Stereoselective iterative one-pot synthesis of N-glycolylneuraminic acid-containing oligosaccharides

The use of an N-acyloxazolidinone-protected S-adamantanyl thiosialoside allows the highly stereoselective, one-pot multicomponent synthesis of alpha-sialoside-based oligosaccharides.     

Stereocontrolled synthesis of unnatural cyclic dipeptides containing an l-valine unit

Stereoselective synthesis of unusual nonproteinogenic dipeptides 7a,b,d,e and h and 13b and i, containing an l-valine unit and a cyclic unnatural α-amino acid, has been accomplished starting from the l-valine derived chiral synthon 1. The absolute configurations of the new stereocentres were assigned on the basis of ¹H NMR spectra.     

Novel amphiphilic cyclic oligosaccharides: synthesis and self-aggregation properties

Novel amphiphilic cyclic disaccharide analogues, in which the saccharide units are connected through stable phosphodiester linkages (CyPLOS, Cyclic Phosphate-Linked OligoSaccharides) and decorated with long lipophilic tentacles at the 2- and 3-OH moieties, have been synthesized. Their propensity to self-aggregation has been investigated by means of 1H and 31P NMR experiments, making it possible to determine for these macrocycles critical aggregation concentration values in the millimolar range.     

Chemoenzymatic iterative synthesis of difficult linkages of oligosaccharides on soluble polymeric supports

[reaction: see text]. A trisaccharide donor containing a cis-Galpalpha(1-->4)Galp linkage was prepared using a synthetic strategy based on chemoenzymatic oligosaccharide synthesis on a soluble polymeric support. Significantly, only retaining glycosyltransferases gave complete reactions, whereas inverting enzymes showed little or no activity with poly(ethylene glycol) (MPEG)-bound lactose as an acceptor. The MPEG-attached trisaccharide was shown to bind to Verotoxin-1 by transfer NOE studies through the Galpalpha(1-->4)Galp portion of the molecule.     

Cyclic phosphate-linked oligosaccharides: synthesis and conformational behavior of novel cyclic oligosaccharide analogues

CyPLOS (cyclic phosphate-linked oligosaccharides), that is, novel cyclic oligosaccharide surrogates, consisting of two, three, and four phenyl-beta-D-glucopyranoside units, 4,6-linked through stable phosphodiester bonds, were prepared by a straightforward and efficient solid-phase protocol. The assembly of the linear precursors was achieved by standard phosphoramidite chemistry on an automated DNA synthesizer, using a suitably protected 4-phosphoramidite derivative of D-glucose as the building block. For the crucial cyclization step a phosphotriester methodology was exploited, followed by a mild basic treatment releasing the desired cyclic molecules in solution in a highly pure form. The cyclic dimer and trimer were also independently prepared by classical solution synthesis, basically following the same approach. The solution structural preferences of the cyclic dimer and trimer, obtained by detailed NMR analysis, are also reported.     

Ribosomal synthesis of unnatural peptides

Combinatorial libraries of non-biological polymers and drug-like peptides could in principle be synthesized from unnatural amino acids by exploiting the broad substrate specificity of the ribosome. The ribosomal synthesis of such libraries would allow rare functional molecules to be identified using technologies developed for the in vitro selection of peptides and proteins. Here, we use a reconstituted E. coli translation system to simultaneously re-assign 35 of the 61 sense codons to 12 unnatural amino acid analogues. This reprogrammed genetic code was used to direct the synthesis of a single peptide containing 10 different unnatural amino acids. This system is compatible with mRNA-display, enabling the synthesis of unnatural peptide libraries of 10(14) unique members for the in vitro selection of functional unnatural molecules. We also show that the chemical space sampled by these libraries can be expanded using mutant aminoacyl-tRNA synthetases for the incorporation of additional unnatural amino acids or by the specific posttranslational chemical derivitization of reactive groups with small molecules. This system represents a first step toward a platform for the synthesis by enzymatic tRNA aminoacylation and ribosomal translation of cyclic peptides comprised of unnatural amino acids that are similar to the nonribosomal peptides.     

