The two most abundant classes of protein glycosylation are N- and O-glycans. These glycans are distinguished by the very atoms by which they are attached to polypeptide, in the biosynthetic pathways through which they pass, and in the structural constraints they impose on the mature glycoprotein. The N-glycan is assembled in the endoplasmic reticulum by en bloc transfer of a pre-assembled oligosaccharide lipid precursor to asparagine side-chains, in a reaction so tightly coupled to translation that the nascent chain remains threaded in the ribosomal tunnel. In contrast, O-glycosylation is a post-translational, monosaccharide-by-monosaccharide process that begins in the Golgi and targets serine or threonine hydroxyls, without need for a lipid carrier or general consensus motif. Divergent glycan formation processes result in distinct core topologies, size distributions and conformational dynamics which influence the way each glycan family regulates protein folding as well as receptor binding and immune recognition.
N-glycans are assembled on a conserved pentasaccharide core and can take on high-mannose, hybrid, or complex branching patterns at the first architectural level. O-glycans have no universal core and are instead added to a patchwork of regionally specific scaffolds elaborated from GalNAc, GlcNAc, mannose, fucose, or xylose. The former are limited by the stereochemistry of the dolichol-linked donor and the specificity of the ER's quality-control lectins and are thus relatively predictable, if microheterogeneous. The latter are generated by competing glycosyltransferase cascades that are sensitive to substrate supply, cell lineage, and environmental cues, and are generally shorter but compositionally more diverse. The structural differences are propagated at the level of the glycoprotein: N-glycans often stand out from the surface as stiff, antenna-like sentinels, while O-glycans form clusters in mucin-like domains that make up long, bottle-brush filaments, able to absorb compressive forces and modulate accessibility.
N-glycans have a common ancestry, they start with an obligatory chitobiose unit that holds the reducing-end GlcNAc in a β-1,4 linkage with Asn in a reaction catalyzed by oligosaccharyltransferase (OST) in a single, vectorial step. The asparaginyl-(GlcNAc)2 platform, in turn, is flanked by an invariant trimannosyl core to which additional antennae are added by β-1,2, β-1,4, and α-1,3 linkages in a hierarchy of transferases that compete for the same acceptor and operate according to a timed program. The lack of such a primordial uniformity allows O-glycans to entertain a more anarchic linkage portfolio. The most prevalent mucin-type, for instance, starts with an α-1,0 attachment of GalNAc to Ser or Thr, but other cores can be derived from β-linked GlcNAc (nucleocytosolic O-GlcNAc), α-linked fucose (Notch-type), or β-linked xylose (proteoglycan-type), which, in turn, determines the grammar of elongation. GalNAc-type initiators are elongated in a core 1 to core 8 series, while the xylose-type is extended in tetrasaccharide repeats. The net result is O-glycans which are shorter but compositionally more diverse, and which may incorporate sulfated galactose, sialylated Lewis antigens or blood-group determinants. The chemical vocabulary to which they have access is consequently both richer and more context-dependent, while that of N-glycans is more constrained, and its opening clause conserved even as its closing lines diversify.
N-glycosylation is a semiregular affair: the Asn residue must be part of an Asn-X-Ser/Thr sequence motif, where X is not proline, and the tripeptide must be on the surface and flexible enough to fit into the active site of oligosaccharyltransferase. As a consequence, N-glycans are found in specific topological regions, typically β-turns or loop regions on which the oligosaccharide can extend laterally without perturbing secondary structure elements. In contrast to N-glycosylation, O-glycosylation has no obvious sequence motif, but is dependent on the local density of Ser/Thr, the neighboring presence of proline residues to kink the protein backbone, and a transient exposure of the hydroxyl sidechains to Golgi-localized transferases. The combination of these factors yields a stochastic and often clustered decoration, which can turn unstructured peptides into extended and rigid stalks or protect protease cleavage sites by steric crowding. As O-glycans are typically added after protein folding, the mature protein surface is directly exposed to the modification. This may be of particular consequence in modifying pockets for ligand binding, metal coordination spheres or antibody paratopes, in a manner that N-glycans, which decorate the protein before folding is completed in the ER, rarely affect. Thus, while N-glycans function as global folding gatekeepers communicating with ER-resident chaperones, O-glycans are contextual modulators of protein–protein interactions extracellularly.
