). Engineered organisms such as yeast have been used for the production of N-glycosylated proteins 24,25 . A library of glycoengineered cell lines is expected to yield a plethora of specific glycovariants that have previously been unobtainable in mammalian cells.

a, Automated solution-phase synthesis using the Optimer-based one-pot approach for a pentasaccharide. A computer program selects appropriate monosaccharide building blocks according to their relative reactivity values in order to achieve best yields for oligosaccharide assembly. b, Automated solid-phase assembly using five monosaccharide building blocks on a polymer resin (black circle), attached by means of a linker. The cycle — consisting of activation and deprotection steps — is performed five times for the assembly of a pentasaccharide. Finally, the linker is cleaved to procure the desired oligosaccharide. Coloured triangles and squares represent different sugar monomer building blocks. P, P′, temporary protecting groups R, hydrocarbon residue to be functionalized X, leaving group.

The development of a fully automated oligosaccharide synthesis process by chemical means has been viewed with considerable scepticism in light of the complexity of carbohydrates, the large number of monomers needed and possible connections between monosaccharide units. Recent bioinformatics studies explored the diversity of mammalian oligosaccharide connectivities using a comprehensive database of isolated N-linked and O-linked glycans, and glycosphingolipids (D.B.W., R. Ranzinger, S. Herget, A. Adibekian, C.-W. von der Lieth and P.H.S., unpublished observations). Data mining has revealed that nature uses only a small proportion of the theoretically possible connections, so the complexity of glycospace (that is, the body of different structures that can, in principle, be constructed) is significantly reduced. According to the results of this database analysis (D.B.W., R. Ranzinger, S. Herget, A. Adibekian, C.-W. von der Lieth and P.H.S., unpublished observations), just 36 building blocks are needed to assemble 75% of known mammalian oligosaccharides by chemical methods. The challenge of synthesizing these 36 building blocks is surmountable when considering that about 100 different amino-acid monomers are commercially available for peptide synthesis.

Each monosaccharide building block is synthesized at a multi-gram scale and can be used for the assembly of various targets. The feasibility of assembling most carbohydrates using a limited, defined set of building blocks is still questioned, but example structures of increasing complexity are being reported. Temporary protective groups mark sites of further glycosylation, and permanent protective groups mask hydroxyl groups to be unveiled at the end of the synthesis. Besides controlling regioselectivity by orthogonal protective groups to account for branching of the carbohydrate chain, the stereochemistry at the anomeric carbon must be controlled. Placement of participating protective groups at the C2 hydroxyl or amine groups ensures the formation of trans-glycosidic linkages, whereas non-participating groups are needed for the preferential installation of cis-glycosides.

Automated solid-phase oligosaccharide synthesis (Fig. 1b) has been developed from insights gained from oligopeptide and oligonucleotide assembly 26 . The first building block is added to a polystyrene resin equipped with an easily cleavable linker containing a free hydroxyl group 27 . An activating agent induces couplings involving glycosyl phosphate and glycosyl trichloroacetimidate building blocks 26 . Unlike oligonucleotide and peptide couplings, glycosidic bond formation occurs mostly at low temperatures and requires a reaction chamber that can be cooled. Excess building blocks (that is, a 5–10-fold molar excess, sometimes applied twice) are added to the chamber for each coupling. Mass action to drive coupling reactions to completion and to achieve high yields is also common to peptide and oligonucleotide syntheses. Washing and filtration remove any side products or remaining reagents before selective removal of a temporary protective group readies the next hydroxyl group for subsequent coupling. Coupling efficiencies can be assessed by spectrometric read-out after protecting-group removal when temporary protecting groups that absorb ultraviolet radiation, such as 9-fluorenylmethyloxycarbonyl (Fmoc), are used 28 . Originally, this coupling–deprotection cycle was automated using a converted peptide synthesizer 26 . An automated oligosaccharide synthesizer prototype with parallel synthesis capability is currently being tested.

After completion of the oligosaccharide sequence, the fully protected product is cleaved from solid support. After global deprotection, the oligosaccharide is purified and its structure verified. A series of increasingly complex oligosaccharides has been assembled, each within 1 day or less, using the automated oligosaccharide synthesizer. This compares favourably with the weeks to months taken using solution-phase methods 28 . Initial syntheses contained only the synthetically less challenging trans-glycosidic linkages, but cis-glycosides such as α-galactoses have now also been selectively incorporated 29 .

