Chemical synthesis of polysaccharides

Here we summarize the chemical syntheses of glycans containing over 20 monosaccharide units, with the recent syntheses of ultra-long glycans, including a 92mer arabinogalactan, a 100mer and a 151mer mannan, and a 128mer rhamnomannan, being highlighted. The assembly strategies, glycosylation methods, and protecting group manipulations, which are crucial to the successful synthesis, are discussed to give guidance for the future synthesis of various polysaccharides.     

Synthesis of a tetrasaccharide related to the O-antigen from Azospirillum lipoferum SR65

Concise synthesis of a tetrasaccharide repeating unit of the LPS isolated from Azospirillum lipoferum SR65 has been accomplished through suitable protecting group manipulations and stereoselective glycosylation starting from commercially available l-rhamnose and d-glucose. The target oligosaccharide in the form of its p-methoxyphenyl glycoside is suitable for further glycoconjugate formation via selective cleavage of the OMP glycoside. Plant growth-promoting bacteria (PGPB) of genus Azospirillum plays important roles in the growth and development of plants. The interaction between the roots of the plants and the microbes is governed by the cell surface carbohydrate polymers (CPS, LPS, etc.). The present synthetic-based study elucidates aspects of plant-microbe interaction and future biofertiliser design.     

Synthesis and reactivity of novel γ-phosphate modified ATP analogues

We hereby present a simple yet novel chemical synthesis of a family of γ-modified ATPs bearing functional groups on the γ-phosphate that are amenable to further derivatization by highly selective chemical manipulations (e.g., click chemistry, Staudinger ligations). A preliminary screen of these compounds as phosphate donors with a typical wild type protein kinase (cdk2) and one of its known substrates p27^kip1 is also presented.     

Recent trends in the synthesis of O-glycosides of 2-amino-2-deoxysugars

The discovery of new methods for stereoselective glycoside synthesis and convergent oligosaccharide assembly has been critical for the area of glycosciences. At the heart of this account is the discussion of the approaches for stereoselective synthesis of glycosides of 2-amino-2-deoxysugars that have emerged during the past two decades. The introductory part provides general background information and describes the key features and challenges for the synthesis of this class of compounds. Subsequently, major approaches to the synthesis of 2-amino-2-deoxyglycosides are categorized and discussed. Each subsection elaborates on the introduction (or protection) of the amino functionality, synthesis of glycosyl donors by introduction of a suitable leaving group, and glycosidation. Wherever applicable, the deprotection of a temporary amino group substituent and the conversion onto the natural acetamido functionality is described. The conclusions part evaluates the current standing in the field and provides a perspective for future developments.     

Non-polymerase-cycling-assembly-based chemical gene synthesis: strategies, methods, and progress

Chemical gene synthesis is a powerful tool for basic biological research and biotechnology applications. During the last 30 years, major advances have been made in the chemical synthesis of DNA sequences ranging from fragments of <1 kb to multi-gene sequences of >30 kb. There is a need for simple, reproducible, less error-prone and cost-effective methods that guarantee successful synthesis of the desired genes and are amenable to automation. Many polymerase chain reaction (PCR)-based and non-polymerase-cycling-assembly (PCA)-based strategies have been developed for chemical gene synthesis. The PCR-based method has been the subject of several recent reviews. Here, we provide an overview of the progress in non-PCA-based chemical gene synthesis using different strategies and methods, including enzymatic gene synthesis, annealing and ligation reaction, simultaneous synthesis of two genes via a hybrid gene, shotgun ligation and co-ligation, insertion gene synthesis, gene synthesis via one strand of DNA, template-directed ligation, ligase chain reaction, microarray-mediated gene synthesis, Blue Heron solid support technology and Sloning building block technology. The fundamental principle underlying each strategy, an example where applicable, and the advantages and disadvantages are discussed. The emphasis is on discussion of the most recent technologies and their potential applications, particularly for microarray-based genomics research.     

