Challenges in Chemical Synthesis of Glycans and the Possible Problems Relevant to Condensed Matter Chemistry

Peng Xu; Biao Yu

doi:10.7536/PC220231

Progress in Chemistry ›› 2022, Vol. 34 ›› Issue (7) : 1548-1553 DOI: 10.7536/PC220231

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Challenges in Chemical Synthesis of Glycans and the Possible Problems Relevant to Condensed Matter Chemistry

Peng Xu ^*
, Biao Yu ^*

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Abstract

Glycans are the most abundant organic polymers in nature, and vital biomaterials for structural support and energy storage in living organisms. Meanwhile, glycans play an important role in cell recognition, differentiation, development, carcinogenesis and immunity. Compared with nucleic acid and protein, the specific role of glycans in many biological processes is still unknown, which is related to the difficulty of accessing well-defined glycans and the lack of precise tools for manipulating glycans in vivo. Synthetic methods in carbohydrate chemistry have been developed rapidly in recent decades, providing a powerful weapon for the study of synthetic glycans, especially oligosaccharides. Nevertheless, compared with the synthesis of nucleic acids and proteins, the synthesis of structurally well-defined glycans remains an unsolved chemical challenge with many unexpected problems. There are various factors that may affect the efficiency and stereoselectivity of glycosylation profile. Furthermore, glycans could be assembled into ordered aggregates through intermolecular non-covalent forces, then affecting the synthesis. For instance, in the process of removal protecting groups, the great change of solubility of glycan has a decisive effect on the reaction. The effect of aggregation formation on reactivity has not been thoroughly studied. Therefore, it is still necessary to complete the synthesis of well-defined glycan through trial-and-error experiments. In addition, glycans and glycoconjugates play an important role in living organisms by forming supramolecular structures. In conclusion, it is of great significance to study the condensed matter chemistry in glycans and their synthesis.

Contents

1 Structure,function and synthesis of glycans in living systems

2 Challenges and advances in the chemical synthesis of glycans

3 Some uncertainties in the chemical synthesis of glycans

3.1 The solvent effect

3.2 The concentration effect

3.3 The temperature effect

3.4 Problems in heterogeneous reactions

3.5 Solubility problem

4 Possible condensed matter chemistry problems

5 Prospect

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Keywords

glycans / chemical synthesis / aggregation / solubility / condensed matter chemistry

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Peng Xu, Biao Yu. Challenges in Chemical Synthesis of Glycans and the Possible Problems Relevant to Condensed Matter Chemistry. Progress in Chemistry, 2022, 34(7): 1548-1553 DOI:10.7536/PC220231

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1 Structure, Function and Synthesis of Glycans in Living System

Sugars are the most abundant organic molecules in nature. They exist not only in the form of monosaccharides or oligosaccharides such as glucose, ribose, fructose and lactose, but also in the form of polysaccharides or glycoconjugates covalently linked to other molecules (such as proteins)^[1]. Among them, high molecular weight starch and glycogen are the main energy storage substances of life, while cellobiose and chitin are the main structural support substances of plants and insects. More importantly, glycans on the cell surface are involved in various recognition processes of cells and are the molecular basis of cell differentiation, proliferation, adhesion, inflammation, and immune response (fig. 1)^[2].