At present, automated oligosaccharide synthesis resembles the early days of automated peptide and oligonucleotide assembly: many carbohydrate structures, both simple and complex, can be synthesized by automation. The problems and drawbacks — such as the excess of buildings blocks used, the difficulties in incorporating certain monosaccharides such as sialic acid, and the double bond in the linking moiety that restricts the deprotection conditions — have been recognized. Some of these limitations have now been addressed, but certain challenges remain. Although commercially available monomeric building blocks are quite expensive, it seems likely that the cost of these reagents will decrease with increasing demand. One of the reasons why automated solid-phase synthesis of oligosaccharides is not currently performed by a larger number of groups is the fact that the synthesis instrument is not yet commercially available. However, the strength of the chemical approach to incorporating unnatural linkages nicely complements the evolving enzymatic technologies. A combination of improved isolation and purification strategies of naturally occurring carbohydrates, enzymatic approaches, the use of engineered organisms and chemical synthetic approaches such as automated solid-phase synthesis is expected to provide scientists with rapid access to defined oligosaccharide libraries for glycomics investigations in the near future.

Several methods have been established to study the interactions of carbohydrates with various other molecules (Table 1). The DNA microarray has been a key tool in genomics research 30 , and protein arrays are used to identify protein–protein interactions and potential inhibitors 31 . Similarly, carbohydrate microarrays have been used in glycomics research to examine the interactions of carbohydrates with other molecules. The chip-based format of microarrays offers important advantages over common screening techniques such as enzyme-linked immunosorbent assays (ELISAs), because several thousand binding events can be screened on a single glass slide and only minuscule amounts of analyte and ligand are required. Assay miniaturization is particularly suitable for glycomics, because access to pure oligosaccharides is the limiting factor. The first carbohydrate microarrays relied on isolated saccharides that were non-covalently attached to membranes 32,33 . A flurry of methodological studies evaluated different aspects of microarray design and adopted oligonucleotide and protein array techniques for carbohydrate chips (Fig. 2). Synthetic monosaccharides and oligosaccharides were covalently attached via different linkers to glass 34 , plastic 35 and gold surfaces 36 , or placed on beads in fibre-optic wells 37 . Initial proof-of-principle studies focused on known interactions between lectins (carbohydrate-binding proteins) and sugars. Current screening efforts rely on carbohydrate arrays in which chemically or enzymatically synthesized and isolated oligosaccharides with a linker on the reducing terminus are covalently attached to glass slides 38 . Standard DNA printing and scanning equipment is used to produce and analyse the carbohydrate microarrays 39,40 .

a, Carbohydrate microarrays can be constructed from synthetic or isolated oligosaccharides that contain a primary amine. The glycans are covalently attached to the glass surface of a microscope slide that has been functionalized with a reactive group (for example, N-hydroxysuccinimide, NHS). b, Incubation of the carbohydrate microarray with a protein, antibody or cell that has been labelled (for example, with a fluorescent group) can be used to determine which oligosaccharide binds to that protein, antibody or cell. In this particular example, the protein represented by a blue wedge specifically binds to the oligosaccharide represented by the yellow triangle, but not to the other oligosaccharides that appear on the glass slide.

Initially, carbohydrate–protein interactions important to the process of HIV infection were analysed, and epitope mapping of HIV-related antibodies was performed 40,41 . The knowledge gained from the microarray experiments was essential to efforts directed at the creation of carbohydrate-based HIV vaccine candidates 42,43 (see page 1038).

During the past 3 years, a host of carbohydrate–protein interactions has been studied using oligosaccharide arrays 44 . National and regional consortia such as the Consortium for Functional Glycomics (NIH, USA) and the ETH Zürich Glycomics Initiative are making this technology widely accessible to life science researchers.

Carbohydrate microarrays can be used to address the interactions of sugars with other types of molecule, as well as entire cells. Carbohydrate–RNA interactions were screened by incubating labelled RNA with aminoglycoside microarrrays 45 . Mechanisms responsible for antibiotic resistance were studied using these arrays together with resistance-causing enzymes 46 .

The binding of cells to microarray surfaces allows the detection and typing of bacteria in blood 47 . The interaction of eukaryotic cells with carbohydrate arrays has also been demonstrated 48 . The oligosaccharide binding preferences of different types of avian and human influenza strains can be determined using carbohydrate microarrays 49 .

