Approaches toward High‐Mannose‐Type Glycan Libraries

Asparagine‐linked (N‐linked) sugar chains are widely found in the rough endoplasmic reticulum (ER), which has attracted renewed attention because of its participation in the glycoprotein quality control process. In the ER, newly formed glycoproteins are properly folded to higher‐order structures by the action of a variety of lectin chaperones and processing enzymes and are transported into the Golgi, while terminally misfolded glycoproteins are carried into the cytosol for degradation. A group of proteins related to this system are known to recognize subtle differences in the high‐mannose‐type oligosaccharide structures of glycoproteins; however, their molecular foundations are still unclear. In order to gain a more precise understanding, our group has established a strategy for the systematic synthesis of high‐mannose‐type glycans. More recently, we have developed “top‐down” chemoenzymatic approaches that allow expeditious access to theoretically all types of high‐mannose glycans. This strategy comprehensively delivered 37 high‐mannose‐type glycans, including G1M9–M3 glycans, and opened up the possibility of the elucidation of structure–function relationships with a series of high‐mannose‐type glycans.     

Construction of a High‐Mannose‐Type Glycan Library by a Renewed Top‐Down Chemo‐Enzymatic Approach

A comprehensive method for the construction of a high‐mannose‐type glycan library by systematic chemo‐enzymatic trimming of a single Man9‐based precursor was developed. It consists of the chemical synthesis of a non‐natural tridecasaccharide precursor, the orthogonal demasking of the non‐reducing ends, and trimming by glycosidases, which enabled a comprehensive synthesis of high‐mannose‐type glycans in their mono‐ or non‐glucosylated forms. It employed glucose, isopropylidene, and N‐acetylglucosamine groups for blocking the A‐, B‐, and C‐arms, respectively. After systematic trimming of the precursor, thirty‐seven high‐mannose‐type glycans were obtained. The power of the methodology was demonstrated by the enzymatic activity of human recombinant N‐acetylglucosaminyltransferase‐I toward M7–M3 glycans, clarifying the substrate specificity in the context of high‐mannose‐type glycans.     

Chemoenzymatic Synthesis of a Phosphorylated Glycoprotein

The majority of lysosomal enzymes are targeted to the lysosome by post‐translational tagging with N‐glycans terminating in mannose‐6‐phosphate (M6P) residues. Some current enzyme replacement therapies (ERTs) for lysosomal storage disorders are limited in their efficacy by the extent to which the recombinant enzymes bear the M6P‐terminated glycans required for effective trafficking. Chemical synthesis was combined with endo‐β‐N‐acetylglucosaminidase (ENGase) catalysis to allow the convergent synthesis of glycosyl amino acids bearing M6P residues. This approach can be extended to the remodeling of proteins, as exemplified by RNase. The powerful synergy of chemical synthesis and ENGase‐mediated biocatalysis enabled the first synthesis of a glycoprotein bearing M6P‐terminated N‐glycans in which the glycans are attached to the peptide backbone by entirely natural linkages.     

Facile Chemoenzymatic Synthesis of O‐Mannosyl Glycans

O Mannosylation is a vital protein modification involved in brain and muscle development whereas the biological relevance of O‐mannosyl glycans has remained largely unknown owing to the lack of structurally defined glycoforms. An efficient scaffold synthesis/enzymatic extension (SSEE) strategy was developed to prepare such structures by combining gram‐scale convergent chemical syntheses of three scaffolds and strictly controlled sequential enzymatic extension catalyzed by glycosyltransferases. In total, 45 O‐mannosyl glycans were obtained, covering the majority of identified mammalian structures. Subsequent glycan microarray analysis revealed fine specificities of glycan‐binding proteins and specific antisera.     

Asymmetric Chemoenzymatic Synthesis of (−)‐Podophyllotoxin and Related Aryltetralin Lignans

(?)‐Podophyllotoxin is one of the most potent microtubule depolymerizing agents and has served as an important lead compound in antineoplastic drug discovery. Reported here is a short chemoenzymatic total synthesis of (?)‐podophyllotoxin and related aryltetralin lignans. Vital to this approach is the use of an enzymatic oxidative C?C coupling reaction to construct the tetracyclic core of the natural product in a diastereoselective fashion. This strategy allows gram‐scale access to (?)‐deoxypodophyllotoxin and is readily adaptable to the preparation of related aryltetralin lignans.     

