Synthesis of complex glycans is considered to be one of the most challenging tasks in chemical synthesis. The difficulty in the synthesis of complex carbohydrates compared to peptides or nucleic acids is largely attributed to the fact that sugars are more recalcitrant to elegant, convergent synthetic methods. In a glycan molecule, there are several hydroxyl groups on each monosaccharide with similar reactivity that differ by only a few kilocalories in activation energy, thus necessitating the careful design of a protecting-group strategy that can be multiple steps removed from the final deprotection step. In addition, there is no solid-phase synthesis equivalent in glycan synthesis such that each glycosidic bond needs to be activated and each branch point needs to be controlled for stereochemistry with each deprotection step running the risk of global failure. Another challenge in glycan synthesis is that many biologically important glycans are only functional when they have non-stoichiometric microheterogeneity (such as microheterogeneity of sialylation, fucosylation or sulfation). These challenges have led to the development of hybrid synthetic approaches that combine automated solid-phase glycan elongation with enzymatic processing steps, in situ monitoring, and machine-learning guided optimization in order to transform glycan synthesis from an artisanal pursuit to a predictable, engineering discipline, without forgoing the structural complexity required for biological activity.
Fig. 1 The chemical biology study of homogeneous N-glycans.1,5
The overall pace of progress in the development of coupling reagents and protecting-group strategies has not kept up with the growing ambition of glycansynthesis. The practitioner is still all too frequently faced with a triad of familiar challenges, including low reaction yields (resulting in high raw material prices), unpredictable regio- and stereochemical outcomes (requiring chromatographic "miracles") and unavoidable molecular heterogeneity (hindering analytical characterization and downstream biological validation). All these problems are related to each other: low conversion usually requires more forcing conditions with detrimental effects on selectivity, and poor selectivity often requires deprotection procedures in which the entire architecture is subjected to potential undesired rearrangements and/or oxidative destruction. This chapter addresses each of these issues in turn and discusses some recent approaches that help to improve the risk–reward balance in complex glycansynthesis.
Yield erosion in glycan synthesis is most often not the result of one fatal misstep, but a consequence of compounding inefficiencies that manifest first during the glycosylation step and are then carried over and compounded in each subsequent manipulation. Steric hindrance at the anomeric center inhibits nucleophilic attack, while competing elimination often yields glycal by-products that are challenging to remove and are susceptible to further decomposition. Protecting groups introduced to enforce regioselectivity can also shield the reaction centre and inadvertently require higher temperatures or longer activation, which in turn hasten orthoester formation and anomeric epimerization. Water that enters the reaction from hygroscopic solvents or incomplete inertion quenches activated donors and forms hemiacetal intermediates that prematurely terminate chain elongation. Many of these losses are addressed by incorporating iterative coupling cycles in which a sequence is built by accepting lower, but still complete, conversion per cycle in favor of unpredictable high-yield events; by capping unreacted acceptors at the end of each cycle, truncated sequences cannot propagate, simplifying downstream purification. Continuous-flow microreactors also provide additional handle: short residence times penalize side reactions that take minutes to form, while segmented flow entraps moisture and oxygen in discrete droplets that can be scavenged before they enter the reaction channel. Glycosyltransferase enzymes, once engineered to tolerate more diverse donors, offer orthogonal reactivity windows that can be stitched in sequence with chemical steps to rescue linkages that classical Lewis-acid catalysis fails to deliver with sufficient efficiency. These and other strategies do not just raise individual yields, but flatten the risk profile of an entire synthetic campaign, making multi-milligram quantities of defined glycans accessible without requiring an inordinate investment.