Glycan is a linear or branched polymer molecule with monosaccharide as the basic unit and connected by glycosidic bonds. Among them, there are disaccharides with the smallest size and simple structure, such as sucrose, maltose and lactose. There are also oligosaccharides with more complex structures but not more than 20 monosaccharides. There are also a large number of natural polysaccharides. Subtle changes in the stereochemistry of monosaccharide units, glycosidic bonds, or linkage positions can cause significant changes in the physicochemical properties and functions of glycans. For example, amylose and cellulose are both glucans with (1 → 4) linked glucoses as repeating units, which differ only in the stereochemistry of the glycosidic linkage. Amylose is linked by α-glucosidic bonds, and the more curved α-glucosidic bonds are beneficial to the formation of helical structure. Cellulose, on the other hand, is linked by β-glucosidic bonds and is prone to form extended structures. This small difference in the stereochemistry of the glycosidic bond not only results in clear differences in physicochemical properties, such as water solubility, but also demonstrates completely different biological functions. Amylose is used to store chemical energy obtained from the process of photosynthesis; Cellulose is used as the structural material of plants. Other β-glucans with different linkages (such as 1 → 3 and 1 → 6 linkages) and branching are widely distributed in bacteria, algae, fungi and cereals, and have diverse biological functions and medicinal activities^[3]. For example, β-glucan in intestinal commensal bacteria Bifidobacterium can induce the production of regulatory immune cells and better inhibit colitis^[4]. Oral β-glucan can increase insulin secretion and stabilize blood glucose levels to achieve diabetes control^[5].

2 Challenges and Advances in Chemical Synthesis of Glycans

Compared with nucleic acids and proteins, the role of glycans in many life processes has not yet been unveiled, which is closely related to their unique and complex molecular structure. Nucleic acids and proteins are linear molecules connected by phosphodiester bonds and amide bonds respectively, while glycan compounds are connected by glycosidic bonds and have alpha and beta configurations, and monosaccharides are polyhydroxy aldehydes or ketones, which can form branched structures. In addition, there are hundreds of monosaccharides in nature, which are the basic units of glycans. Moreover, a large number of hydroxyl groups in glycan structures are modified in different ways, such as acetylation, methylation, phosphorylation and sulfation. The invention and application of technologies such as automatic sequencing, automatic synthesis and gene expression of proteins and nucleic acids have greatly accelerated the research process of genomics and proteomics. However, in the research of glycans, the automatic synthesizer of sugar chains can only be used for the synthesis of some simple glycans at present^[6].

The chemical synthesis of glycans with precise structures remains an unsolved challenge, and the main reasons include the difficulty of controlling the regioselectivity of multiple hydroxyl groups with similar activities; It is difficult to control the stereochemistry of the glycosidic bond; High molecular weight glycans are difficult to obtain. There are currently three methods for the synthesis of high molecular weight glycans: (1) stepwise glycosidation; (2) condensation polymerization; (3) ring-opening polymerization^[7]. Polycondensation and ring-opening polymerization can only synthesize mixtures of glycans; Stepwise glycosidation can synthesize structurally defined glycans, but the synthesis efficiency is very low, and only a few types of glycans can be synthesized. For the precise synthesis of glycans, chemists have developed a large number of orthogonal protecting groups, glycosidation methods, and synthetic strategies. The orthogonal protection strategy is used to selectively shield the hydroxyl groups on the sugar ring, leaving only the hydroxyl groups at specific sites for subsequent glycosidation reactions, but the preparation process of the required sugar building blocks with orthogonal protecting groups is time-consuming and laborious. On the other hand, stereoselective control of glycosidic bond formation is also a great challenge. Although selective glycosidation reactions have been extensively studied, the universality of these methods is still not ideal^[8]. In the past few decades, a large number of new glycosidation methods (including glycosyl donors and promoters) have been reported, and glycochemists have begun to study the synthesis of natural or unnatural polysaccharides^[9]. At present, it is still very difficult to synthesize longer and larger glycans by chemical total synthesis, because the molecular weight of structurally defined glycans is usually less than 10 000 G/mol (degree of polymerization less than 60)^[7].

3 Some Uncertainties in Chemical Synthesis of Glycans

There is no universal method for the chemical synthesis of glycans, and unexpected experimental results are often encountered^[6,10]. For example, glycosidation is a key reaction in glycan synthesis, and its reaction mechanism is usually that the glycosyl donor is activated to form a glycosyl oxonium ion, which is then attacked by a nucleophile to form a glycosidation product^[11]. Among them, many factors may affect the efficiency and stereoselectivity of glycosidation, such as solvent, concentration of donor and acceptor, temperature, etc.

