Glycan synthesis is a central theme in modern chemical biology and is fundamental to translating structural glycomics to functional interrogation, in that it affords access to pure, well-defined carbohydrate molecules. In contrast to other templated biopolymers, glycans are formed by non-templated enzymatic pathways. As such, the endogenous glycome is microheterogeneous and insufficiently pure for mechanistic interrogation. Synthetic methods are therefore required for obtaining homogeneous glycoforms. These are both chemical and enzymatic in nature, but are driven by the same fundamental principles, i.e., stereochemical control, regioselective protection and reaction orthogonality. Chemical approaches are based on the use of protecting groups to mask the reactive hydroxyls and to direct glycosidic bond formation. In contrast, enzymatic methods utilize the inherent regio-, stereo- and substrate specificities of glycosyltransferases to install monosaccharide residues one at a time under mild aqueous conditions, in chain elongation reactions. Developments in automated solid-phase synthesis and one-pot multienzyme systems have made the rapid synthesis of complex structures a practical reality. The applications of synthetic glycans are varied and include defined antigens for vaccines, tools to probe host-pathogen interactions and glycoengineered therapeutics with enhanced pharmacokinetics. In addition, glycans also serve as functional materials for nanoarchitectonics. Glycan synthesis thus moves the glycome from a descriptive catalog of structures to a toolkit of synthetic manipulables to probe structure-function relationships and design carbohydrate-based therapeutics against disease.
Synthetic glycans are chemically or enzymatically synthesized carbohydrates. The direct chemical synthesis of glycan structures is necessary because glycans, as produced naturally, are found in mixtures rather than as defined structures amenable to in-depth studies of their biological function. Classical approaches of carbohydrate chemistry employ synthetic routes in solution, using multiple protection and deprotection steps. They were time consuming and often involved low-throughput processes. During each synthetic step the products and intermediates must be separated and purified. In recent years this field has advanced and solid-phase synthesis procedures have become more common. In this type of synthesis, a building block is attached to a solid, insoluble support and a series of glycosylation reactions can be performed in rapid succession. The synthetic glycan is typically cleaved from the solid support and deprotected at the end of the synthesis. The solid phase method has been optimized for efficiency, and synthetic glycans can now be constructed with relative ease in a manner similar to peptide synthesis. In addition to chemical methods, glycan synthesis by glycosyltransferases has become increasingly important. Recombinant glycosyltransferases can be used to produce glycosidic linkages in a regio- and stereo-selective fashion. In addition, enzyme catalyzed reactions are in principle more environmentally friendly, and in practice can often be easier to scale-up. Chemical and biochemical methods have been combined to produce complex glycans (chemoenzymatic synthesis). For example, chemically synthesized glycans can be further elongated by a series of glycosyltransferases. Glycan synthesis now provides a powerful platform to produce key materials needed for vaccine development, diagnostic tools and therapeutics. The materials produced by glycan synthesis have been critical to translating glycomic research to the clinic.
Synthetic glycans are an important tool in glycobiology because they can supply a pure and structurally homogeneous material to overcome a major obstacle. Glycans are not made according to a template, and their structures are the result of a combinatorial process. It is often not possible to isolate pure glycans from natural sources in amounts suitable for study. Glycan synthesis has, therefore, solved this problem in many cases, and synthetic glycans are often used for research. In vaccine development, synthetic glycans have been used to prepare defined immunogens such as bacterial capsular polysaccharides or tumor associated carbohydrate antigens, in order to elicit protective antibodies without contamination from immunosuppressive contaminants from whole organism extracts. Synthetic glycans have also been used to help define minimal antigenic epitopes for T-cell glycopeptide vaccines. In conjugate vaccines, the glycan hapten is conjugated to a carrier protein, and it is important that the conjugation is well defined and uniform across the vaccine batch. Glycan synthesis is also a powerful tool to study host recognition of pathogens, for example by placing pathogen-associated glycans on microarrays or cell surfaces in order to identify host binding partners or pathways of infection. In drug development, glycans can be synthesized with optimized properties (e.g. added sialic acids for longer half-life in serum, changed branching, or sulfation to tune affinity or clearance). Synthetic glycans can be used as standards for quantitative analysis and for glycomic profiling in diseases, and rare or unnatural glycans can be made as new starting points for the development of glycomimetics as antagonists of pathological lectins. Many of the fundamental roles of glycans in development, immunity, and pathology could not be understood without the availability of synthetic glycans.
