Carbohydrates have been hard to assemble chemically, but the increasing awareness of the biological importance of this class of complex structure has pushed efforts to accelerate their synthesis. This paper describes how the synthesis of oligosaccharides can be automated by using the solid-phase synthesizer. And how various oligosaccharides can be synthesized using a protecting scheme that is unique to each structure.
Oligosaccharides are an important class of biomolecules, and are involved in many biochemical processes3. In biological organism, post-translation modification of certain proteins includes the linkage of various oligosaccharides to amino acids side chains to form glycoproteins. For examples, N-Glycans are attached to the side chain of asparagine via an N-glycosidic linakge, and are present in 90% of all glycoproteins4. O-Glycans are attached to the side chains of threonine or serine.
Carbohydrates also participate in the modification of proteins to form proteoglycans, which is another class of biologically essential marcomolecules. Proteoglycans are located in the extracellular matrix in the cells. The basic proteoglycan unit consists of a core protein with multiple linear sugar chains known as Glycosaminoglycans (GAGs) chain covalently attached. Examples of GAGs includes chondroitin sulfate, keran sulfate, or heparin sulfate that interact with various enzymes, growth factors, cytokines, morphogens, and cell adhesion molecules and play a central role in cell signaling6.
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In biological systems, cell surface glycoconjugates such as glycolipids participate in cell-cell communication. Glycolipds such as Glycosylphosphatidylinositol (GPI) anchors facilitate the transduction pathway and cellular recognition processes. GPI anchors attached to the C-terminus of a peptide chain during post-translation modifications that serve to attach protein to the cell surface. GPI anchors are mainly found in eukaryotic cells and protozoan parasites5 and play a role in Plasmodium falciparum infections that cause malaria.
However, a third major class of biopolymer behind peptides and oligonucleotides, oligosaccharides are not as extensively studied despite the importance of these complex carbohydrates in the biological systems.
Access to pure carbohydrates remains challenging and has impeded biological investigations2. The main reason why carbohydrates are not as extensively studied upon is due to the lack of pure, structurally defined carbohydrates and glycoconjugates1. Oligosaccharides in biological system are usually found in miniscule concentration and in microheterogeneous form in nature. Isolation and identification of oligosaccharides from nature sources for biological studies is therefore difficult and inefficient. Other alternative includes the cultivation of eukaryotic cells for oligosaccharide isolation, however such method is expensive, tedious and low yielding. The only way to obtained sufficient amount of oligosaccharides for biophysical and biochemical studies is through efficient synthetic methods.
However, classical solution phase synthesis of oligosaccharides is inefficient and tedious as a different synthetic strategy is required for each molecule. Oligosaccharides pose such synthetic challenge unlike peptides or oligonucleotides, which only form linear chains; the monomers of each oligosaccharide chain can be linked in many different ways. For the consideration of an efficient oligosaccharides synthetic methodology, automated solid-phase synthesis of oligosaccharides was developed.
Difficulty of oligosaccharide synthesis
As compared to peptides and oligonucleotides, oligosaccharides are more difficult to synthesize owing to its branched nature and the need to control its stereochemistry of the formation of its glycosidic bond. Peptides and oligonucleotides are strictly linear biopolymers assembled from four nucleotide building blocks or 20 amino acids respectively. In mammals, simple carbohydrate can consists of 10 or more different types of monosaccharides. In biological systems, oligonucleotides are typically connected lipid or a protein to form glycocojugates, and normally the structures of the oligosaccharides are more complicated than the peptides and oligonucleotides itself.
The synthesis of oligosaccharides can be mainly controlled in two manners. By functionalizing the hydroxyl groups on monosaccharide moiety during oligosaccharides formation, the regiochemistry of the oligosaccharides can be controlled. A protection scheme using of permanent and temporary hydroxyl protection is implemented. Permanent protecting groups, such as benzyl ethers can be implemented at hydroxyl groups not involved during the formation of glycosidic bonds. Temporary protecting groups, such as ester can be implemented at hydroxyl groups that eventually need to form bonds during the synthesis. Deprotecting the hydroxyl group can allow hydroxyl to act as nucleophilic acceptor during glycosidic bond formation.