Mass spectrometry characterization for chemoenzymatic glycoprotein synthesis

The current project describes the chemoenzymatic modification of bovine ribonuclease B (RNase B) to contain a single glycosylation site with a known glycan. A reactive disaccharide oxazoline derivative was synthesized and stereospecifically added to deglycosylated RNase B through endo-β-N-acetylglucosaminidase M catalyzed chemoenzymatic transglycosylation. Oxazoline formation conditions were optimized using mass spectrometry, and the product verified based on its collision-induced dissociation (CID) mass spectrum. Enzymatic removal of native glycans as well as formation of the desired homogeneous product was also monitored using mass spectrometry. LC-MS(n) using four sequential rounds of CID was used to verify that the original glycosylation site had been reorganized to contain the new glycan. The techniques described herein are not limited to this analyte or glycan and should be amenable to the synthesis of numerous homogeneous glycoconjugates with judicious choice of enzyme/substrate combinations. The combined use of chemoenzymatic synthesis and mass spectrometry-based characterization shows promise for the development of homogeneous glycoprotein reference materials. A well-defined glycoprotein standard containing a single glycan of known composition, linkage and stereochemistry would be of great value for the comparison and evaluation of glycoprotein analysis techniques.     

Stereoselective Construction of β‐Mannopyranosides by Anomeric O‐Alkylation: Synthesis of the Trisaccharide Core of N‐linked Glycans

A new and efficient approach for direct and stereoselective synthesis of β‐mannopyranosides by anomeric O‐alkylation has been developed. This anomeric O‐alkylation of mannopyranose‐derived lactols is proposed to occur under synergistic control of a kinetic anomeric effect and metal chelation. The presence of a conformationally flexible C6 oxygen atom in the sugar‐derived lactol donors is required for this anomeric O‐alkylation to be efficient, probably because of its chelation with cesium ion. In contrast, the presence of a C2 oxygen atom plays a minor role. This glycosylation method has been successfully utilized for the synthesis of the trisaccharide core of complex N‐linked glycans.     

Synthesis of a core trisaccharide building block for the assembly of N-glycan neoconjugates

A short and high yielding synthesis of a core trisaccharide 1 as the key building block in the assembly of a library of N-glycan neoconjugates is presented. The β-d-Manp-(1→4)-d-GlcpNAc linkage was introduced by inversion of the C-2 position of a β-glucoside. The glucosyl donor was efficiently synthesised following a recently published one-pot strategy. 2-Naphthylmethyl and benzylidene-acetal protection in the terminal mannose permitted selective liberation of main branching sites for subsequent glycosylation. A C5 azido linker attached to the anomeric position, which is stable throughout the synthesis, will allow for the posterior immobilisation of deprotected glycans on a microarray surface.     

Total Synthesis of Isodaphlongamine H: A Possible Biogenetic Conundrum

Herein we describe the first synthetic efforts toward the total synthesis of isodaphlongamine H, a calyciphylline B‐type alkaloid. The strategy employs a chemoenzymatic process for the preparation of a functionalized cyclopentanol with a quaternary center. This molecule is elaborated to form an enantiopure 1‐aza‐perhydrocyclopentalene core, representing rings A and E of all calyciphylline B‐type alkaloids. Further transformations involve the formation of a cyclic enaminone, 1,4‐conjugate addition with a cyclopentenyl subunit, and intramolecular aldol cyclization to achieve a pentacyclic intermediate, ultimately forming isodaphlongamine H in a total of 24 steps from the commercially available compound 2‐carbethoxycyclopentanone. Isodaphlongamine H exhibits promising inhibitory activity against a panel of human cancer cell lines.     

A Short,Efficient Synthesis of the Octamannan Residue of High Mannose Type Sugar Chains

Abstract

An efficient route for the synthesis of octamannan 1, found in high mannose type sugar chains, is described. to construct octasaccharide 1 by as few synthetic steps as possible, we employed a chemoenzymatic strategy: the enzymatic synthesis of oligosaccharide blocks using glycosidases followed by chemical coupling to form a branched structure. By use of this methodology, many synthetic steps were eliminated and 1 was easily synthesized.     