Glycan synthesis is underpinned by a number of concepts that provide the framework for the selective construction of glycosidic bonds with defined stereo- and regiochemistry. Protecting group strategy is a major consideration since carbohydrate monomers are equipped with multiple hydroxyl groups of comparable reactivity and chemoselective protection is necessary to prevent side reactions from forming bonds at undesired positions. Acetyl, benzoyl and benzyl ethers are among the most common protecting groups, each of which have orthogonal deprotection conditions allowing for stepwise elaboration of a target structure. Stereocontrol is another key consideration: the α/β-configuration of a glycosidic linkage can be controlled by neighboring group participation from ester substituents on C2 (trans-glycosides) or solvent effects/anomeric effect in non-participating donors. Choice of glycosyl donor, whether trichloroacetimidate, thioglycoside, or phosphate, for example, also impacts the conditions for activation and coupling efficiency and each donor class exhibits different reactivity. In enzymatic synthesis, donor specificity of glycosyltransferases and their requirement for an activated sugar nucleotide (UDP-GlcNAc, CMP-sialic acid, GDP-fucose) must be matched with the acceptor substrate tolerances of the enzyme. In chemoenzymatic synthesis these paradigms are mixed and matched such that chemical methods are used to construct core structures followed by enzymatic extension for the regioselective installation of terminal modifications. Automated solid-phase synthesis is based on the concept of a solid support, with cleavable anchors allowing for iterative cycles of glycosylation without intermediate purification. One-pot multienzyme systems are an example of process integration that combines multiple glycosyltransferases in a single vessel to streamline production. These concepts and others are prerequisites for designing efficient synthetic routes from simple disaccharides to more complex branched polysaccharides, and for systematically interrogating glycan structure-function relationships.
Glycans can be synthesized by using the chemical, enzymatic, or chemoenzymatic strategy. Chemical synthesis depends on protecting group manipulations, activation of glycosyl donors, and generation of glycosidic linkages in a predefined stereochemical outcome. This is the most flexible approach in synthesizing unnatural or highly-modified structures. Enzymatic synthesis takes advantage of the regioselectivity and stereoselectivity of glycosyltransferases and glycosidases under mild aqueous reaction conditions to synthesize native linkages with high efficiency. This approach, however, requires activated sugar nucleotide donors and is often limited by the availability of enzymes with the desired activity and broad substrate scope. The chemoenzymatic strategy combines the chemical and enzymatic strategies to take advantage of the flexibility and diversity of chemical synthesis and the regioselectivity and stereoselectivity of enzymatic reactions. Chemoenzymatic synthesis has thus become the leading strategy for the synthesis of complex targets. In general, chemical synthesis is used to build up the core structure followed by an enzymatic step to append the more sensitive terminal decorations that are not stable under chemical conditions. The selection of the strategy for glycan synthesis is usually determined by the complexity of the target, the level of stereochemical control needed, the scalability, and the intended use, with new automated strategies and one-pot cascade reactions emerging for both chemical and enzymatic synthesis to streamline these processes and reduce the need for intermediate purifications.