Glycosyltransferase activity can be selectively modulated by chemical modifications of acceptor substrates

A range of N-acetyllactosamine derivatives, which are modified by a wide range of functionalities at C-2(') and C-6, have been synthesised and the kinetic parameters of transfer catalysed by recombinant alpha-2,6-sialyltransferase and alpha-1,3-fucoyltransferase VI determined. Several of the chemical modifications led to selective modulate the activity the enzymes and offer promising lead compounds for the development of oligosaccharide primers for selective metabolic inhibition of oligosaccharide biosynthesis.     

Towards tailor-made oligosaccharides—chemo-enzymatic approaches by enzyme and substrate engineering

Carbohydrate structures have been identified in eukaryotic and prokaryotic cells as glycoconjugates with communication skills. Their recently discussed role in various diseases has attracted high attention in the development of simple and convenient methods for oligosaccharide synthesis. In this review, recent approaches combining nature’s power for the design of tailor made biocatalysts by enzyme engineering and substrate engineering will be presented. These strategies lead to highly efficient and selective glycosylation reactions. The introduced concept shall be a first step in the direction to a glycosylation toolbox which paves the way for the tailor-made synthesis of designed carbohydrate structures.     

Efficient synthesis of a 6-deoxytalose tetrasaccharide related to the antigenic O-polysaccharide produced by Aggregatibacter actinomycetemcomitans serotype c

Concise synthesis of a 6-deoxy-α-l-talose tetrasaccharide, 6-deoxy-α-l-Talp-(1→3)-6-deoxy-α-l-Talp-(1→2)-6-deoxy-α-l-Talp-(1→3)-6-deoxy-α-l-Talp, the dimer of the disaccharide repeating unit of the OPS from Aggregatibacter actinomycetemcomitans serotype c, has been accomplished through suitable protecting group manipulations and stereoselective glycosylation starting from commercially available l-rhamnose. The target oligosaccharide in the form of its p-methoxyphenyl glycoside is suitable for further glycoconjugate formation via selective cleavage of this group.     

New chemical trends in ganglioside research

A report is given of recent progress in the methodology for isolation of gangliosides from natural sources, for the preparation of molecular species of gangliosides homogeneous in both the oligosaccharide and ceramide portions of the molecule, for chemical manipulation and derivatization of gangliosides, and for the preparation of gangliosides radiolabelled in different parts of the molecule. Particular emphasis has been given to: high performance liquid chromatographic procedures capable to separate gangliosides on the basis of their oligosaccharide or ceramide moieties and yielding completely homogeneous compounds, that is gangliosides with a single oligosaccharide, a single long chain base and a single fatty acid; two-dimensional thin-layer chromatographic procedures, provided with a fully computerized quantification system, particularly suitable to identifying gangliosides containing alkali-labile linkages, including ganglioside lactones; chemical procedures of high yield for reducing gangliosides at the double bond of long chain base, for selective removal of the fatty acyl moiety and replacement with a novel fatty acid, and for the synthesis of ganglioside lactones; chemical procedures for inserting fluorescent, paramagnetic or photoreactive probes at the fatty acyl part of the ganglioside molecule; procedures for chemical isotopic radiolabelling of gangliosides at the level of sialic acid acetyl group and at the fatty acid moiety. Examples are provided evidencing the significance and potential use of a variety of ganglioside derivatives in the study of ganglioside metabolism and functional implications.     

Synthesis of two trisaccharides related to the triterpenoid saponins isolated from Solanum lycocarpum

Chemical synthesis of two trisaccharides related to the triterpenoid saponins isolated from Solanum lycocarpum from commercially available d-Glc, d-Gal and l-Rha have been achieved by following concise and high-yielding route. The target trisaccharide 1 has been made by following a bis-glycosylation approach that has minimized the protecting group manipulations up to great extent. The trisaccharides have been synthesized in the form of their p-methoxyphenyl (OMP) glycosides to leave the scope for further glycoconjugates formation by the selective removal of the OMP glycoside and trichloroacetimidate chemistry. La(OTf)₃ has been used successfully as the promoter for the NIS mediated activation of thioglycosides.     