3.1 Solvent effect

Solvent, as the most abundant substance in the glycosidation system, has a significant effect on the reaction. There may be an interaction between the oxonium ion intermediate formed after the dissociation of the leaving group of the glycosyl donor and the solvent under promoter activation conditions. Therefore, according to the polarity of the solvent and the ability to provide lone pair electrons, it can be divided into: (1) solvents with low polarity and weak coordination, such as benzene and toluene; (2) Moderately polar weakly coordinating solvents, such as dichloromethane, nitromethane, and 1,2-dichloroethane, are the most commonly used reaction solvents, which have excellent solubility for organic substrates even at low temperatures and do not cause side reactions; (3) Solvents with high polarity and strong coordination, such as acetonitrile and propionitrile, which have strong solvation effect, can form positive ion complexes with glycosyl oxonium intermediates and significantly affect the stereoselectivity of glycosidation^[12]; (4) Solvents with low polarity and strong coordination, such as diethyl ether, dioxane, and tetrahydrofuran. Unlike highly polar solvents, although they have strong coordination ability, they have very low dissociation ability for ions, and the stability of ion pairs in these solvents is higher than that in nitriles. When they are used as solvents for glycosidation, they also significantly improve the selectivity of glycosidation.

In the synthesis of polysaccharide polycondensation, the reaction is a stepwise growth process, and the high molecular weight of the polycondensation product can be achieved only when the conversion rate is more than 90%, the theoretical degree of polymerization is only 10 when the conversion rate is 90%, and the theoretical degree of polymerization can reach 100 when the transformation rate is 99%^[13]. Du's research group used dichloromethane as a solvent and tetrabutylammonium iodide as an accelerator to prepare polysaccharide polyorthoester by polycondensation of 2,3,4-triacetylglucobromoside under reflux conditions, and the molecular weight of the prepared polysaccharide reached 6600 ~ 6900 G/mol (Figure 2)^[14]. However, when the solvent was changed to benzene, toluene, and pyridine, the molecular weights of the glycans were all less than 1700 G/mol. Due to the high sensitivity of orthoester structures to acidolysis, such glycans may be used to synthesize highly pH-responsive materials that can rapidly and selectively deliver drugs to malignant tissues with low extracellular pH.

3.2 Concentration effect

In 1997, Boons' group found that the concentration of donor and acceptor had a slight effect on the selectivity of [1 + 1] glycosidation reaction when they studied the glycosidation of perbenzylglucosinolate donor and dipropylidene galactose acceptor^[15]. A 5-fold reduction in concentration increased the α/β selectivity from 5.1: 1 to 7.0: 1. Many research groups have encountered similar concentration effects^[16]. For example, Gao Jian's research team of Shandong University realized the specific selective synthesis of branched α-glucan decasaccharide in 2020 (Figure 3). Interestingly, when the pre-activation strategy [3 + 3] and [6 + 4] glycosidation reactions were carried out, when the reaction concentration of donor and acceptor was reduced from 20 mM to 5 mM, the α/β selectivity could be increased from 4:1 to 1:0, which greatly improved the efficiency of glycan synthesis^[17].

Kononov group of Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, found that when studying the glycosidation reaction of all-acetyl-protected sialic acid thioglycoside donors,The introduction or concentration change of exogenous amide/imine can affect the yield and stereoselectivity of sialylation reaction, which is speculated to be due to the change of hydrogen bonding mode of sialoglycosyl donor in solution^[18]. Later, they used supramolecular polymer analysis to prove that 2,3,5-tri-O-benzyl-arabinofuranose bromo glycoside existed in acetonitrile by dynamic light scattering.Nanoscale and mesoscale superpolymers exist in 1,2-dichloroethane, and the formation of these superpolymers has a certain influence on the selectivity of glycosidation^[19].