In peptides, by comparison, the amide bond is always planar and chemically inert. Glycosidic linkages, by contrast, face the additional challenge of the axial/equatorial ambiguity of the pyranose ring; one anomeric mis-assignment will invert the presentation of all of the peripheral hydroxyl groups and the molecule will no longer be recognizable by its cognate lectin or receptor. Classical neighboring-group participation mechanisms give at least partial stereochemical insurance, but the necessary 2-O-acyl protecting group restricts the branching and can migrate during prolonged activation to give orthoester side-products that appear identical to the desired β-product. Regioselectivity is another challenge: the four secondary hydroxyls on a typical gluco-configured acceptor are within a pKa unit of each other in nucleophilicity, and solvent, counter-ion and even rate of agitation can sway glycosylation outcomes. Remote protecting groups are now being used as conformational locks: placement of a 4,6-O-benzylidene acetal forces the glucoside into a skew-boat conformation that presents the 3-OH and sterically shields the 2-position, templating regioselectivity without relying on the formation of transient stannylene intermediates. Photocaged donors that liberate glycosylating equivalents only on spatially patterned illumination are being used for orthogonally addressable couplings on microarray surfaces, bypassing the combinatorial headache of solution-phase competition. In the meantime, directed-evolution efforts on glycosyltransferases are now yielding mutants with active-site loops that can recognise the steric fingerprint of specific acceptor hydroxyls, and transfer the regio- and stereochemical selectivity from the biological to the chemist's flask. These and other convergent innovations do not obviate the fundamental challenge of carbohydrate coupling, but rather transform it from an unpredictable art into a design space where the outcomes can be modelled, simulated, and ultimately engineered.
In some cases, even if each glycosylation step is of satisfactory conversion and selectivity, the final product may be a micro-population of closely related isomers whose chromatographic separation may require gradient steepness and stationary phase selectivity beyond the typical capabilities of preparative chromatography. In addition, truncated sequences which may form as a result of incomplete couplings, protecting-group migration, or acid-catalysed hydrolysis co-elute with the desired glycan, confounding both MS quantification and biological validation. This is further complicated by the fact that oligosaccharides tend to form anomeric mixtures if they are stored for long periods, especially when the reducing end is susceptible to trace contaminants present in the buffers used. These problems are being addressed by chemists by embedding real-time release assays in which photocleavable linkers are used to liberate aliquots at different stages of the synthesis for analysis by micro-LC–MS while leaving the solid-phase elongation cycle uninterrupted; if an immediate deviation in retention time or isotopic envelope is detected, this will trigger a change in parameters and the defective sequences will not be allowed to propagate. Post-synthetic purification is also being transformed through the development of mixed-mode resins that combine size exclusion with boronate affinity, thus making use of the unique vicinal diol fingerprint of different glycoforms. Most importantly, there is now also recognition that complete chemical homogeneity may not always be required for biological activity; instead, controlled heterogeneity libraries in which pre-defined ratios of sialylated and asialylated (or other known variants) are mixed, can be screened directly for their functional readout, thereby allowing the purification effort to be focussed only on the biologically active subset. Through the integration of analytical feedback at every synthetic node and an acceptance of strategic, design-driven heterogeneity, the field is beginning to recast purity from an unquantifiable ideal into a quantitative, risk-managed parameter.
The overall efficiency of a glycan assembly campaign is seldom limited by a single kinetic barrier; rather, it is the product of a convolution of electronic, steric, and environmental factors that influence the reaction during each elongation cycle. The protecting-group mosaic tunes donor ionization rates, the solvent phase determines ion-pair separation, and the temperature window preconditions the manifold toward kinetic or thermodynamic glycosides. These parameters are often intimately intertwined (electron-withdrawing esters require higher activation energy, which in turn reduces stereocontrol unless the solvent polarity is increased), and their optimization must be approached holistically rather than in isolation. Recent trends make it possible to iterate data-rich: microflow reactors ensure rapid heat transfer while inline analytics report conversion and anomeric ratios in real time, allowing multivariate algorithms to find sweet spots that classical one-factor-at-a-time screens would miss. The following sections describe the most influential levers and explain how contemporary strategies are turning what was once an empirical craft into a predictable design exercise.