Comparative Study of Chemical Approaches to the Solid-Phase Synthesis of a Tumor-Seeking α-MSH Analogue

The synthesis of a cyclic melanocortin analogue (H-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂), where the Boc-protected derivative of a metal-chelating pyrazolyl ligand (pz) was inserted as N-terminal residue, was addressed by several different Fmoc/tBu and Boc/Bzl solid-phase strategies. On-resin cyclization was achieved immediately following incorporation of Asp, by condensation of the Asp side chain carboxyl with the Lys side chain primary amine after selective and simultaneous removal of side chain protecting groups. The success of the synthesis was highly dependent on the chemical strategy employed, with Boc/Bzl chemistry giving the best results. On the light of our findings, Fmoc/tBu strategies are not advantageous for the solid-phase synthesis of this particular type of lactam-bridged peptides. Last, but not least, the target peptide was recently found to have promising tumor-seeking properties (J Biol Inorg Chem 13:449–459, 2008).     

Tools and ingredients for the biocatalytic synthesis of carbohydrates and glycoconjugates

The presence of multiple functional groups and stereocentres in carbohydrates and glycoconjugates make them challenging targets for synthesis. Although progress in chemical synthesis and engineering is impressive, there is still a need to selectively introduce and remove protecting groups in the total synthesis of target molecules of increasing complexity. Multiple hydroxyl-groups with similar reactivities have to be differentiated in order to form the desired glycosidic bonds in a regio- and stereospecific way. To complement the existing chemical tools and ingredients, biocatalysts for selective carbon–carbon bond formation and glycosylation reactions have been developed. The availability of auxiliary ingredients like transfer reagents is a prerequisite for the development of viable biocatalytic process steps. In the case of dihydroxyacetone-phosphate-dependent aldolases, e.g. fructose-1,6-bisphosphate aldolase (EC 4.1.2.13), the large-scale availability of dihydroxyacetone-phosphate (DHAP) eliminates the need to synthesize the donor DHAP. For the pyruvate-dependent aldolases, e.g. the N-acetylneuraminic acid aldolase (EC 4.1.3.3) and acetaldehyde-dependent aldolases like the 2-deoxy-d-ribose-5-phosphate aldolase (4.2.1.4), the donors pyruvate and acetaldehyde are also available on a large scale. A broad range of natural and recombinant aldolases have been produced in stable lyophilized form. Recombinant transketolase together with a new synthesis of hydroxypyruvates has provided a platform technology for the preparation of monosaccharides, whereby the carbon backbone is extended by a two-carbon unit (C2-elongation). Natural and recombinant glycosyltransferases have been prepared on a large-scale to establish biocatalytic glycosylations in water as highly regio- and stereospecific reaction methodologies without the need for laborious protecting group manipulations, solubility adaptations and complex synthetic schemes. In order to simplify the synthetic manipulations for specific glycosylations, toolkits for β-1,4-galactosylations, α-1,3-galactosylations and α-1,3-fucosylations have been developed for rapid quantitative conversions. The introduction of matched pairs of biocatalysts and transfer reagents as ingredients together with the optimized reaction methodology as tool provide an important starting point for biocatalytic glycomics.     

Synthesis, analysis, purification, and intracellular delivery of peptide nucleic acids

Peptide nucleic acids (PNAs) are nonionic DNA mimics. Their novel chemical properties may facilitate the development of selective and potent antisense and antigene strategies for regulating intracellular processes. Described herein are procedures for the synthesis, purification, handling, and characterization of PNAs. A simple protocol for the lipid-mediated introduction of PNAs into in vitro cultures of mammalian cells is provided.     