The structure–activity relationship of glycosaminoglycan polysaccharides, including heparin and chondroitin sulphate, is poorly understood. Carbohydrate microarrays containing synthetic 50 or isolated 51 heparin oligosaccharides have served to identify specific sequences recognized by different fibroblast growth factors 52 . Immobilization of a series of chondroitin tetrasaccharides has aided the identification of a novel tumour necrosis factor-α antagonist 53 and the exact chondroitin sequences bound by proteins 54 .

In addition to the application of carbohydrate arrays to the study of biomolecular interactions, oligosaccharide microarrays are beginning to be used as diagnostics and to conduct epidemiological studies. Hundreds of human sera have been screened for antibodies against the malaria toxin glycosylphosphatidylinositol (GPI) anchor 55 , and a correlation between the presence of specific antibodies and resistance to severe malaria has been established (F. Kamena, M. Tamborrini, X. Liu, G. Pluschke and P.H.S., unpublished observations). The search for serological markers of autoimmune diseases has yielded results for Crohn's disease 56 . Epitope mapping of antibodies against tumour-associated antigens is also feasible 57 .

The presence of certain oligosaccharide structures in a glycoprotein sample can be determined using arrays of immobilized lectins 58 . These carbohydrate-binding proteins recognize terminal saccharide units and have been used to analyse the dynamic bacterial glycome 59 .

Oligosaccharide therapeutics

Given the prevalent role of carbohydrates in a wide range of biological processes it may seem surprising that there are few carbohydrate-based therapeutics and diagnostics on the market. In addition to monosaccharide-inspired drugs such as the influenza virus treatment Tamiflu 60,61 (oseltamivir phosphate Roche) two blockbuster drugs, acarbose (Precose, Glucobay Bayer) and heparin, stand out. Both oligosaccharides were derived by isolation and reached the clinic before a detailed structure–activity relationship had been established. In addition, aminoglycosides — naturally occurring pseudo-oligosaccharides — have been used clinically to treat infectious diseases induced by a variety of Gram-negative bacteria 62 . The antibiotic activity of aminoglycosides is due to their inhibition of protein synthesis, which results from their binding to bacterial ribosomes 62 .

The oldest carbohydrate-based drug is isolated from animal organs and has been used clinically as an antithrombotic agent since the 1940s. Heparin activates the serine protease inhibitor antithrombin III, which blocks thrombin and factor Xa in the coagulation cascade 63 . This drug is a highly heterogeneous mixture of polysaccharides and is associated with severe side effects, including heparin-induced thrombocytopenia, bleeding and allergic reactions. Chemically or enzymatically fragmented heparins (low-molecular-weight heparins, LMWHs) are also heterogeneous, but are more bioavailable, with a longer half-life, a more predictable anticoagulant activity and fewer side effects in vivo.

After the specific pentasaccharide responsible for the anticoagulant property was identified in the early 1980s (ref. 64 Fig. 3a), a herculean effort lasting more than 10 years was begun to establish a structure–function relationship using synthetic oligosaccharides 64 . As a result of this drug-development effort, a synthetic pentasaccharide known as Arixtra (fondaparinux sodium GlaxoSmithKline) has been available since 2002 (ref. 65). However, Arixtra does have some clinical shortcomings, such as an exceedingly long half-life in vivo and little to no dose-dependent activity in certain indications 66 . Thus, LMWHs still have the highest market share of all antithrombotics, and the need for additional synthetic heparin molecules with specific activities persists.

a, A pentasaccharide sequence of heparin. This sequence is responsible for binding to antithrombin III. b, Synthetic spore surface tetrasaccharide of B. anthracis. This molecule was used for the generation of anticarbohydrate antibodies to detect anthrax spores and is currently being used in vaccine development.

Recent advances in heparin sequencing 16 , heparin synthesis 67,68,69,70 and heparin microarray technology 50,51 have provided the tools to identify specific sequences or sequence families that interact with proteins such as chemokines. The chemical synthesis of a broad range of heparin analogues should allow researchers to study the molecular mechanism of angiogenesis and to modulate wound healing and other medically relevant processes (see page 1030).

Carbohydrates such as starch and sucrose are principal components of food, and have to be enzymatically broken down in the intestinal tract. Acarbose 71 , a pseudo-oligosaccharide of microbial origin, is produced by fermentation. This α-glucosidase and α-amylase inhibitor interferes with and regulates intestinal carbohydrate digestion, controls the rate of absorption of monosaccharides and influences the intermediary carbohydrate metabolism. It is used to treat type 2 diabetes.