Chemical glycan synthesis is based on the use of protecting groups to selectively mask the multiple hydroxyl groups of the carbohydrate monomers, allowing for regioselective activation and coupling. The general approach starts with per-acetylation or benzoylation to protect most hydroxyl groups, followed by selective deprotection of the anomeric center to install the leaving group, usually a trichloroacetimidate, thioglycoside, or phosphate ester. The donor is then activated under Lewis acidic conditions (generally trimethylsilyl triflate or boron trifluoride etherate), to generate an oxocarbenium ion, which can be trapped by a nucleophilic acceptor alcohol, leading to the formation of the glycosidic bond. Stereocontrol is exerted through neighboring group participation from ester substituents at C2 (to give trans-glycosides), or through solvent and the anomeric effect in non-participating systems. The coupling of smaller oligosaccharide blocks, which are often prepared independently, to larger targets in convergent assembly strategies is a common way of reducing the number of synthesis steps on advanced intermediates. The downside to this flexibility is that the overall yields tend to be low because of the multiple protection-deprotection sequences involved, anomeric stereocontrol can be challenging for certain sugar combinations, and the solubility of the protected intermediates in organic solvents is often poor. Ether-type solvent systems have been introduced more recently, leading to increased coupling yields for large substrates, while also adding N-acetyl protection as NAc2 to increase reactivity by interrupting intramolecular hydrogen bonding. These and other new approaches continue to expand the chemical toolbox to provide access to structures such as core-fucosylated N-glycans that remain challenging targets for purely enzymatic synthesis.
Summary of biocatalytic methods used to deliver building blocks appropriate for chemical glycan synthesis.1,5
Chemical synthesis of glycans has been long established, but the regio- and stereospecificity of glycosyltransferases have also been used to generate carbohydrates by "templated" enzymatic synthesis under aqueous, mild, and protecting group-free conditions. Activated sugar nucleotide donors (UDP-GlcNAc, GDP-fucose, CMP-sialic acid) are used in these systems; each is recognized by a family of transferases with specific acceptor substrate specificities and defined linkage formation. In one-pot multienzyme (OPME) systems, a mixture of transferases is combined in one reaction vessel, and sugar nucleotides are recycled in situ by pyrophosphatases and other cofactor recycling enzymes, without requiring purification of the expensive donor nucleotides. Benefits of these methods include atom economy and limited waste production, orthogonal synthesis conditions, and regio- and stereospecific linkages that are either challenging or inaccessible to form by chemical means, and native linkages in particular. Drawbacks of the enzymatic methods have included limited access to unnatural analogs due to narrow glycosyltransferase substrate tolerance, and the difficulty of expressing active recombinant enzymes. However, discovery of promiscuous transferases with relaxed substrate specificities has allowed access to a broader chemical space, such as modified sugars with azido, fluoro, or amino substituents for subsequent functionalization by click or other chemistry. Immobilization of enzymes on solid supports or within hydrogels has improved stability and reusability of the enzymes, and flow-based systems have also been demonstrated for these enzymatic glycan production systems. Mutant glycoside hydrolases that lack hydrolytic activity, referred to as glycosynthases, have been engineered to use activated sugar fluorides as donors for enzymatic glycan synthesis, with no nucleotide requirement. Enzymatic synthesis of glycans is especially useful for complex, biologically relevant targets that are difficult to synthesize chemically and for which sensitive functional groups must be preserved, such as sialylated Lewis antigens and sulfated glycosaminoglycan fragments.
In a chemoenzymatic synthesis, chemical and enzymatic reactions are combined. In many cases, this allows taking advantage of the strengths of both synthetic methodologies to rapidly build complex glycans. Chemical synthesis is used to construct the scaffold or introduce non-natural functionalities that are not tolerated by enzymes, and enzymatic extension is then used to add more labile terminal modifications such as sialic acid or fucose residues. The synthesis of thioglycoside building blocks for automated glycan assembly often uses a chemoenzymatic route: the galactose moiety is installed by enzymatic transglycosylation using a galactosyltransferase and a thiol-containing acceptor, which is then chemically acetylated to afford a protected donor for polymerization. In a one-pot chemoenzymatic cascade, a sequence of glycosyltransferases are used with minimal purification in between, made possible by in situ regeneration of the sugar nucleotides and removal of inhibitory byproducts using a set of auxiliary enzymes. Flow chemistry platforms enable the integration of enzymatic and chemical reactions, with enzymes immobilized on solid supports and substrates flowing through, to construct continuous integrated processes that take simple sugars and convert them to complex products. The use of engineered enzymes with relaxed substrate specificities also enables the incorporation of modified sugars to expand the structural space accessible through chemoenzymatic synthesis. This is particularly attractive for the synthesis of glycoproteins: the peptide backbone is chemically synthesized, and homogeneous glycans are then enzymatically installed to afford glycoprotein therapeutics with defined glycoforms that can have improved stability and activity.