Travelling‐wave ion mobility mass spectrometry and negative ion fragmentation of hybrid and complex N‐glycans

Nitrogen collisional cross sections (CCSs) of hybrid and complex glycans released from the glycoproteins IgG, gp120 (from human immunodeficiency virus), ovalbumin, α1‐acid glycoprotein and thyroglobulin were measured with a travelling‐wave ion mobility mass spectrometer using dextran as the calibrant. The utility of this instrument for isomer separation was also investigated. Some isomers, such as Man₃GlcNAc₃ from chicken ovalbumin and Man₃GlcNAc₃Fuc₁ from thyroglobulin could be partially resolved and identified by their negative ion fragmentation spectra obtained by collision‐induced decomposition (CID). Several other larger glycans, however, although existing as isomers, produced only asymmetric rather than separated arrival time distributions (ATDs). Nevertheless, in these cases, isomers could often be detected by plotting extracted fragment ATDs of diagnostic fragment ions from the negative ion CID spectra obtained in the transfer cell of the Waters Synapt mass spectrometer. Coincidence in the drift times of all fragment ions with an asymmetric ATD profile in this work, and in the related earlier paper on high‐mannose glycans, usually suggested that separations were because of conformers or anomers, whereas symmetrical ATDs of fragments showing differences in drift times indicated isomer separation. Although some significant differences in CCSs were found for the smaller isomeric glycans, the differences found for the larger compounds were usually too small to be analytically useful. Possible correlations between CCSs and structural types were also investigated, and it was found that complex glycans tended to have slightly smaller CCSs than high‐mannose glycans of comparable molecular weight. In addition, biantennary glycans containing a core fucose and/or a bisecting GlcNAc residue fell on different mobility‐m/z trend lines to those glycans not so substituted with both of these substituents contributing to larger CCSs. Copyright © 2016 John Wiley & Sons, Ltd.     

Fragmentation of negative ions from N‐linked carbohydrates,Part 4. Fragmentation of complex glycans lacking substitution on the 6‐antenna

Negative ion CID spectra of N‐linked glycans released from glycoproteins contain many ions that are diagnostic for specific structural features such as the detailed arrangement of antennae and the location of fucose residues. Identification of such ions requires reference glycans that are often difficult to acquire in a pure state. The recent acquisition of a sample of N‐glycans from a patient lacking the enzyme N‐acetylglucosaminyltransferase‐2 provided an opportunity to investigate fragmentation of glycans lacking a 6‐antenna. These glycans contained one or two galactose‐N‐acetylglucosamine‐chains attached to the 3‐linked mannose residue of the trimannosyl‐chitobiose core with and without fucose substitution. The spectra from the patient sample clearly defined the antenna distribution and showed striking differences from the spectra of isomeric compounds obtained from normal subjects. Furthermore, they provided additional information on previously identified antenna‐specific fragment ions and indicated the presence of additional ions that were diagnostic of fucose substitution. Glycans obtained from such enzyme‐deficient patients can, thus, be a valuable way of obtaining spectra of specific isomers in a relatively pure state for interpretation of mass spectra. Copyright © 2010 John Wiley & Sons, Ltd.     

基于Endo-M N175Q糖链转移反应与丙酮富集糖肽的LC-MS分析糖蛋白N-糖链

基于化学酶标记和丙酮富集糖肽方法,建立了一种可靠、有效、简单的糖蛋白N-糖链分析方法。以唾液酸糖肽(SGP)为模型糖肽,比较了样品中丙酮加入量对SGP的富集效果,最终选择加入样品体积5倍量的丙酮。用丙酮富集经胰蛋白处理的核糖核酸酶B(RNase B)酶解液中的糖肽,以富集分离得到的糖肽(糖基供体)和PDPZ-Boc-Asn-GlcNAc(糖基受体)作为酶反应底物,进行Endo-M N175Q的转糖基反应,得到PDPZBoc-Asn-GlcNAc-N-糖链标记物。采用YMC C18色谱柱为分析柱,10 mmol/L甲酸铵-乙腈为流动相梯度洗脱,经液相色谱-串联质谱(LC-MS)检测得到5种高甘露糖型糖链。结果表明,丙酮可有效地富集大量肽和少量糖肽混合溶液中的糖肽,Endo-M N175Q可将天然糖肽的糖链转移到PDPZ-Boc-Asn-GlcNAc受体上。将该方法应用于胎球蛋白N-糖链分析,检测到5种复杂型N-糖链。该研究为各种糖蛋白N-糖链检测提供了新的分析方法。     