Carbohydrate-based vaccines

The cell surfaces of bacteria, parasites and viruses exhibit oligosaccharides that are often distinct from those of their hosts. Specific types of glycoconjugate are often more highly expressed on the surface of tumours than on normal cells 72 . Such cell-surface carbohydrate markers are the basis for carbohydrate-based detection systems and vaccines. An immune response against the carbohydrate antigens that results in the death of target cells is required for a carbohydrate-based vaccine. Such vaccines have been widely used against a host of diseases for several decades 73 . The carbohydrate antigens for antibacterial vaccines were isolated from biological sources. Recently, intense efforts focused on the use of defined carbohydrate antigens that are synthesized rather than isolated. A carbohydrate-based approach has also been pursued for anticancer vaccine candidates 74,75,76 (see page 1000). However, one of the early carbohydrate-based anticancer vaccine candidates recently failed in a Phase III clinical trial.

Antibacterial vaccines

Polysaccharide capsules, glycoproteins or glycolipids cover the cell surfaces of many bacteria. Capsular polysaccharides are either homopolymers or made up of between two and six repeating sugar units. Capsular polysaccharides elicit type-specific protective immune responses in adults but not in infants, who do not respond with antibodies that confer protection. Conjugate vaccines consisting of a carbohydrate antigen and an immunogenic protein can overcome this immunogenicity problem and produce high titres of protective antibodies.

Improved analytical tools have helped to identify the exact chemical structure of carbohydrate antigens and have aided the development of new vaccines. Several vaccines based on purified capsular polysaccharides or on neoglycoconjugates are now commercially available, such as vaccines against Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenza type b (Hib) and Salmonella typhi 77 . Meningitis caused by Hib has essentially been eradicated in areas where national vaccination programmes using protein conjugate vaccines have been implemented.

Vaccine development could benefit greatly from the new glycomics technologies. The identification of specific oligosaccharide antigens has been aided substantially by sequencing and carbohydrate arrays. The procurement of defined oligosaccharides using improved solution- and solid-phase methods has become fast enough to be used reiteratively in drug-development efforts.

A synthetic oligosaccharide-based conjugate vaccine is now used in Cuba, where the large-scale synthesis, pharmaceutical development and clinical evaluation of a conjugate vaccine composed of a synthetic capsular polysaccharide antigen of Hib was achieved. Long-term protective antibody titres compared favourably with products prepared with the Hib polysaccharide extracted from bacteria 78 .

A tetrasaccharide has been discovered on the surface of spores of the biowarfare agent Bacillus anthracis 79 . Once the durable form of the pathogen has been inhaled it will kill most victims if treatment is not commenced immediately. Synthesis of a species-specific tetrasaccharide antigen 80,81 (Fig. 3b) allowed the production of antibodies that specifically recognize B. anthracis in the presence of the closely related opportunistic human pathogen Bacillus cereus 82 . Challenge experiments to create a conjugate vaccine against anthrax are ongoing.

Antiparasite vaccines

Like bacteria, many parasites have unique glycoconjugates on their surfaces. The specific carbohydrates may serve as a starting point for the creation of conjugate vaccines, but efforts towards this goal have been hampered by the fact that the parasites are very difficult to culture and because glycoconjugates cannot be obtained in pure form or sufficient quantity by isolation.

Plasmodium falciparum is the most pathogenic of the single-celled parasites of the genus Plasmodium that are responsible for malaria. Malaria infects 5–10% of humans worldwide and kills more than 2 million people each year. Infected mosquitoes transmit the parasite, which leads to the common symptoms of chills and fever. Drug resistance is a growing problem at a time when there is still no effective vaccine.

P. falciparum expresses a large amount of GPI on its cell surface 83 . This glycolipid triggers an inflammatory cascade that is responsible for much of malaria's morbidity and mortality. When a protein conjugate of a synthetic hexasaccharide GPI malaria toxin was administered to mice before infection, this resulted in a highly reduced mortality rate of only 10–20%, compared with 100% without vaccination 55 . Cross-reactivity of the antibodies with human GPI structures was not observed owing to the differences between human and P. falciparum GPI. Immunization of mice did not alter the infection rate or overall parasitaemia, indicating that the antibody against the GPI neutralized toxicity without killing the parasites 55 .

Preclinical studies involving protein conjugates of synthetic GPI antigens are currently underway. To support such vaccine development efforts, methods for the large-scale synthesis of oligosaccharide antigens have been developed by taking advantage of the latest advances in carbohydrate synthesis technology. Very small amounts of synthetic antigen (10 −9 –10 −7 g per person) are required, and the production of several kilograms of antigen per year will suffice.