N-glycans are more constrained structurally as a result of the chitobiose-asparagine anchor. The chair conformation of the reducing-end GlcNAc, locked by hydrogen-bonding to the peptide backbone at its C2 acetamido group, imposes torsional constraints on the remainder of the glycan chain and orients the antennae of branched N-glycans into a pseudo-planar structure. Additional motion is added with further α-1,6 branching, but the resultant structure is still largely constrained such that the sialic acids and other termini are presented at a relatively consistent height above the surface of the glycoprotein, and this property is leveraged by both influenza hemagglutinin and Siglec receptors. In contrast to N-glycans, O-glycans do not have the topological restraints of a chitobiose anchor and their dynamics are those of a polymer brush; each α-linked GalNAc in the O-glycan chain is a ball-and-socket joint, permitting the chain to access a broad distribution of backbone dihedrals. Crowded together on mucin regions, these glycans self-interact by H-bonding and hydrophobic stacking and they form extended rods that extend or collapse with changes in pH, ionic strength, or shear stress. The resulting conformational plasticity provides the shock absorption needed to clear the airways and lubricate the gastrointestinal tract, but also creates binding pockets that can transiently sequester cytokines or serve as a presentation platform to specific receptors in a concentration gradient. In this sense the lack of order of O-glycans provides functional robustness, while the regimented nature of N-glycans provides structural certainty—two different strategies to encode biological information in carbohydrates.
At a conceptual level, two broad strategies exist for the construction of glycans. In the first of these (the enzymatic approach), all linkages are formed by glycosyltransferases or other biocatalysts in the conditions of the living cell. This strategy gives access to fully unprotected and regio- and stereochemically accurate glycans, but it is limited to building linkages for which there is an evolutionary precedent. The alternative (chemical) strategy is not bound by the human genome, and can be used to incorporate unnatural linkages, isotopic labels or any kind of spatially addressable functional handle into glycans. It requires the full panoply of protecting-group strategies, however, and is usually accomplished using stoichiometric promoters. These strategies do not exclude each other, and indeed are converging: the lipid-linked oligosaccharides which are formed on dolichol in the endoplasmic reticulum are often further processed chemically in the Golgi, and synthetic chemists are increasingly using enzymes to either "proof-read" or further elongate synthetic fragments. A working knowledge of the mechanisms of each approach, including dolichol-driven N-glycan assembly, the stepwise extension of O-glycans, and the strategic advantages and disadvantages of a purely chemical approach at different stages is therefore a prerequisite to a rational design for engineered glycans for therapeutic, diagnostic, or materials purposes.
N-glycan biogenesis begins on the cytosolic side of the endoplasmic reticulum. Monosaccharides are added in a stepwise fashion to a dolichol pyrophosphate carrier lipid that is tethered to the membrane. The first committed step in this process is a strictly universal, near-algebraic reaction. GlcNAc-1-P is added from UDP-GlcNAc to dolichol-P to form a pyrophosphate bridge, setting the lipid up for a second GlcNAc and five mannose residues, all added from soluble nucleotide sugars. The lipid-linked heptasaccharide is flipped across the ER membrane by a scramblase that neither accepts truncated intermediates nor mis-ordered sugars. Gaining four more mannoses and three glucoses from dolichol-P-mannose and dolichol-P-glucose donors synthesized by cytosolic enzymes and then flipped, the complete Glc3Man9GlcNAc2-pyrophosphate-dolichol is now ready for oligosaccharyltransferase. The enzyme found in the ER binds to the ribosome while detecting Asn-X-Ser/Thr sequons during protein synthesis and attaches the glycan structure to the polypeptide in one complete transfer. Since this reaction is vectorial and irreversible, any misfolded glycoprotein is condemned to ER-associated degradation, so only properly folded clients proceed to the Golgi where trimming and remodeling begins. The cellular machinery removes phosphate groups from the dolichol scaffold and then recycles it. This lipid-centric process explains why N-glycans have a universal core: The lipid carrier functions as both a template and tether to confine the three-dimensional structure of growing chains while establishing a quality-control checkpoint that soluble glycosyltransferases cannot establish.