糖苷内切酶法合成带有均一糖链的糖蛋白和糖肽

糖基化作用是真核生物蛋白翻译后修饰的重要环节,糖链对于蛋白质的结构和功能有重要影响。目前,合成带有均一糖链的糖蛋白和糖肽的策略主要有:(1)利用糖基化的氨基酸进行固相或液相合成。(2)将氨基化的寡糖链直接与预先合成的带有糖基化位点的多肽相结合。(3)利用糖基转移酶和糖苷酶的化学酶法合成策略。以上三种方法,都有各自的优点和不足。相对而言,利用微生物来源的β-N-乙酰氨基葡萄糖苷内切酶(ENGase)合成策略是目前发展较快且更具实践意义的方法。糖苷内切酶法合成策略的研究进展包括:(1)ENGase催化机制的研究。(2)糖基供体的研究。(3)ENGase突变体的研究。(4)糖苷内切酶法的应用。     

Engineering disulfide bonds of the novel human beta-defensins hBD-27 and hBD-28: differences in disulfide formation and biological activity among human beta-defensins

Human beta-defensins comprise a large number of peptides that play a functional role in the innate and adaptive immune system. Recently, clusters of new beta-defensin genes with predominant expression in testicular tissue have been discovered on different chromosomes by bioinformatics. beta-Defensins share a common pattern of three disulfides that are essential for their biological effects. Here we report for the first time the chemical synthesis of the new fully disulfide-bonded beta-defensins hBD-27 and hBD-28, and compare the results with synthetic procedures to obtain the known hBD-2 and hBD-3. While hBD-27 was readily converted into a product with the desired disulfide pattern by oxidative folding, hBD-28 required a selective protective group strategy to introduce the three disulfide bonds. The established synthetic processes were applied to the synthesis of hBD-2, which, like hBD-27, was accessible by oxidative folding, whereas hBD-3 required a selective strategy comparable to hBD-28. Experimental work demonstrated that trityl, acetamidomethyl, and t-butyl are superior to other protection strategies. However, the suitable pairwise arrangement of the protective groups can be different, as shown here for hBD-3 and hBD-28. Determination of the minimum inhibitory concentration against different bacteria revealed that hBD-27, in contrast to other beta-defensins tested, has virtually no antimicrobial activity. Compared to the other peptides tested, hBD-27 showed almost no cytotoxic activity, measured by hemoglobin release of erythrocytes. This might be due to the low positive net charge, which is significantly higher for hBD-2, hBD-3, and hBD-28.     

Free-radical mediated synthesis of enantiomerically pure, highly functionalized inositols from carbohydrates.

We report the synthesis, free-radical cyclization of precursors 1,2,7-trideoxy-7-iodo-3,4:5,6-di-O-isopropylidene-D-gluco-hept-1-enitol (1), methyl 7-O-acetyl-6-O-benzyl-8-bromo-2,3,8-trideoxy-4,5-O-isopropylidene-D-gluco-oct-2-enonate (2) and 5-O-acetyl-4-O-benzyl-6-bromo-6-deoxy-2,3-O-isopropylidene-D-glucose-O-benzyloxime (3), readily prepared from D-glucose, and some selected transformations of the carbocycles obtained from these intermediates. In compound 1 we have installed a terminal double bond and an iodide as radical acceptor and leaving group, respectively. Compounds 2 and 3 are epsilon-bromo aldehydes substituted with alpha,beta-unsaturated ester and oxime ether functions as radical traps, respectively. The tributyltin hydride mediated ring closure of these radical precursors have afforded a series of interesting, diverse and highly functionalized carbocycles which can be considered useful building blocks for the synthesis of branched-chain cyclitols, aminocyclitols and aminoconduritols. In these processes, a good chemical yield and high stereoselectivity has been found in the newly formed stereocenters. Particularly interesting has been the finding that the stereochemical outcome of the free-radical cyclization is independent of the ratio of isomers (E or Z) in oxime ether 3. These results show the power and the state of art of this strategy for the stereocontrolled synthesis of enantiomerically pure inositols from carbohydrates.     