Protecting groups thus have a two-fold responsibility: shielding functionality that should be innocent and setting up reactivity gradients that allow iterative coupling. Electron density is hyper-conjugated away from the anomeric centre by alkyl ethers, "arming" the donor and lowering activation enthalpy, while acyl substituents inductively "disarm" the sugar by withdrawing electron density and thus allow orthogonal coupling sequences without self-condensation. The arm–disarm paradigm is complicated by the position of the substituents however: a C-2 benzoyl ester can also act as a leaving group, triggering reaction via a dioxolenium ion and biasing the reaction towards 1,2-trans glycosides, while a C-2 benzyl ether is fully "disarming" and leaves the oxocarbenium completely exposed on both faces, leading to anomeric mixtures if solvent or temperature biases are not used. Silyl ethers remote from the anomeric center can lock the sugar into pre-organized reactive skew-boat conformations if bulky enough, speeding up glycosylation while also protecting adjacent hydroxyls from unwanted acylation side reactions. A too armed donor, on the other hand, may ionize prematurely during storage or work-up, leading to glycal by-products that terminate chain growth. The state of the art of synthesis thus approaches protecting groups as tunable resistors in an electronic circuit: their placement and electronic signature are now routinely modelled in silico before bench execution, and iterative microflow glycosylations are used to feedback conversion data to the design loop and fine tune the protecting-group code for subsequent elongations. The result is a risk-managed palette of reactivity that can be dialed up or down with molecular granularity, effectively elevating protecting-group selection from an empirical art to a quantitative variable in an optimization algorithm.
Solvent and catalyst can be thought of as both ends and means. Solvent mediates between mechanism and medium. It is instrumental in the stabilization of charged species, in determining the dynamics of ion-pair separation, and in controlling the path of nucleophilic approach. Ethers of low polarity are preferred for the generation of intimate ion pairs, as this can be utilized in intramolecular aglycon delivery to give 1,2-cis linkages with high fidelity. Nitrile solvents, in contrast, stabilize solvent-separated oxocarbenium ions and thus shift the manifold to 1,2-trans products that are thermodynamically more stable. The emerging class of deep-eutectic co-solvents offers interesting design possibilities, as for example, hydrogen-bond donors like choline chloride create a structured solvation shell that suppresses the hydrolytic pathway while at the same time is fluid enough for microfluidic device operation. The catalyst is a likewise crucial component. Lewis acids like silver or tin salts are well established for their activation of the anomeric leaving group, but may also cause side reactions such as orthoester formation or trans-esterification of acyl protecting groups. Cationic gold or platinum complexes, by contrast, are new-generation promoters that open a milder activation window and allow glycosylations at sub-ambient temperatures, where anomeric selectivity is determined kinetically. Hydrogen-bond donor/acceptor organocatalysts are bifunctional, and can thus simultaneously activate donor and acceptor to organize a pre-transition-state complex that compensates for the entropic penalty of bond formation. If these catalysts are immobilized on porous carriers, they can be employed in continuous-flow microreactors, which permit iterative glycosylations without cumulative metal contamination.
The temperature determines the kinetic stereoselectivity and the thermodynamic relaxation but in the particular case of glycosylations, the kinetics is complex enough that an Arrhenius intuition is often confounded. As a general trend, a low temperature will favor α-triflate intermediates, due to the anomeric effect stabilizing the axial conformation. This is one reason why lower temperatures might be favorable for these reactions and 1,2-cis glycosides, in case the acceptor is nucleophilic enough, result. However, too low a temperature will kinetically trap the system in a metastable ion-pair situation that upon warming results in an anomeric mixture. At higher temperatures, the donor ionization is faster, but so is the rate of the unwanted trans-glycosylation that wipes out the earlier stereochemical gain and leads to mixtures that are chromatographically inseparable. Fast infrared thermography is nowadays emerging for microflow reactors and allows to map the spatial temperature profile along the reaction channel. Transient exothermic hot spots can initiate side reactions like orthoester formation or glycal elimination. If the temperature profile of such a microflow reactor is adjusted by microwave irradiation or resistive heating in a real-time feedback loop, these hot spots are suppressed in milliseconds, and the desired kinetic selectivity can be maintained at high throughput. Isothermal calorimetry can provide information about the activation entropy of each glycosylation step and is used to parameterize kinetic models that can then predict the optimal temperature profile rather than a constant set point. This profile can be made a dynamic variable using the associated machine-learning algorithm, which ingests the calorimetric and spectroscopic data of each microbatch iteration, and self-optimizes across microbatch cycles to focus on a narrow thermal window where the reaction yield and selectivity are simultaneously maximized.