The synthesis of glycans is associated with several challenges. Stereoselectivity in glycosidic bond formation, or synthesis with the desired anomeric stereochemistry, is a critical issue. Since changes in reaction conditions can lead to different α/β linkages, reactions that result in mixtures can be difficult to separate and purify. In addition, the need for regioselective protection of hydroxyl groups that have similar reactivities means that multiple protecting groups are often needed. This adds steps and lowers the overall yield. Protection/deprotection of each sugar unit can be associated with side reactions, loss of products and epimerization of adjacent centers. Due to the lack of predictive models for the stereochemistry of glycosylation, reaction parameters such as solvent, temperature, activation method, and protecting group patterns typically need to be screened, which is often a tedious process. The costs and time associated with glycan synthesis are also non-trivial. Rare sugars building blocks are often expensive, and some may not be readily available from commercial suppliers. Multistep sequences can take weeks to complete in the lab. The net result is that the synthesis of even a single complex glycan can be a major undertaking, limiting the throughput and accessibility of glycan synthesis. These challenges have hindered the broader adoption of glycan synthesis as a routine tool, and significant efforts are still required in the areas of automation, catalysis, and process integration to make synthetic carbohydrates more widely accessible.
A key difficulty with chemical glycan synthesis is controlling stereoselectivity. There is a small energetic preference between α- and β-glycosidic linkages. Steric and electronic interactions also determine the anomeric configuration, but are challenging to predict. Glycosylation generally proceeds by activation of a glycosyl donor to form an electrophilic oxocarbenium ion species, which is then attacked from either face to produce a mixture of diastereomers. In trans-glycosides (linkages where the substituent and the C2 atom are on opposite sides of the ring), neighboring group participation from the C2 ester or amide protecting group generally delivers high β-selectivity by anchimerically directing the nucleophile from the opposite face of the ring. The lack of such anchimeric assistance in the formation of cis-linkages (linkages where the substituent and the C2 group are on the same side of the ring) leads to very poor stereocontrol. Effects of remote protecting groups are also known, which can bias selectivity through preorganization, but these are highly substrate-specific. A similar challenge arises in the case of 1,1'-glycosylations, where control over stereochemistry at two anomeric centers needs to be exerted simultaneously. This often leads to only modest selectivity, with mixtures of diastereomeric side-products. Choice of solvent and additives can bias the result, but general models are lacking. The combined difficulty in predicting the stereochemical outcome of glycosylation reactions thus means that extensive empirical optimization is often required for each new glycosidic linkage being introduced. Synthesis of complex glycans with multiple cis-linkages is therefore typically very laborious and low-yielding. The lack of predictive computational tools to aid in the selection of reaction conditions further leads to trial-and-error cycles and hinders throughput/scalability in cases where therapeutics require a single, homogenous glycoform.
To make glycan synthesis more efficient and scalable, strategies that minimize the number of synthetic steps, reduce reaction times, and allow for continuous processing should be adopted. Automation of glycan assembly on solid supports, inspired by solid-phase peptide synthesis, is one approach. Here, monosaccharide building blocks are added iteratively to an insoluble support, enabling rapid cycles of chain elongation without intermediate purification, substantially decreasing manual labor and increasing reproducibility. Flow chemistry, wherein reagents are continuously fed through columns of immobilized enzymes or microfluidic reactors, provides consistent reaction conditions and can be monitored in real time to fine-tune yields. Cost-effectiveness can be achieved by in situ regeneration of sugar nucleotides in enzymatic cascades to lower donor costs, as well as by using cheap starting materials and recyclable catalysts to minimize reagent expenses. Scalability may be improved by designing protecting group strategies that are more robust to extended reaction times and higher temperatures, without degradation. Standardization of protocols and building block libraries would facilitate the transfer of methods between laboratories, which is essential for the industrial production of glycoconjugates as biologics, where glycan consistency is often a regulatory requirement. While significant progress has been made, challenges in substrate scope and the need for specialized expertise persist in the field. Future advancements will likely involve the development of artificial intelligence-driven reaction optimization to predict reaction conditions that lead to the highest yield and selectivity, advancing glycan synthesis towards a reproducible manufacturing process to meet the demands of therapeutic and diagnostic applications.