Leishmaniasis, which is caused by another protozoan parasite, is transmitted by sandflies and affects more than 12 million people worldwide. Leishmania resides in macrophages, making them difficult to treat. In a search for a potent vaccine, the lipophosphoglycans (LPGs) 84 that are ubiquitous on the cell surfaces of the parasites and are composed of a GPI anchor, a repeating phosphorylated disaccharide and different cap oligosaccharides became a target. The cap tetrasaccharide has been the focus of efforts towards a conjugate vaccine based on a synthetic antigen. The branched tetrasaccharide was assembled by automated solid-phase synthesis 85 and conjugated to a virosomal particle to enhance immunogenicity. These highly immunogenic conjugates yielded antibodies that selectively recognize parasite-infected livers 86 . Challenge studies in an animal model are currently underway.

Recent advances and future development

For many years the lack of tools for studying glycobiology prevented biologists and medical researchers from addressing research problems that involve carbohydrates. During the past decade, sequencing and synthesis technologies that are commonly used to study nucleic acids and proteins have become available for glycomics as well. Now, carbohydrate sequencing of glycoconjugates is often possible even though sample preparation is complicated by carbohydrate microheterogeneity and the absence of amplification procedures. Automated solid-phase synthesis, improved methods for solution-phase oligosaccharide assembly, enzymatic methods and the use of engineered cells have complemented each other, allowing oligosaccharide synthesis to take a big step forward by granting access to different classes of glycoconjugate. In turn, these methods have helped procure oligosaccharides and their non-natural analogues for the creation of high-throughput screening methods such as carbohydrate arrays.

The identification of specific oligosaccharides, by sequencing followed by comparison with synthetic oligosaccharides, has yielded insight into the interactions of carbohydrates and proteins. Oligosaccharide involvement at key positions of signalling pathways is beginning to emerge and a molecular understanding of carbohydrate binding to proteins is evolving. Detailed structural studies — including studies of protein–carbohydrate interactions — using X-ray crystallography will become commonplace in the near future. Further improvements in the methods by which oligosaccharides are sequenced and synthesized will be needed to make their routine use possible for non-specialists.

A better understanding of the biological roles of carbohydrates and improved sequencing and synthesis techniques are beginning to influence the design of diagnostic and therapeutic approaches. Carbohydrate arrays help to define new disease markers by screening the sera of patients. Bacterial and viral detection and typing can be achieved using carbohydrate microarrays. Synthetic access to oligosaccharides of infectious agents that are hard to culture and isolate (for example, B. anthracis and P. falciparum) facilitates antibody production for specific detection of these pathogens. These anticarbohydrate antibodies may become important for passive immunization. The first conjugate vaccine candidates containing synthetic oligosaccharide antigens are reaching preclinical and clinical trials against bacterial (for example, Hib), viral (for instance, HIV) and parasitic (for example, malaria and leishmaniasis) infections. The trend to produce defined vaccine antigens using chemical and enzymatic methods, as well as engineered cells, is likely to increase, and synthetic vaccines are expected to complement already existing vaccines containing purified polysaccharides.

As our understanding of carbohydrate involvement in signalling cascades — in particular of those that involve glycosaminoglycans — expands, carbohydrate-mediated processes will become the target of drug-development efforts using small organic molecules. Glycomics has just gone beyond the initial proof-of-principle studies for diagnostics and therapeutic candidates. Improved tools and a better molecular understanding should convince those biologists and medical researchers who previously avoided carbohydrates to address questions involving this class of molecule. The excitement of glycomics is just beginning, with many discoveries to be made and applications to be developed.

The financial support provided by TUBITAK through project 114M239 is gratefully acknowledged.

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Keywords: EPS, microbial production, exopolysaccharides, systems biology, xanthan, levan, pullulan, dextran

Citation: Ates O (2015) Systems Biology of Microbial Exopolysaccharides Production. Front. Bioeng. Biotechnol. 3:200. doi: 10.3389/fbioe.2015.00200

Received: 20 August 2015 Accepted: 30 November 2015
Published: 18 December 2015

Alvaro R. Lara, Universidad Autónoma Metropolitana-Cuajimalpa, Mexico

Alessandro Giuliani, Istituto Superiore di Sanità, Italy
Adelfo Escalante, Universidad Nacional Autónoma de México, Mexico

Copyright: © 2015 Ates. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.