The key to improving the efficiency of glycan assembly is increasingly shifting from point reaction optimizations to a holistic re-engineering of the design–make–purify feedback loop. Modern workflows are end-to-end: optimizing algorithms run on microfluidic platforms with engineered biocatalysts whose kinetics and fidelity can be tuned in silico and validated in real-time. These systems reduce the cost of experimentation to almost zero as every microgram of material is converted to data for training. The outcome is a self-learning workflow where yield, stereoselectivity, and impurity profiles are predicted, validated, and improved within a single day, a fraction of the time required for traditional batch-based approaches. The next sections will cover the three big knobs—automation, biocatalyst tuning, and in situ feedback—that drive this shift from a bespoke endeavor to a robust synthetic routine.
Improving glycan synthetic output has reached a point where systemic re-engineering of the entire design–make–purify workflow is required, rather than simply adjusting reactions in isolation. The latest approaches now include optimization algorithms running within microfluidic platforms, engineered biocatalysts whose speed and fidelity can be tuned in silico, and closed-loop process control based on in-line analytics that translate each µg of product into training data. This creates a self-correcting process pipeline in which the trajectories of yield, stereoselectivity and impurities can be predicted, validated and refined within a single day, dramatically reducing the cycles that previously could take weeks. We review the three main control knobs (automation, enzymatic tuning and in-line feedback) that underpin the rapid shift of carbohydrate assembly from an artisanal process to a robust unit operation.
While chemical glycosylation is powerful for the installation of unnatural linkages, its aqueous robustness is often wanting, as is its ability to effectuate late-stage sialylation under mild conditions. Glycosyltransferases, whose active sites have been engineered to accommodate donor analogues equipped with photocaged, azido, and fluorinated reporter groups, are beginning to plug this void, expanding the accessible chemical space without compromising the native-like fidelity of the enzymatic process. Ten-thousand-cell lysates are screened per hour in droplet microfluidics platforms, with fluorogenic acceptors that only become fluorescent upon correct formation of the glycosidic linkage; positive hits are sequenced and re-subjected to structure-based algorithms that prioritize second-generation mutations most likely to increase kcat without compromising Km. In addition to active-site tuning, surface-charge re-patterning also improves the solubility of the enzymes in co-solvent systems that render hydrophobic acceptors soluble, without the need for amphiphilic micellar additives that muck up downstream purification. Perhaps most impactful, however, is the recent development of tandem biocatalytic cascades in which sulfatases, fucosidases, and sialyltransferases have been co-immobilized on porous carriers, where the substrates percolate through the particle bed and exit the other side as fully elaborated glycans, without the need for intermediate work-ups that would otherwise dilute the yield and risk metal contamination. As each enzyme is labelled with a unique short peptide barcode, the individual turnover numbers in the mixture are deconvoluted by quantitative mass-spectrometry, and the ratios can be tuned to maximize the overall flux while minimizing side-product formation. The result is a continuously improved catalytic ecosystem whose performance is steered by genetic, computational, and process-level inputs, to synthesize glycan sequences that can rival natural products in sequence complexity but which can be accessed under benign and scalable conditions.