3.3 Temperature effect

The reaction temperature has a very important effect on the glycosidation yield, stereoselectivity, and degree of polymerization^[20]. In general, the solubility of the reactant increases and the reactivity of the reagent is enhanced at higher temperatures. Valverde's group in Spain found that the temperature had a great influence on the stereoconfiguration of the product during the template-directed synthesis of cyclic glycosides (Fig. 4)^[21]. In the intramolecular glycosylation of disaccharides, there was no stereoselectivity in the formation of cyclic disaccharide products at room temperature (β/α = 1 ∶ 1). When the temperature is lowered, the β selectivity appears, and at -50 ℃, the selectivity is slightly improved (β/α = 1.7 ∶ 1); When the temperature was lowered to -78 ℃, the β selectivity increased to 5.5 ∶ 1. Sharkey et al. Of the State University of New York also found a similar phenomenon when synthesizing glycans by 1,2-epoxy ring opening glycosylation. Ring-opening polymerization using a Lewis acid such as phosphorus pentafluoride as an initiator under high temperature conditions has poor stereoselectivity and is prone to the formation of a considerable number of isomers. At low temperature, the polymerization proceeds mainly through the trialkyloxonium mechanism to give 1,2-trans glycans. However, at high temperature, the oxonium intermediate is dominant and the thermodynamically stable α-configuration glycan is easily formed. Taking 3,4,6-O-tribenzyl-1,2-epoxyglucose as an example, the reaction was initiated by phosphorus pentafluoride at -60 ℃ for 4 H, and the molecular weight could reach 10000 G/mol, with 90% of β- (1 → 2) glycosidic bonds and 10% of α glycosidic bonds^[22]. Both the degree of polymerization and the stereoselectivity decrease when the temperature is raised or the promoter is changed.

3.4 Problems in heterogeneous reactions.

In the glycosidation reaction, it is generally necessary to add a drying agent to remove the trace water contained in the solvent and the reactants, so the reaction system becomes heterogeneous. Mukaiyama's team at Tokyo University of Science found that adding different desiccants to the same glycosidation reaction had different effects (Figure 5)^[23]. The glycosidation reaction failed to proceed with the addition of molecular sieves of 3.3Å or 4.4Å. However, the glycosidation reaction could be carried out smoothly with the addition of 55Å molecular sieve, and the desired disaccharide (α/β = 88/12) was obtained in 98% yield. The addition of calcium sulfate as a drying agent slightly decreased the glycosidation yield (88%, α/β = 90/10). Kakuchi's research group at Hokkaido University found that when 2-butenylthiophenium hexafluoroantimonate was added as an initiator, the ring-opening polymerization of 1,6-deoxymannose was homogeneous in the first 5 min, and then gradually turned into a heterogeneous reaction to form non-gel-like highly branched mannan^[24].