Endohexosaminidase-catalysed glycosylation with oxazoline donors: fine tuning of catalytic efficiency and reversibility

A complete series of oxazoline di-, tri-, tetra-, and hexasaccharides, corresponding to the core sections of N-linked glycoprotein high mannose glycans, together with the corresponding oligosaccharides containing a central glucose unit, were synthesised and tested as glycosyl donors for glycosylation of a GlcNAcAsn glycosyl amino acid catalysed by the endohexosaminidases M (Endo M), A (Endo A) and H (Endo H). Whilst Endo H did not catalyse any glycosylation reactions, both Endo M and Endo A efficiently catalysed glycosylations that were not limited to donors containing the Manbeta(1-->4)GlcNAc linkage. Precise structure activity relationships and time course studies have revealed fine-tuning of the efficiency of the synthetic processes which correlated both with the enzyme used and the precise oxazoline structure. Efficient irreversible glycosylation was achievable with both Endo M and Endo A, further demonstrating the use of structurally modified oxazoline donors as transition state mimics in order to promote enzyme-catalysed synthesis, whilst precluding product hydrolysis; enzymes in these cases display "glycoligase" activity.     

Identification of N‐glycans from Ebola virus glycoproteins by matrix‐assisted laser desorption/ionisation time‐of‐flight and negative ion electrospray tandem mass spectrometry

The larger fragment of the transmembrane glycoprotein (GP1) and the soluble glycoprotein (sGP) of Ebola virus were expressed in human embryonic kidney cells and the secreted products were purified from the supernatant for carbohydrate analysis. The N‐glycans were released with PNGase F from within sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS‐PAGE) gels. Identification of the glycans was made with normal‐phase high‐performance liquid chromatography (HPLC), matrix‐assisted laser desorption/ionisation mass spectrometry, negative ion electrospray ionisation fragmentation mass spectrometry and exoglycosidase digestion. Most glycans were complex bi‐, tri‐ and tetra‐antennary compounds with reduced amounts of galactose. No bisected compounds were detected. Triantennary glycans were branched on the 6‐antenna; fucose was attached to the core GlcNAc residue. Sialylated glycans were present on sGP but were largely absent from GP1, the larger fragment of the transmembrane glycoprotein. Consistent with this was the generally higher level of processing of carbohydrates found on sGP as evidenced by a higher percentage of galactose and lower levels of high‐mannose glycans than were found on GP1. These results confirm and expand previous findings on partial characterisation of the Ebola virus transmembrane glycoprotein. They represent the first detailed data on carbohydrate structures of the Ebola virus sGP. Copyright © 2010 John Wiley & Sons, Ltd.     

A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di‐N‐acetyllegionaminic Acid‐Containing Glycosides

A chemoenzymatic synthon was designed to expand the scope of the chemoenzymatic synthesis of carbohydrates. The synthon was enzymatically converted into carbohydrate analogues, which were readily derivatized chemically to produce the desired targets. The strategy is demonstrated for the synthesis of glycosides containing 7,9‐di‐N‐acetyllegionaminic acid (Leg5,7Ac₂), a bacterial nonulosonic acid (NulO) analogue of sialic acid. A versatile library of α2‐3/6‐linked Leg5,7Ac₂‐glycosides was built by using chemically synthesized 2,4‐diazido‐2,4,6‐trideoxymannose as a chemoenzymatic synthon for highly efficient one‐pot multienzyme (OPME) sialylation followed by downstream chemical conversion of the azido groups into acetamido groups. The syntheses required 10 steps from commercially available d ‐fucose and had an overall yield of 34–52 %, thus representing a significant improvement over previous methods. Free Leg5,7Ac₂ monosaccharide was also synthesized by a sialic acid aldolase‐catalyzed reaction.     

Highly Efficient Synthesis of Multiantennary Bisected N‐glycans Based on Imidates

The occurrence of N‐glycans with a bisecting GlcNAc modification on glycoproteins has many implications in developmental and immune biology. However, these particular N‐glycans are difficult to obtain either from nature or through synthesis. We have developed a flexible and general method for synthesizing bisected N‐glycans of the complex type by employing modular TFAc‐protected donors for all antennae. The TFAc‐protected N‐glycans are suitable for the late introduction of a bisecting GlcNAc. This integrated strategy permits for the first time the use of a single approach for multiantennary N‐glycans as well as their bisected derivatives via imidates, with unprecedented yields even in a one‐pot double glycosylation. With this new method, rare N‐glycans of the bisected type can be obtained readily, thereby providing defined tools to decipher the biological roles of bisecting GlcNAc modifications.     