One of the greatest challenges in glycan synthesis has been the inability to monitor the reaction in real time: neutral substrates and products, chromatographic peak coalescence and low stability of protecting groups can lead to a delayed discovery of a failed reaction only after costly work-up. Now, emerging on-line monitoring using mid-infrared fiber probes, miniaturized mass spectrometry and fast evaporative light-scattering detectors, provide multivariate output every few seconds without sampling. Machine-learning based on previous campaigns can identify spectral changes that predict undesirable orthoester formation, glycosyl donor hydrolysis or glycosyl acceptor elimination, at which point the control system can compensate by altering activator flow-rate or temperature set-point within safe operational limits. Because the feedback loop is closed through a Bayesian estimator, the algorithm learns with each cycle, continuously tightening the window of safe operation and reducing batch-to-batch variability. For photoglycosylations, inline UV–vis spectrometry can be used to monitor depletion of photocaged promoters, and LED intensity adjusted dynamically to keep photon flux constant even as solution turbidity changes. The overall effect is a provable reduction in batch failure rate, reduced solvent consumption, and an electronic batch record that is compliant with regulatory authorities by recording all operational deviations and associated corrective action, turning process monitoring from a post-mortem activity to a pre-emptive action.
The conversion of a beautiful retrosynthetic drawing to a gram-scale glycan often is the proving ground where many synthetic plans fall short in the face of a persistent reality. In both industrial production facilities and academic-scale laboratory reactors, synthetic approaches are converging on three general non-exclusive strategies—one-pot cascades that avoid losses from intermediate isolations, enzymatic modifications that impose orthogonal regio- and stereochemical control, and chemoenzymatic iterations that interchange catalysts. These methods have the potential to address issues of metal contamination, batch consistency and cost of goods that ultimately will determine whether a therapeutic glycan will move forward into clinical development or remain on the computer screen. The following examples highlight some of the challenges, such as poor yields, undesirable stereochemistry, and mid-sequence protecting-group failure that have been overcome through a creative, data-driven, iterative process among synthetic chemists, enzymologists and process engineers.
Linear glycosylations are hampered by multi-step loss of material. Intermediate purifications between reagents necessitate waste of reagents and elongated timelines. Single-pot multi-reaction or One-pot programming (OPP) protocols sequence the relative windows of reactivity for each donor in a single reaction vessel, with armed donors coupling first under soft Lewis-acidic conditions to provide the glycan core, then with the disarmed thioglycosides protected from the initial promoter the addition of a stronger promoter chemoselectively extends the chain. Photolabile protecting groups are removed by LED light between each coupling to avoid acid/base quenches which could hydrolyze sialic acid donors. Inline SPE cartridges remove excess promoter and freed aglycones to avoid them from taking part in transglycosylation side-reactions. Proof of concept synthetic case studies show that a tetrasaccharide that was available in 18 % isolated yield over four linear steps can be assembled in a single afternoon with approximately 3x higher throughput and achieving purity to serve as the basis for animal immunization. Translating to a continuous flow process is through the use of segmented flow coils with immiscible fluorinated plugs between each "mini-batch" where each plug's reaction temperature, residence time and promoter strength can be independently controlled without the entire stream being segmented. With no user intervention between glycosylations oxidative degradation and anomeric equilibration are eliminated to transform the academic concept to a robust reproducible process amenable to regulatory qualification.
Chemical methods of stereocontrol break down when the linkage to be formed is sterically congested or when the aglycon is substituted with electron-withdrawing groups, which lower the nucleophilicity. Mutant glycosyltransferases that reverse the native regioselectivity while retaining anomeric selectivity were isolated in directed-evolution experiments performed in emulsion droplets that package a single E. coli cell expressing the mutant enzyme. The hotspot mutations that defined the phenotype reside in a loop that regulates access to the acceptor-binding pocket and point mutations saturating the hotspot enlarge the cavity such that β-1-4 glucosylation of sterically hindered C-2 carbonyl-substituted glucosamine derivatives that are resistant to Lewis-acid catalysis becomes possible. Commercial partners of this work immobilize the biocatalyst in silica monoliths within stainless-steel cartridges, which are easily integrated in GMP facilities as pre-packaged modules. The biotransformation is performed at slightly elevated temperature, which increases the reaction rate while avoiding aggregation; surface pegylation of lysines improves the stability in this context. Inline LC–MS analysis allows for monitoring of the product distribution on a timescale of a few minutes; when the content of the desired stereoisomer starts to drop below target levels the residence time or donor feed is adjusted by the control system in order to keep the quality of the product within a narrow range. In this way the group has been able to produce a tumor-associated carbohydrate antigen whose α-2-3 sialic acid cap was previously only accessible via low-temperature chemical glycosylation at sub-gram scale. Replacing the cryogenic step by aqueous biocatalysis lowers the overall energy input and eliminates the need for chromatographic enrichment, since the reaction provides the required stereoisomeric excess to meet API-grade criteria.