Chemically and enzymatically synthesized glycans can be used to directly translate the fields of carbohydrate chemistry to therapy, vaccines, and diagnostics. Chemical and enzymatic synthesis is used to prepare homogeneous and structurally well-defined glycoforms of glycans that are not available by biological means. Synthetic glycans are often used to engineer functionally improved glycans as therapeutics and vaccines and to serve as standards for diagnostic assays. In the case of therapeutics, synthetic glycans allow for fine tuning of protein pharmacokinetics, including the introduction of sialic acids to prolong half-life, increased branching to improve receptor binding or the introduction of sulfate groups to improve clearance. Vaccine design has taken advantage of synthetic glycans to produce well-defined antigens that can drive strong and specific immune responses that are not confounded by the immunosuppressive contaminants found in natural extracts. In diagnostics, synthetic standards of glycans are used as calibration standards in order to detect disease associated glycan signatures in patient samples. In all of these cases, the production of rare or unnatural glycans using synthetic methods can provide functional capabilities not present in naturally synthesized glycans, including mimetics of natural glycans that are able to antagonize pathological lectin binding or that are used as stable probes for imaging applications. As the field of synthetic glycobiology advances, many of these applications are moving from being scientific curiosities into clinical workhorses.
Synthetic glycans are used in the development of therapeutic glycoproteins, to improve their pharmacokinetics and reduce immunogenicity. The production of glycoproteins in cell-free expression systems with homogeneous glycosylation has proven to be a viable method to produce homogeneous glycoproteins in vitro. These systems allow synthetic glycans to be engineered into therapeutic glycoproteins to improve their serum half-life and reduce dosing frequency (e.g. by adding elongated sialylation or a specific branching pattern). Chemoenzymatic glycoprotein synthesis enables the synthesis of the glycan core chemical and the addition of sensitive terminal groups enzymatically. This has been used to create afucosylated antibodies with improved antibody-dependent cell cytotoxicity towards tumor cells. Glycan remodeling is used to modify existing therapeutic proteins through the selective removal and reconstruction of surface glycans in order to improve their efficacy and safety. Synthetic glycans can also be attached to enzyme therapeutics used for enzyme replacement therapy to shield the enzyme from immune detection while targeting the mannose receptor to improve cellular uptake in metabolic disorders. The production of glycoproteins with a defined glycoform is essential for the production of reproducible glycoprotein therapeutics, an important consideration in their approval by the FDA and other regulatory agencies. As the production of synthetic glycans becomes more accessible it will also allow the distributed manufacture of such therapeutics at a lower cost, which will become an important strategy for diseases prevalent in low-income countries. Glycan synthesis therefore enables a new paradigm in therapeutic design where the carbohydrate content of a molecule can be explicitly designed to fit its therapeutic purpose instead of being a byproduct of its mode of production.
Synthetic glycans have been used to produce glycoconjugate vaccines. These vaccines use well-defined carbohydrate antigens to generate protective immunity to a variety of bacterial pathogens. They are traditionally produced by isolation of capsular polysaccharide from cultured bacteria and are associated with poor batch-to-batch consistency, laborious purification processes, and high production costs. Synthetic chemical approaches to glycoconjugate vaccines have involved assembling the oligosaccharide epitope de novo and chemically conjugating it to the carrier protein. Protein glycan coupling technology (PGCT) is a method to streamline glycoconjugate vaccine production by instead coupling carbohydrates to proteins in vivo in bacteria through an enzymatic process. Bacteria express oligosaccharyltransferases that mediate the transfer of a lipid-linked glycan to a carrier protein in a single step without requiring purification of the components. This can simplify the manufacturing process and make vaccines easier to scale, particularly for use in low-resource environments. Synthetic approaches to glycoconjugate vaccines also allow for multivalent vaccines that contain multiple serotype-specific glycan epitopes on a single carrier protein to increase coverage. Defined synthetic antigens also allow for the incorporation of non-natural modifications to increase immunogenicity or stability. Production of homogeneous glycoconjugates also can improve quality control over vaccine batches, which was a large problem of traditional methods. In addition, as computational tools for glycan antigen design are further developed, these tools will allow for the in silico selection of immunogenic epitopes, which can streamline vaccine design. Synthetic glycans are advancing vaccinology by providing pure, well-characterized antigens that can be used to elicit protective and durable immunity with improved safety compared to naturally derived antigens.