Fig. 1 Schematic drawing of N-glycan processing.1,5
O-glycan synthesis uses no lipid carrier or consensus sequon, but instead is dependent on sequential addition by a series of Golgi-localized glycosyltransferases with specific acceptor and sugar preferences (all reactions are against the hydroxyl group of serine or threonine). Because the first of these transferases, polypeptide GalNAc-transferase, is a family of closely related proteins with some overlapping but generally distinct peptide preferences, there is a kinetic competition that depends on protein folding (or localized unfolding) to determine which hydroxyl groups are decorated. Once the first GalNAc is added, the chain can be extended by a galactose on the beta-1,3 linkage by core 1 synthase or a GlcNAc on the beta-1,3 linkage by core 3 synthase (the choice is specific to cell type and the glycans can be removed by glycosidases). The galactose or GlcNAc can in turn be extended with blood-group antigens, sialyl Lewis epitopes, or sulfated sugars in a stepwise process that is dependent on the relative expression of the enzymes as well as availability of the sugar-nucleotide donors and the pH of the lumen, which gets increasingly acidic as vesicles mature along the secretory pathway. As the full sugar chain is not added en bloc, the addition process can be stopped or branched at any point, so different glycoforms can be added to the same polypeptide depending on the cell's metabolic state, inflammatory status, etc. The lack of lipid anchoring also allows for more conformational breathing. The peptide chain can reorient in a way that exposes previously hidden hydroxyl groups, and this reorientation is influenced by the glycosylation status (feedback loop with local structure). In this way, O-glycans contrast with the templated, rigid assembly of N-glycans, but instead can function as fast, context-sensitive switches to regulate cell adhesion, immune recognition, and receptor signaling.
Fig. 2 O-glycan synthesis pathways used for the generation of selectin ligands.2,5
Chemical glycosylation is less constrained by limitations of natural products than enzymatic glycosylation is, in that the former can place an arabinose β to an adjacent 1-keto group, and can introduce "unnatural" bonds that are not proofread by enzymatic hydrolysis, such as thio-glycosides or C-glycosyl bonds. This freedom is purchased by requiring a protecting group strategy that ensures only the desired nucleophile is available for reaction. This often doubles the number of steps in a synthesis, and can carry an environmental load if stoichiometric promoters or heavy-metal salts are needed. Stereocontrol is not absolute but conditional: the same donor will not give a single anomer or might give orthoester side-products if the topology of the acceptor or solvent polarity is varied beyond a certain range. In the enzymatic case, the structural freedom is traded for structural fidelity: a glycosyltransferase will add one, and only one, sugar in one, and only one, linkage, under aqueous, near-neutral conditions without protecting groups, making multi-step, one-pot cascades possible in flow reactors. In return, the enzyme is exacting in its substrates: what it has not evolved to encounter will not enter the active site, whether it be a non-natural epimer, a sulfate in an unexpected position, or an aglycon that is too hydrophobic or too long. Active, soluble transferases are also difficult to come by, and typically need mammalian expression and a narrow regime of detergents that are difficult to scale. Chemo-enzymatic strategies thus try to have the best of both worlds, using chemical steps to introduce orthogonal handles or non-natural sugars, then finishing the structure enzymatically to endow it with native-like selectivity, but require solvent transfers and more delicate pH control. The decision of whether to proceed chemically or enzymatically is thus more of a continuum than an either/or decision.
To start, the choice of the best glycan depends not on one ideal structure, but on the context of what one needs. For example, the presence of a terminal sialic acid can enhance plasma half-life but can mask antibody-dependent cellular cytotoxicity (ADCC) that one may want for cancer applications. Furthermore, the presence of high-mannose residues can promote cellular uptake when used in vaccine applications but can lead to undesired clearance when used on, for example, therapeutic enzymes. Thus, first, the list of "must-have" characteristics of a given glycan should be written, including (but not limited to) attributes such as extended circulation, low immunogenicity, target receptor engagement, stability to folding changes, etc. The list is then cross-referenced with glycosylation types and methods available to achieve the desired glycan profile. For instance, N-glycans are a highly-conserved glycan type with a defined core that can be more predictably and specifically modified either in vivo or in vitro, and are thus often chosen when regulatory standards require batch consistency; O-glycans on the other hand are smaller in size and offer more flexibility, with the ability to tolerate higher levels of clustered O-glycosylation and faster cell-surface switching. Furthermore, hybrid glycans, which consist of a synthetic non-natural linker with enzymatically attached natural sugars on each end, are being increasingly selected when both natural-like structure and orthogonal properties are desired. At the end of the day, the "correct" glycan is the one with synthesis costs, analytical accessibility, and immunogenic profile that fits with the intended clinical indication, manufacturing approach, and patient population.