Enterolosaponins A and B,novel triterpene bisdesmosides from Enterolobium contortisiliquum,and evaluation for their macrophage-oriented cytotoxic activity

Two novel triterpene bisdesmosides, designated as enterolosaponin A (1) and B (2), were isolated from Enterolobium contortisiliquum. The chemical structures of 1 and 2 were determined by analysis of their extensive spectroscopic data, as well as hydrolysis followed by chromatographic study. Enterolosaponins have a 2-amino-2-deoxy-D-glucosyl unit (D-glucosamine) as one of the monosaccharides constituting their oligosaccharide moieties, which have been rarely found in natural product research. Enterolosaponin A (1) exhibited a highly selective cytotoxicity against BAC1.2F5 mouse macrophages, and it should be notable that the macrophage death caused by 1 was shown to be neither necrotic nor due to induction of apoptosis from morphology of the died cells, whose cytosol occurred in vacuolation.     

Chemical and enzymatic synthesis of glycopolymers

The development of efficient, fast, flexible and general synthetic routes to glycopolymers is an ongoing challenge and much progress has been made in recent years. Chemical coupling methods have become increasingly sophisticated to fine-tune reactivity of reagents by fortuitous choices of anomeric activating group and protecting groups. As a result, oligosaccharide synthesis has become more predictable and reliable even to the extent that first examples of saccharide library syntheses in solution and on the solid phase have been published. In biology, the repertoire of biocatalysts that can be used for glycoside synthesis is ever-increasing, and enzyme-catalysed glycosylation steps have been successfully incorporated into synthetic strategies.     

Catalysis by nucleoside hydrolases

Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Because of their vital role in the protozoan purine salvage pathway, nucleoside hydrolases from parasitic protozoa in particular have been studied extensively by X-ray crystallography, kinetic methods and site-directed mutagenesis. An elaborate network of conserved interactions between the metalloenzyme and the ribose enables steric and electrostatic stabilisation of the oxocarbenium-ion-like transition state. Activation of the leaving group by protonation before the formation of the transition state is a recurring catalytic strategy of enzymes that cleave N-glycosidic bonds. However, the mechanisms underlying leaving group activation are still the subject of debate for the nucleoside hydrolases.     

Expanding the Scope of Native Chemical Ligation in Glycopeptide Synthesis

Glycoprotein is one of the important biopolymer in a biological system. In order to understand the complex correlation between the exact oligosaccharide structure of the glycoprotein and its function, preparation of homogeneous glycoprotein is to be essential. For such a purpose, chemical synthesis is one of the most promising methods to obtain homogeneous glycoproteins. Glycopolypeptide, which is a backbone of glycoprotein and an essential intermediate for glycoprotein synthesis, can be obtained through coupling of peptide and glycopeptide segments because straightforward synthesis of such a long glycopolypeptide is still a challenging task. Native chemical ligation (NCL) is one of the powerful methods for the coupling reaction of peptides, however, despite extensive investigation, NCL has site limitation for the coupling. In this context, we discovered NCL at serine site, where is a highly conserved amino acid residue in glycoproteins. This reaction strategy is owed to conversion reaction of cysteine residue to serine residue after conventional NCL. This conversion reaction is consisted of three steps; S-methylation of cysteine, CNBr reaction to afford O-ester linked peptide, and O to N acyl shift to get native peptide linkage with serine residue. During extensive investigation of the strategy, we found new reaction media for CNBr reaction, which is the key reaction in the strategy. This enabled us to synthesize not only N-linked glycopeptides but also O-linked sialyl glycopeptides. Thus we could demonstrate the usefulness of this new glycopeptide ligation strategy. In this short review, we will introduce our newly developed cysteine to serine conversion reaction which will expand the application of NCL in peptide as well as glycopeptide synthesis.