3.5 The problem of solubility

In recent years, the preparation of glycans with precise structures and more than 20 monosaccharides by stepwise synthesis has been reported, in which the removal of sugar chain protecting groups is a major difficulty^[25]. Glycan synthesis involves a large number of protecting groups, and their complete removal requires more drastic reaction conditions and longer reaction times. The reaction intermediate after partial removal of the protecting group or the final product after complete removal of the protecting group has a great change in solubility in the solution system, and aggregation occurs due to the change of the weak interaction force. Aggregates may precipitate from the solution and affect the progress of the reaction. The stability, crystallinity and poor water solubility of cellulose glucans are caused by their dense intermolecular and intramolecular hydrogen bonding network and hydrophobic interaction, including the hydrogen bonding between the oxygen atoms of the 3-position hydroxyl and the 5-position hydroxyl of the adjacent glucose residues. Intramolecular and intermolecular hydrogen bonds between the 6-hydroxyl group and the 2-hydroxyl group of the sugar ring; Hydrophobic interactions between polar surfaces of sugar rings rich in C-H bonds as well as van der Waals forces. Seeberger and Delbianco of Max Planck Institute for Colloid and Interface in Germany synthesized a series of cellulose and its derivatives, and found that the yield of protection group removal of glycans was related to the length of sugar chain, modification mode and functionalization degree^[26]. For example, cellohexaose and dodecasaccharide have aggregation effect in solution system and solubility change of intermediate when the protecting group is removed; The yield of cellohexaose protective group removal was 18%, while the yield of cellododecose removal was reduced to 2%. The introduction of a methyl protecting group at the 3-position of the sugar ring of the cellododecasaccharide moiety destroys the corresponding hydrogen bonding, and the efficiency of deprotection can be increased by more than 3.5 times. Yu Biao's research group of Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, also encountered similar problems in the synthesis of bacteroid lipopolysaccharide O-antigen 128 sugar. The final protecting group removal process involves the cleavage of 258 protecting groups and 322 carbon-oxygen bonds. The solubility of the glycan substrate changes with the progress of the reaction. The removal of 64 benzylidene and 64 benzyl protecting groups by reductive hydrogenation requires a change in the composition of the reaction solvent. The total yield of the removal of protecting groups is only 15%, which is much lower than the 52% deprotection yield of octasaccharide (Figure 6)^[25f].

4 Possible Condensed Matter Chemistry Problems

Glycan self-assembles into aggregates through intermolecular noncovalent interactions, which is the structural basis of biomolecular interactions to form supramolecules. In which weak interaction forces manipulate glycan self-assembly to form functional higher order structures. In 2019, Seeberger and Delbianco's team used a series of chemically synthesized oligosaccharides to study the influence of sugar chain length and structure on aggregation mode^[27]. Spherical nanoparticles (diameter 40 ~ 60 nm) were formed by dialysis of β1 → 6-linked glycans, while self-assemblies with different morphologies (such as rods, needles, nanoparticle aggregates, spheres, etc.) Were formed by rapid solvent displacement method; β1 → 4-linked oligosaccharides did not form ordered supramolecular self-assemblies due to their rigid structure. After that, the authors used disaccharides to study the effect of assembly conditions on the structure of saccharide self-assemblies. Nanofibers were formed by dialysis at high concentrations, and needle-like self-assemblies were easily formed by solvent displacement. Acetone is used as a solvent to obtain a gel-like micron-wire structure. The authors surmise that these self-assembly behaviors are related to prolonged π-conjugated systems and/or charge delocalization.

Among them, four disaccharides will emit different fluorescence under the excitation light of four channels due to self-assembly. Using X-ray powder diffraction experiments, they found that the interaction with water can disturb the morphology of aggregates, and the higher crystallinity in self-assemblies is related to the quenching of emission light, while verifying that one of the disaccharides has unique optical properties different from fluorescent molecules.The absorption range is much larger than that of the free material, the emission range is between 350 nm and 500 nm, and the luminescence quantum yield is about 0. 85 under 360 nm excitation. Most surprisingly, the maximum emission wavelength of this disaccharide is red-shifted with increasing excitation wavelength.

5 Outlook

More than 120 years have passed since Fisher, a Nobel Prize winner in chemistry, first determined the chemical structure of monosaccharides. Thousands of carbohydrate molecules have been discovered and synthesized, and these carbohydrates can exist in different forms of aggregation. In biological systems, the aggregation state of glycans shows different biological effects and participates in various physiological functions under normal conditions. Under abnormal conditions, the formation and change of aggregation may become a key link in pathological or toxicological processes^[28]. In the application of natural glycans, a large number of materials with various properties have been prepared by chemical modification to change the aggregation morphology. In the process of glycan synthesis, the change of aggregation state of glycan synthesis intermediates and end products affects the progress and selectivity of the reaction. However, the characterization of the aggregation state and its influence on the reaction are still unknown, resulting in "unexpected" results in glycan synthesis. Therefore, it needs to be paid attention to and studied in depth in future work.

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