In the clinic, glycans serve as multifunctional excipients covalently linked to the API itself. A single N-glycan at the periphery of an Fc domain can sterically shield proteases, lowering clip-rate in muscle and mask hydrophobic patches that would otherwise nucleate particles at high-concentration in storage. The same oligosaccharide can increase hydrodynamic radius, slowing renal filtration and prolonging exposure without increasing the protein scaffold past the size-of-entry threshold for extravascular tissue. But the core architecture matters too: high-mannose glycans can bind to mannose receptors on reticulo-endothelial cells, speeding clearance and potentially shortening half-life, while α-2,3-sialylated complex types are spared and can additionally reduce systemic inflammatory tone by binding inhibitory Siglecs. For enzymes used in replacement therapy, O-glycans localized to the catalytic domain can serve as molecular bumpers that repel proteolytic nicking without occluding substrate access; if too proximal to the active cleft, however, they can warp the Michaelis complex and lower Kcat. Computational loop grafting now enables new N-glycosylation sequons to be introduced at solvent-exposed turns that experience the highest B-factors, in effect converting thermal breathing sites into rigid, carbohydrate reinforced struts. The resulting increase in melting temperature is often additive, so iterative introduction of two or three sequons can shift the onset of aggregation several degrees without changing the primary therapeutic interface. Finally, glyco-engineering cell lines can be screened for relative expression of bisecting GlcNAc-transferase vs. core fucosyltransferase, a ratio that determines both serum longevity and effector-function potency, such that a single amino-acid substitution can pivot an antibody from an ADCC-enhanced phenotype to a silent scaffold suitable for enzyme delivery.
Vaccine glycobiology presents a host of unique challenges and opportunities. On the one hand, we are tasked with instructing the immune system to respond to carbohydrate epitopes that are generally not immunogenic; on the other hand, we often find that these very glycans are used by pathogens to hide from host immune surveillance. Synthetic oligosaccharides address this challenge by providing only the minimal protective epitope (without contaminating endotoxin or host-mimicking glycoforms) while the conjugation site, density and carrier protein are independently optimized to provide maximal T-cell help. High-mannose oligomers can for example be added enzymatically to surfaces of viral spikes to form a "self-adjuvanting" nanoparticle that preferentially recruits mannose receptors on dendritic cells, thereby boosting cross-presentation without the need for exogenous adjuvant. Masking native high-mannose patches on the other hand by appending complex-type glycans can downregulate off-target binding to mannose receptors and redirect the antibody response toward neutralizing surfaces. Site-selective installation of non-natural linkers in turn allows controlled orientation of the carbohydrate away from carrier epitopes, potentially reducing the carrier-induced suppression that has historically dogged glycoconjugate vaccines. Terminal α-1,3-linked galactose for example can be used to engage the complement lectin pathway if opsonizing IgG2a is to be favored over IgG1, thereby steering the immune response toward Th1-biased, bactericidal phenotypes. Synthetic glycans also allow analytical closure: same lots can be used from pre-clinical to clinical, thereby avoiding the batch-variability that has historically complicated correlation of glycoform with efficacy. Dual-hapten constructs in which two serotype-specific oligosaccharides are presented on the same carrier molecule have also become feasible using chemo-enzymatic tools, allowing valency complexity to be reduced while simultaneously broadening serotype coverage and streamlining regulatory dossiers.