Chemoenzymatic synthesis and Fcγ receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcγIIIa receptor

Structurally well-defined IgG-Fc glycoforms are highly demanded for understanding the effects of glycosylation on an antibody's effector functions. We report in this paper chemoenzymatic synthesis and Fcγ receptor binding of an array of homogeneous IgG-Fc glycoforms. The chemoenzymatic approach consists of the chemical synthesis of defined N-glycan oxazolines as donor substrates, the expression of the Fc domain in a CHO cell line in the presence of an α-mannosidase inhibitor kifunensine, and an endoglycosidase-catalyzed glycosylation of the deglycosylated Fc domain (GlcNAc-Fc homodimer) with the synthetic glycan oxazolines. The enzyme from Arthrobacter protophormiae (Endo-A) was found to be remarkably efficient to take various modified N-glycan core oxazolines, including the bisecting sugar-containing derivatives, for Fc glycosylation remodeling, resulting in the formation of the corresponding homogeneous Fc glycoforms. Nevertheless, neither Endo-A nor the Mucor hiemalis endoglycosidase mutants (EndoM-N175A and EndoM-N175Q) were able to transfer full-length complex-type N-glycan to the Fc domain, implicating the limitations of these two enzymes in Fc glycosylation remodeling. Surface plasmon resonance (SPR) binding studies with the synthetic IgG-Fc glycoforms unambiguously proved that the presence of a bisecting GlcNAc moiety could significantly enhance the binding of Fc to FcγRIIIa, the activating Fcγ receptor, independent of Fc core-fucosylation. Interestingly, the Fc glycoforms carrying an unusual bisecting sugar moiety such as a mannose or a LacNAc moiety also demonstrated enhanced affinity to FcγRIIIa. On the orther hand, the presence of a bisecting GlcNAc or core-fucosylation had little effect on the affinity of Fc to the inhibitory Fcγ receptor, FcγRIIb. Our experimental data also showed that the α-linked mannose residues in the pentasaccharide Man3GlcNAc2 core was essential to maintain a high affinity of Fc to both FcγRIIIa and FcγRIIb. The synthetic homogeneous Fc glycoforms thus provide a useful tool for elucidating how a fine Fc N-glycan structure precisely affects the function of the Fc domain.     

A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides and the Assembled Multivalent Glycopeptides

By displaying different O‐glycans in a multivalent mode, mucin and mucin‐like glycoproteins are involved in a plethora of protein binding events. The understanding of the roles of the glycans and the identification of potential glycan binding proteins are major challenges. To enable future binding studies of mucin glycan and glycopeptide probes, a method that gives flexible and efficient access to all common mucin core‐glycosylated amino acids was developed. Based on a convergent synthesis strategy starting from a shared early stage intermediate by differentiation in the glycoside acceptor reactivity, a common disaccharide building block allows for the creation of extended glycosylated amino acids carrying the mucin type‐2 cores 1–4 saccharides. Formation of a phenyl‐sulfenyl‐N‐Troc (Troc=trichloroethoxycarbonyl) byproduct during N‐iodosuccinimide‐promoted thioglycoside couplings was further characterized and a new methodology for the removal of the Troc group is described. The obtained glycosylated 9‐fluorenylmethoxycarbonyl (Fmoc)‐protected amino acid building blocks are incorporated into peptides for multivalent glycan display.     

Chemoenzymatic probes for detecting and imaging fucose-α(1-2)-galactose glycan biomarkers

The disaccharide motif fucose-α(1-2)-galactose (Fucα(1-2)Gal) is involved in many important physiological processes, such as learning and memory, inflammation, asthma, and tumorigenesis. However, the size and structural complexity of Fucα(1-2)Gal-containing glycans have posed a significant challenge to their detection. We report a new chemoenzymatic strategy for the rapid, sensitive detection of Fucα(1-2)Gal glycans. We demonstrate that the approach is highly selective for the Fucα(1-2)Gal motif, detects a variety of complex glycans and glycoproteins, and can be used to profile the relative abundance of the motif on live cells, discriminating malignant from normal cells. This approach represents a new potential strategy for biomarker detection and expands the technologies available for understanding the roles of this important class of carbohydrates in physiology and disease.