Purely chemical or purely enzymatic approaches can occasionally become untenable for intrinsic reasons. Protecting-group gymnastics can create unmanageable steric congestion while enzymes can be intolerant of non-natural monosaccharides or hydrophobic aglycons in their active sites. As illustrated below, hybridization can provide a way out by toggling between catalytic regimes during sequence assembly. One reported example is the synthesis of a sulfated Lewis X hexasaccharide. The first core was assembled chemically under flow conditions to install a 6-O-sulfate group that would be incompatible with sulfatase activity, whereas galactosylation and sialylation were performed by a mutated β-1-4 galactosyltransferase and α-2-3 sialyltransferase, respectively, because these linkages required higher stereochemical fidelity than can be achieved by neighboring-group participation alone. A facilitating strategy was the use of a cleavable PEG tag that only rendered the elongating glycan water soluble after the enzymatic steps so that it could be phase-transferred between organic and aqueous media without intermediate chromatography. Another industrial example is a branched high-mannose epitope for a broadly neutralizing antibody programme. Chemical mannosylation was used to establish the β-1-4 chitobiose core, followed by a mannosidase-catalyzed trans-glycosylation reaction that added the terminal D1 and D3 arms, structural motifs that are difficult to distinguish using classical arm–disarm logic. Process analytics showed that a slight excess of the GDP-mannose donor was sufficient to suppress hydrolytic back-reaction and thus drive the equilibrium toward product, circumventing the need for large solvent volumes. The final hybrid sequence achieved a 10-fold improvement in throughput compared with the fully chemical benchmark while meeting heavy-metal and endotoxin specifications for human use, showing that chemoenzymatic integration is no longer an esoteric academic exercise but a viable and regulatorily endorsed manufacturing paradigm.
Encountering glycan targets that laugh in the face of conventional coupling strategies with either insoluble branched topology or sensitive chemical functional groups, we approach each project as an engineering challenge not a synthetic headache. The team has created a feedback loop rich environment where reactions are scaled to the micro-liter level and run under inline monitoring conditions with next iteration design decisions being made within minutes of analytical review and bespoke protocols being issued to customers as "living documents" to scale and shift with regulatory demands. By pairing high-throughput automated flow chemistry and CRISPR-edited biocatalysts, we are able to routinely accomplish syntheses that previously took months down to a matter of weeks, without sacrificing purity or compatibility with FIH requirements.
Fig.2 Core Synthesis/Enzymatic Extension (CSEE) strategy for N-glycan synthesis.2,5
Struggling with low-yield reactions, stereochemical control, or purification bottlenecks in your glycan synthesis workflow? Our expert team specializes in solving complex carbohydrate synthesis problems-helping researchers and R&D teams optimize their methods, scale production, and achieve high-purity, reproducible results.
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1. Why is complex glycan synthesis so difficult?
Challenges include controlling stereochemistry, minimizing side reactions, and achieving complete purification for highly branched glycans.
2. What causes low yield in glycan synthesis?
Poor coupling efficiency, incompatible solvents, or protecting group interference are common reasons.
3. How can automation help solve synthesis problems?
Automated systems provide precise temperature and timing control, improving reproducibility and reaction efficiency.
4. What are the best strategies for improving glycan synthesis?
Use optimized reaction conditions, enzyme-assisted catalysis, and real-time analytical monitoring to enhance outcomes.
5. Can I get expert help troubleshooting synthesis issues?
Yes. Our glycan chemistry specialists provide consultation and project optimization for challenging carbohydrate synthesis workflows.
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