Analytically, glycans are also used as calibrants, affinity ligands, and reporter tags. Uniformity is a quality attribute necessary for assay reproducibility, as in isotopically labeled N-glycans that are enzymatically remodeled in heavy-acetate conditions then added to complex tryptic digests to enable internal standardization for LC–MS quantitation of biotherapeutic glycosylation, or to account for ion-suppression. Apparent drift in fucosylation from one run to the next can then be distinguished from biological changes. Synthetic standards are also used to build retention-time libraries for specific O-glycans, using chemically defined sialic acid linkages on GalNAc-cores and orthogonal stationary phases, to more accurately assign tumor-associated O-glycan truncations in serum samples. Glycan-coated surfaces have also been used as synthetic receptors; self-assembled monolayers that present clustered sialyl Lewis mimic selectin counter-receptors and capture circulating tumor cells with higher affinity than antibody-based columns and at higher shear stress (as found in microfluidic devices). Photocleavable glycan probes, which link a carbohydrate to a mass-encoded reporter with a nitrobenzyl linker, can also be used to affinity enrich then photocleave in a manner that does not involve harsh acid hydrolysis that would traditionally destroy acid-labile modifications. In diagnostics, synthetic glycans have also been used as competition ligands in lateral-flow assays. If patient antibodies bind to the pathogen-derived glyco-epitope, they will displace the glycan tracer, which is labeled with a gold nanoparticle, creating a visual signal that is proportional to antibody titer, without the batch-to-batch variability of using pathogen lysates. Glycan-based internal standards are also used for cross-laboratory harmonization of certain critical quality attributes for regulatory biosimilar glycoprofiles compared to originator data sets with statistical rigor.
A recent translational program led to the conversion of a standard, non-genetically engineered IgG1 monoclonal antibody (mAb) into a site-specific drug conjugate (ADC) by remodeling its unique Fc glycan. The purpose of this molecular surgery was the selective elimination of receptor-over-expressing tumors, with minimal damage to healthy tissue, a challenge that has long been confounded by stochastic lysine or disulfide conjugation, which can create inhomogeneous mixtures of ADCs with difficult to predict toxicities. In contrast, threading a single azide-bearing monosaccharide onto the biantennary core followed by capture of a cytotoxic payload with a bio-orthogonal triazole led to a homogeneous construct whose drug-to-antibody ratio could be adjusted to an integer value without chromatographic pruning. Here we report this journey in three acts (conceptual framing, practical implementation, and biological outcome) in an effort to show how glycan engineering can simultaneously address stoichiometry, stability, and scalability in the ADC space.
The initial phenotypic screen identified a chimeric antibody candidate that was in late stage oncology development as an ideal carrier due to its low rate of internalisation and high serum stability. However, the highly fucosylated and galactosylated glycan structure lacked a chemical handle for direct conjugation. Random lysine modification led to a drug-to-antibody ratio that ranged from nought to eight and reduction of the hinge cysteine destroyed the inter-chain disulfide scaffold with an ensuing reduction in circulation half-life. A decision tree was therefore established that would reduce the native glycoform to a single, reactive site which could be added enzymatically and then used for traceless payload conjugation. The target product profile included a site-controlled, even-numbered drug loading with retention of Fcγ-receptor affinity to recruit immune-effector function and a manufacturing process that could be performed in a single bioreactor without using organic solvent partitioning. Regulatory issues also required any carbohydrate mimic that was added to be traceable using standard LC–MS instruments and should not induce anti-carbohydrate antibodies that could lead to rapid clearance during repeat dosing. The project scope also set the requirement that the final conjugate should be stable in human plasma for a minimum of one week at thirty-seven degrees and that the payload should not lose its cytotoxic function once released within the lysosome. These requirements led to the selection of a chemo-enzymatic platform that combined the selectivity of glycosyltransferases with the bioorthogonality of copper-free click chemistry to avoid codon re-engineering of the antibody and the additional toxicology packages and CMC re-filings that would have ensued.
Briefly, the process was comprised of four sequential, tightly integrated steps with minimal intermediate isolation. To begin, the antibody was treated with an endoglycosidase that reduced the heterogeneous Fc glycan to a single N-acetylglucosamine (GlcNAc) stub and left the peptide backbone intact; the reaction was carried out in histidine buffer under argon to avoid oxidation of the solvent-exposed methionines. The antibody was then incubated with a recombinant glycosyltransferase and a UDP-activated monosaccharide that had an azide-terminated tetraethylene glycol spacer; the enzyme added the azido sugar to the C4 hydroxyl of the GlcNAc stub, which yielded a homogeneous, click-compatible terminus that was distant from the Fc surface, and therefore did not sterically clash with Fcγ-receptor binding. The crude reaction mixture was then diafiltered into phosphate-buffered saline containing a cyclooctyne-linked maytansinoid; the copper-free strain-promoted cycloaddition was run at room temperature overnight, and resulted in near-quantitative conjugation with no detectable payload hydrolysis or antibody aggregation. The final construct was purified by tangential-flow filtration followed by size-exclusion chromatography, which yielded a monodisperse species whose drug-to-antibody ratio could be confirmed by intact mass spectrometry and whose positional integrity could be mapped by glycopeptide fragmentation. In-line Raman spectroscopy was used to track consumption of the azido sugar and formation of the triazole product, thereby avoiding the need for off-line sampling that could introduce adventitious metal contaminants. The entire process from naked antibody to final conjugate took less than three days, and required only two buffer exchanges, a throughput that is compatible with clinical-scale perfusion bioreactors.