Formation of a stereo specific glycosidic bond also poses some difficulty in the synthesis of oligosaccharides (Fig.1). Glycosidic bond formation requires the activation of an electrophilic glycosyl donor at the anomeric carbon centre of one monosaccharide, to react with the nucleophilic glycosyl donor, which is normally a hydroxyl group. Glycosidic bond formation can occur in two stereospecific pathways, resulting in the formation of either α- or β- anomers. To control the stereospecific formation of glycosidic bond, neighbouring group participation of hydroxyl protecting group at C2 can be used. For instance, ester-protecting group at C2 hydroxyl can form a cyclic oxonium ion intermediate that shields the attack of nucleophilic acceptor at one face, resulting in the formation of trans-glycosidic linkages.
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Fig. 1. The Glycosidic bond formation can occur in two stereospecific pathways, resulting in the formation of either α- or β- anomers. Glycosidic bond can be stereoselectively form by using of neighbouring participating group to limit the nucleophilic attack of glycosidic acceptor at one face leading to the formation of β- anomer.
Implementation of automated solid-phase synthesis
The paradigm shift to solid-phase synthesis of oligosaccharides begins in 1971 after Frechet successfully synthesized di- and trisaccharides on a polymer support7. Although the methodology is constantly improving to allow the synthesis of more complicated oligosaccharides; solid phase synthesis of oligosaccharides still remains unproductive and time consuming. Therefore, by automation of the solid-phase synthesis of oligosaccharides, target oligosaccharide can be synthesized in a more convenient way. Advantages of the automated solid-phase synthesis of oligosaccharides includes high synthesis yield by using excess reagent to drive reaction to completion, improved purity of the obtained product and reduced the amount steps needed for purification.
The automated oligosaccharide synthesizer needs to fulfil certain criteria before it can be implemented successfully:
A device that is able to perform repetitive chemical synthesize at controlled temperature.
Synthetic approach with either the glycosidic acceptor or the glycosidic donor end of carbohydrate attached to the solid support.
Selection of a linker that is chemically inert to all synthetic reactions.
A protection scheme that leads to the effective yield of the target oligosaccharides.
Regio and stereo control of the formation of oligosaccharides.
The machine used for the automated synthesis of peptides was adopted and modified for the automated synthesis of oligosaccharides. The synthetic strategy proposed involves exposing the nucleophilic hydroxyl acceptor of the monosaccharide on the solid support, and additional of a monosaccharide with exposed electrophilic donor in solution for coupling to occur. Successive deprotection of the temporary protection group on the new saccharide will reveal new hydroxyl to continue coupling reaction. Further coupling will yield the targeted oligosaccharides bounded to the solid support which can be easily de-protect, purify and cleaved from the solid support to obtain the desired oligosaccharides.
Synthesis of α-mannosides
In this literature, three oligosaccharides with different structure complex were synthesized to explore the viability of the automated oligosaccharide synthesis methodology. Due to the structural difference of the oligosaccharides, different synthesis and protection scheme was used for each oligosaccharide. The main aim of all syntheses is the development of a general synthetic method to construct any oligosaccharide or oligosaccharide analogue.
The synthesis of α-mannosides was chosen for the automation process as α-mannosides have been synthesized in solution and on solid support before. Moreover, due to the occurrence of α-mannosides in biological structures such as glycolipids, α-mannosides synthesis has been the focus of biological research.
α-mannosides consists of a series of α-(1→2) glycosidic linkage, linking the mannosides together. Trichloroacetimidate donor 2 (Scheme 1), was choose as the synthetic building block due to ease of preparation, and it has ester functional group on carbon 2 that can control the stereo configuration of the glycosidic bond formation via neighbour group participation, hence the formational of α-(1→2) glycosidic linkage. Trichloroacetimidate donor 2 was initially activated with Lewis acid trimethylsilyl trifluoromethanesulfonate (TMSOTf) under acidic condition. The activated donor 2 then coupled with olefin linker 1 bounded to solid support(1% cross-linked polystyrene) to form the first monosaccharide in the oligosaccharide synthesis. Olefin linker 1 was used as it was inert to the coupling cycle conditions and easily cleaved from the solid support via olefin cross metathesis. Acetyl ester protecting group was then removed from Carbon 2 using sodium methoxide under basic conditions to expose the nucleophilic hydroxyl acceptor. The coupling cycle is completed and can be repeated with the addition of another Trichloroacetimidate donor 2 to elongate the oligosaccharide chain with repeating units of Trichloroacetimidate. Using the automated oligosaccharide synthesis methodology, pentamannoside 3 was synthesized in 14 hours. Initial characterisation of the resin bounded pentasaccharide 6 shows high purity even without purification after 9 synthetic steps. Heptamer 4 and decamer 5 were prepared, averaging yield of 90 to 95% per step. Each monomer took about 3 hours to synthesize, therefore the synthesis of heptamannoside 4 was completed in 20 hours with a 42% overall yield, as compared to 14 days and 9% overall yield for manual solid support synthesis. Therefore, automated oligosaccharide synthesizer can construct linear oligosaccharide at a faster rate and higher yield.