Synthetic glycans also provide invaluable standards and tools for the discovery and validation of glycan-based biomarkers, which are based on the detection of changes in the glycosylation of cells that are associated with diseases. For example, in cancer, tumor cells often display altered O-glycans and highly branched N-glycans that can be targeted using immobilized synthetic mimics on microarrays or diagnostic chips. Synthetic glycan microarrays allow for the high-throughput analysis of serum antibodies that bind to tumor-associated carbohydrate antigens, and can be used to identify antibody signatures that are associated with the stage and metastatic potential of cancers. Synthetic oligosaccharides that mimic the repeating units of bacterial lipopolysaccharide or capsular polysaccharides are also used as defined antigens in serological assays to differentiate between infection and colonization with certain bacterial pathogens. Rare glycans that are not found in nature can also be synthesized for use as standards in the detection of low-frequency changes in glycosylation that are diagnostic of early stages of inflammation, such as changes in sialic acid linkages or fucosylation patterns. Synthetic glycans are also widely used as internal standards in quantitative clinical glycomics applications using mass spectrometry, and glycan-coated nanoparticles are being developed as contrast agents for imaging that use disease-specific glycan recognition to concentrate the imaging probe in a diseased tissue. Precise control of glycan spacing and multivalency through chemical synthesis can also increase the sensitivity of these assays to allow for the detection of low-abundance biomarkers from small sample volumes. Synthetic glycan libraries will also be needed in the future as we move toward personalized medicine, where they can be used to identify patient-specific glycan signatures that can inform treatment decisions and be used to track responses to therapy.
Main classes of glycans modulating cancer hallmarks.2,5
Synthesis of complex glycans and glycoconjugates is rapidly moving away from the traditional, laborious, small scale methods towards a more process-oriented and standardized approach. Currently, the synthesis of one complex oligosaccharide typically takes several weeks of bench work, and has precluded systematic, large-scale structure-function studies. Many of the new strategies are beginning to address this by providing end-to-end solutions for automated glycan synthesis. These platforms integrate the complete set of protecting group manipulations, glycosyl coupling cycles, and purification steps for one glycan synthesis in a completely automated fashion. Solid-phase glycan synthesis is currently being developed with photocleavable linkers and is being integrated with microfluidic reactors to reduce reagent use and increase reaction speed. Machine learning techniques are also being applied to large datasets of coupling conditions and stereochemical outcomes for each coupling step, to predict optimal conditions to streamline optimization and avoid extensive empirical testing. In-line, real-time analytical monitoring (mass spectrometry and NMR) allows immediate feedback, and the possibility to dynamically change reaction conditions to optimize yields. Many of these efforts are now also focusing on incorporating green chemistry principles, including the development of recyclable catalysts and tags, aqueous phase reactions, and biocatalytic multi-step cascade reactions to remove toxic chemical reagents. Envisioned for the future is also a system that allows even non-experts to be able to synthesize glycans. For this, software packages will need to be designed, which convert the glycan structure of interest into an actual synthesis protocol, and these in turn will be run on automated benchtop synthesis platforms.