Notably, the formed conjugate was uniformly drug-loaded with a payload-antibody ratio of two (confirmed by glycopeptide mapping), a level that is likely near optimal (cytotoxic at high potency against the target expressing cell lines in vitro in the microscale range while sparing antigen-negative cell lines) while remaining safe (i.e., off-target tissue staining in mice revealed a significantly lower incidence of mitotic block by the payload as compared to an orthogonal heterogeneous ADC prepared by traditional lysine maleimide conjugation chemistry). Critically, the pharmacokinetics of this homogeneous ADC in humanized mice was comparable to the unconjugated antibody, so glycan conjugation did not impact clearance or trigger anti-carbohydrate antibodies (monitoring for four weeks). This 1,4-sugar bridge thus can be used as an inert handle for site-specific conjugation without impacting PK. The strategy is also robust as it can be applied to other antibodies with diverse isotypes and antigen targets. As proof-of-concept, site-specific homogeneous ADCs were generated from an anti-CD19 IgG2a and anti-HER2 IgG1 at the same homogeneity with the same enzymatic strategy. From a regulatory point of view, the addition of a single defined sugar into the antibody obviates the need for highly controlled and complex glycoform release specifications; thus, it is anticipated that only intact mass and glycopeptide mapping will be required for this antibody for the in-process and release assays. For the same reason, it should be possible to create orthogonal click handles by engineering additional glycosylation sites at a distinct position and engineer complex ADCs that have more than one type of payload (e.g., a cytotoxic drug and an immune agonist), which can be installed in a rational manner without additional chemical complexity.
Choosing between N-glycan and O-glycan synthesis is more than a structural decision—it determines how your molecules perform in real biological systems. Our custom glycan synthesis services bridge scientific insight with technical precision, helping researchers and biotech teams create high-quality glycans tailored to their specific applications.
Our synthesis team works closely with you to define your project's biological goals and structural requirements. Whether you need N-linked glycans for antibody glycoforms or O-linked glycans for vaccine and diagnostic research, we design a synthesis pathway that maximizes yield, purity, and reproducibility. Each glycan is synthesized to meet exacting research or preclinical standards.
We combine advanced chemical glycosylation methods with enzyme-assisted synthesis to replicate both natural and novel glycan structures. This hybrid approach allows us to produce complex branching patterns, control stereochemistry, and achieve superior site specificity—ideal for glycoengineering and structure–activity studies.
Every synthesized glycan undergoes comprehensive HPLC, LC-MS, and NMR characterization. From monosaccharide composition to linkage verification, our multi-step validation ensures absolute confidence in the structure and performance of your product. We deliver complete documentation suitable for publication or regulatory submission.
From exploratory studies to full-scale production, we provide end-to-end glycan synthesis solutions trusted by global biotech innovators. Our commitment to precision, transparency, and scientific excellence ensures every project meets the highest standards of glycoscience.
Ready to get started? Contact our Custom Glycan Synthesis Team to discuss your N-glycan or O-glycan project and receive a personalized consultation.
1. What is the main difference between N-glycan and O-glycan synthesis?
N-glycans are attached to nitrogen atoms in asparagine residues, while O-glycans attach to oxygen atoms in serine or threonine residues. Their biosynthetic pathways and functions differ accordingly.
2. Which type of glycan is used for antibody development?
N-glycans are typically used for antibody glycoengineering because they affect Fc region stability and immune effector functions.
3. Are O-glycans easier to synthesize than N-glycans?
O-glycans are often simpler structurally but require careful control of linkage types. N-glycans, though more complex, are well-suited for automated synthesis workflows.
4. How do N- and O-glycans influence biological function?
They affect protein folding, signaling, and immune recognition. Structural variation can dramatically change therapeutic efficacy.
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