Synthesis of Phytoalexin elicitor (PE) β-glucan
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Phytoalexin elicitor (PE) β-glucan was chosen and synthesized using the automated oligosaccharide synthesizer due to its biological mode of action with soybean plant. In the presence of fungal β-glucan, soybean plant can be triggered to release antibiotic phytoalexins. This defence mechanism by soybean plant is a very well explored1. These oligosaccharides have been synthesized previously in solution8 and on the solid support8, hence were expected to serve well as a benchmark for the automated oligosaccharide synthesis. The synthesis of the branched β-(1→3)/β-(1→6) glucan structure was carried out using two glycosyl phosphate building blocks 8 and 9. Each phosphate building block was activated with TMSOTf for nucleophilic hydroxyl attack. A levulinoyl ester served as a 6-O temporary protecting group on building block 8 and the 2-O pivaloyl group on building block 9 ensured the complete trans-selectivity in the glycosylation bond formation. Levulinoyl ester can be deprotected using hydrazine solution in pyridine/acetic acid. The alternation of a disaccharide phosphate building block with a monosaccharide will results in the alternating elongation of the branched structure. Using automated oligosaccharide synthesis and the above synthetic scheme, branched hexasaccharide 10 was synthesized in 10 hours with more than 80% yield, measured by HPLC anaylsis. Dodecasaccharide 7 (Fig. 3) was synthesized in 17 hours with more than 50% yield under the same reaction scheme. The results show that automated oligosaccharide synthesis is truly more efficient and less tedious as compared to conventional methods for polysaccharide synthesis.
Fig. 2. Dodecamer phytoalexin elicitor β-glucan.
Synthesis of trisaccharide 13
From the synthesis of the α-mannosides and Phytoalexin elicitor (PE) β-glucan, the effectiveness of glycosyl phosphates and trichloroacetimidates as glycosidic donor were affirmed. In addition, the usage of using leuvulinate ester and acetate in unison seems to serve well as a general temporary protection scheme for hydroxyl in terms of regio and stereo control. Hence, trisaccharide 13 was synthesized to test the generality of this methodology, by incorporating all aspects of the automated chemistry that have been studied so far. The glycosidic bonds of trisacharride 13 are at the C2 position of mannose with glucosamine donor and the C4 hydroxyl of glucosamine with galactose donor. Formation this two challenging linkages in trisaccharide 13, often leads to side reactions9.
Monosaccharide building block 2, 11 and 12 was prepared and used for the automated synthesis of trisaccharide 13. Donor 2 was chosen in accordance to the formation of α-mannosides, where α-(1→2) glycosidic linkage was synthesized. Monomer 11 was designed in a way such that the phthalimide amine-protecting group at C2 can act as a neighbouring participating group that limits the formation of only α-glycosidic bond. Levulinate ester at C4 of monomer 11 permits the rapid deprotection with hydrazine as demonstrated in the synthesis of the PE β-glucan. Scheme 3 shows the automated synthesis of trisaccharide 13 with an overall yield of 60% for the crude product after cleavage from solid support. After the full deprotection of trisaccharide 13, n-pentyl glycoside 14 achieved in 62% yield.
In summary, the examples stated by the literature clearly illustrate the major improvements in time and yield as compared to the conventional synthesis of oligosaccharide. Such as the implementation of a glycosylation/deprotection cycle for the synthesis of decamer consisting of α-(1→2) mannoside. Branched oligosaccharide, such as hexasaccharide 10 and dodecasaccharide 7 synthesized with much faster duration as compared to its conventional synthesis. Lastly, synthesis of complicated trisaccharide 13 using both glycosyl phosphate and trichloroacetimidate donors illustrate in the previous two examples, and using of protecting scheme based on acetate and levulinate esters. Although, automated approach for oligosaccharide produces remarkable improvements over conventional oligosaccharide, it does have its limitations and shortcomings. Hence, in this discussion, the limitations of automated oligosaccharide will be closely examined, as well as the suggestion of possible of improvements. Last but not least, reference will be made to other similar synthetic approach to discuss the possibility of incorporating it into automated oligosaccharide synthesis methodology for better efficiency and yield.