Glycan synthesis is being transformed by automation technologies that are translating time-consuming, multistep manual protocols into streamlined, reproducible, rapid workflows that allow the rapid production of compound libraries for screening or therapeutic development. Automated solid-phase platforms based on peptide synthesizers are anchoring a growing glycan chain to an insoluble support and adding monosaccharides in iterative cycles without the need for intermediate purification. This method shortens the time for synthesizing glycans from weeks to days and enhances reproducibility between batches. Coupled with microfluidic technologies, synthesis can be miniaturized into reactions that occur in picoliter droplets, leading to a massive reduction in reagent volumes and the parallel synthesis of hundreds of structures. High-throughput synthesis is also being enabled through the development of one-pot multienzyme systems in which multiple glycosyltransferases can work sequentially in a single vessel, with sugar nucleotides being regenerated in situ to allow for catalytic turnover. These biocatalytic cascades avoid the need for purification of the expensive donor molecules in each step, and can be integrated with in-line purification steps using affinity chromatography. Liquid transfer, temperature cycling, and reaction monitoring are all being accomplished with robotics, while AI-guided software is being developed to predict coupling conditions to eliminate trial-and-error optimization cycles. Quality control is being enabled by automated LC-MS systems that can rapidly validate each step in the synthetic process to identify any failed reactions before the errors are propagated. These advances are especially relevant for vaccine development, where the ability to rapidly produce diverse glycan antigens is needed to respond to emerging pathogens. As these platforms become more mature, they will enable academic and clinical laboratories to synthesize custom glycans routinely for personalized diagnostics and therapeutic glycoengineering.
Synthetic glycans have the potential to play a key role in the implementation of personalized medicine. The glycan profile of an individual can provide information about the genetic, metabolic and disease state of a patient. This can be determined by screening a synthetic glycan library on a microarray and using data analysis algorithms to determine which biomarker is expressed or most highly expressed by an individual. This approach could be used to select a therapeutic option based on individual patient glycomic profiles. Synthetic glycans are being developed for use in personalized cancer vaccines, where synthetic tumor-associated carbohydrate antigens are used to stimulate an immune response against a patient's tumor, which could account for tumor heterogeneity and immune suppression. Synthetic glycans that can be used to 'normalize' IgG glycosylation have been proposed as therapies to reduce inflammation in autoimmune disease. Production of glycoproteins using cell-free expression systems and synthetic glycosylation pathways could be used to produce patient-specific glycoproteins with optimized pharmacokinetic profiles with reduced immunogenicity and side effects. Glycan-based nanoparticles are being developed as vehicles for drug delivery, with synthetic ligands for functionalization to direct therapeutic payloads to targeted tissues by glycan recognition, sparing healthy cells from exposure to therapeutic agents. Synthetic glycans can also be used as internal standards in glycomics experiments, providing accurate quantitation of glycomic changes as a function of disease progression and treatment. As the cost of glycan synthesis is reduced by automation and the ability to predict patient glycan responses is improved by computational methods, it is expected that synthetic glycans will be used in clinics to pair glycan-targeted therapeutics to the individual glycomic profiles of patients.
Glycan synthesis has evolved from an academic curiosity to a critical enabling technology underpinning major advances in biotechnology, medicine, and materials science. The advent of chemical and enzymatic synthetic methods to access homogeneous, well-defined carbohydrates has transformed glycans from being merely descriptive biomarkers of disease and health to being tools for therapy and diagnostics. Moreover, synthetic methodologies have progressed from painstaking solution-phase chemistry to automated solid-phase and chemoenzymatic cascade processes. The increasing ease, integration, efficiency, and scale of glycan synthesis, together with the recent advances in glycan sequencing and analytical tools such as glycan microarrays and mass spectrometry, have created an integrated approach for interrogating the glycome. The continued development and scale-up of glycan synthetic approaches, alongside advances in automation, machine learning/artificial intelligence, and synthetic biology, will likely lead to on-demand glycan production at the bench level of researchers and clinicians. This development will enable personalized medicine to reach its full potential, with glycan signatures directing treatment decisions and synthetic glycoconjugates as targeted therapeutics. Glycan synthesis also promises to enable new applications in biotechnology beyond medicine, including defined glycans as tools for engineering biomaterials with precise cell-adhesion properties, glycan-based sensing elements for environmental and water quality monitoring, and food ingredients with new or improved nutritional qualities. As the toolbox for glycan synthesis and functionalization continues to expand, the focus in the field will increasingly shift from the synthesis itself to the function of synthetic glycans in complex biological systems. This shift will require close collaboration across disciplines to ensure that glycan synthesis becomes a true enabling technology for the biotechnology of the future, where the sugar code is no longer just read but written and rewritten to address society's needs.
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