Limitation and improvements
From the literature, it can be seen that using of automated oligosaccharide does show significant improvements over in its yield and time as compared to conventional synthetic methods; it is especially significant if compared to classic solution phase synthesis. However, as observed, the percentage yield of the oligosaccharide using automated solid phase synthesizer still varies over a lower range of about 40 to 85%. The overall yield further decrease when the number of oligosaccharide monomer increase in the oligosaccharide chain. Yield also decreases when branching of oligosaccharides increased.
The decrease of yield can be attributed by a few factors. First, the anomeric effect at the anomeric carbon of carbohydrate that favours the formation of α-glycosidic over β-glycosidic bond. Even with the neighbour group participation at C2 by ester protecting group, which confer β-selectivity during glycosylation, some α-glycosidic linkages will still be formed. This effect will be even more prominent when number of glycosidic bonds increase due to the synthesis of longer and branched oligosaccharides. Another side reaction that can occur during glycosylation is the formation side product 16 from oxonium intermediate 15. When a non-bulky ester is used for neighbouring group participation to confer β-selectivity, the oxonium ion might be susceptible to nucleophilic attack by small hydroxyl groups, leading to the formation of side product 16. Hence, decrease in overall yield. To prevent the formation of such side products, bulky O-pivaloyl (as illustrated in this literature) or even O-benzoyl can be used as ester protecting group at C2.
Improvements in the yield of the automated synthesis of the oligosaccharide can be achieved by increasing using excessive reactive electrophilic donor. One of the advantages of solid phase oligosaccharides is the anchoring of the nucleophile which allows for an excess of the reactive donor to be used to drive the reaction to completion1. Excess reactive electrophilic donor can be easily washed off using organic solvent like dichloromethane.
In this literature, the yield of oligosaccharide synthesis was measured at the end of the completion of each oligosaccharide. However, rapid assessment of the success of each coupling reaction will more beneficial to determine the effectiveness of the overall reaction. By recognizing a problematic, incomplete coupling instantaneously, the synthesis can be aborted and valuable building block can be conserved. Therefore, improvements can be made by the use of UV-active protecting groups to allow the real-time monitor of the success of the automated synthesis. By measurements of the absorption the deprotection solutions allows for calculation of the material bound to the polymer support1.
Prospective future of automated oligosaccharide synthesis
The largest challenging problem in the large-scale chemical synthesis of oligosaccharides is the control of the stereo- and regio-chemistry of the bond formation10. As seen in this literature, the specific use of certain protecting groups manipulations are needed to control the stereo- and regio-chemistry of the products. Even with automated oligosaccharide synthesis, this demand for specific protection scheme will still hinder the efficient production of oligosaccharide for biological testing11. Recent developments in the oligosaccharides methodology includes the synthesis of oligosaccharides using enzyme catalyst based on two major classes of enzyme: the glycosyl transferases and the glycosidases. The main advantage of this approach as compared with conventional synthesis is that the region- and stereo-selectivity of the target oligosaccharide can be achieved without the need of protecting functional group. Enzyme can orient the glycosyl donor and acceptor to catalyse the formation of a specific linkage.
The β-D-mannoside linkage is found in several biological structures, most commonly found in N-linked glycoproteins, is one of the most difficult glycosidic bond to synthesized chemically12,13. The enzymatic transfer of β-mannosyl residues was successfully obtained with retaining β-glycisidases. The usage of β-mannosynthase allows the synthesis of β1,3 or β1,4 mannosides with even much higher yields. Therefore, by incorporating the use of enzyme into automated oligosaccharide synthesis in the near future, the selectively use of protecting scheme can be eradicated, further improving the efficiency of automated oligosaccharide synthesis.
Development t in many aspects of oligosaccharide synthesis is essential for the progress of future biological investigations. Such as the adaptation of automated oligosaccharide synthesis, and in the near future the use of enzyme controlled oligosaccharide synthesis. The improvement in acquiring defined structure from a machine will impact the field of glycobiology such that we may one day be able to fully appreciate the importance of oligosaccharides in nature.