Molecular design and synthesis of artificial ion channels based on cyclic peptides containing unnatural amino acids

A series of novel cyclic peptides composed of 3 to 5 dipeptide units with alternating natural-unnatural amino acid units, have been designed and synthesized, employing 5-(N-alkanoylamino)-3-aminobenzoic acid with a long alkanoyl chain as the unnatural amino acid. All cyclic peptides with systematically varying pore size, shape, and lipophilicity are found to form ion channels with a conductance of ca. 9 pS in aqueous KCl (500 mM) upon examination by the voltage clamp method. These peptide channels are cation selective with the permeability ratio P(Cl(-))/P(K(+)) of around 0.17. The ion channels formed by the neutral, cationic, and anionic cyclic peptides containing L-alanine, L-lysine, and L-aspartate, respectively, show the monovalent cation selectivity with the permeability ratio P(Na(+))/P(K(+)) of ca. 0.39. On the basis of structural information provided by voltage-dependent blockade of the single channel current of all the tested peptides by Ca(2+), we inferred that each channel is formed from a dimer of the peptide with its peptide ring constructing the channel entrance and its alkanoyl chains lining across the membrane to build up the channel pore. The experimental results are consistent with an idea that the rate of ion conduction is determined by the nature of the hydrophobic alkanoyl chain region, which is common to all the channels.     

Permethacrylated carbohydrates: synthesis and reactivity in glycosidation reaction

The synthesis of various permethacrylated mono- and disaccharides in acceptable yields is reported. The reactivity of these unusual polymerizable compounds has been investigated in diverse glycosidation reaction conditions. New original polymerizable donor sugars have been prepared successfully in good to excellent yields.     

Toward solution-phase automated iterative synthesis: fluorous-tag assisted solution-phase synthesis of linear and branched mannose oligomers

We report herein the first synthesis of linear and branched mannose oligosaccharides using fluorous-tag assistance with reagents and FSPE protocols that are amenable to automation. The particular fluorous linker proved to maintain solubility of the growing oligosaccharide chain such that identical reaction solvent conditions and purification protocols could be used between glycosylation and deprotection reactions, thereby rendering the procedures amenable to automation.     

Enzyme-catalyzed synthesis of natural and unnatural polysaccharides

Synthesis of natural and unnatural polysaccharide was achieved via “enzymatic polymerization” by utilizing a glycoside hydrolase as catalyst. Particularly, hyaluronan, chondroitin, and their derivatives belonging to glycosaminoglycans have been prepared using sugar oxazoline monomers designed on the basis of the concept “transition-state analogue substrate”. The oxazoline derivatives of N-acetylhyalobiuronate [GlcAβ(1→3)GlcNAc] and N-acetylchondrosine [GlcAβ(1→3)GalNAc], which have the repeating disaccharide structures of hyaluronan and chondroitin, respectively, were successfully polymerized by the catalysis of hyaluronidase, giving rise to synthetic hyaluronan and chondroitin. Their 2-substituted oxazoline derivatives were also polymerized to the corresponding N-acylated hyaluronan and chondroitin derivatives. Furthermore, N-acetylchondrosine oxazoline derivatives sulfated at the C4, the C6, and both the C4 and C6 of the GalNAc unit were catalyzed by hyaluronidase; the monomer sulfated at the C4 was polymerized to chondroitin 4-sulfate with well-defined structure, whereas the other two monomers were exclusively hydrolyzed to the corresponding disaccharides. These different kinds of natural and unnatural polysaccharides having relatively high molecular weights were produced in all cases by the catalysis of hyaluronidase. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5014–5027, 2006     

Chemical modification of hydroxyl groups of poly(vinyl alcohol) by a glycosidation reaction

A new procedure for chemical modification of poly(vinyl alcohol) (PVA) was established by a glycosidation reaction of hydroxyl groups in PVA with triacetylated sugar oxazoline 1 . ¹H and ¹³C NMR analyses indicated that triacetylated N‐acetyl‐D‐glucosamine (GlcNAc) was introduced onto a PVA backbone selectively via a β‐O‐glycoside linkage. Deacetylation of triacetylated GlcNAc‐substituted PVA 2 resulted in GlcNAc‐substituted PVA 3 in good yield. These modified PVAs 2 and 3 exhibited solubilities and thermal properties different from the original PVA.     

Proline-catalyzed stereoselective synthesis of natural and unnatural nocardiolactone

A concise diastereo and enantioselective synthesis of natural and unnatural nocardiolactone is accomplished by proline-catalyzed crossed-aldol reaction as the key step.     

Enzymatic formation of indole-containing unnatural cyclic polyprenoids by bacterial squalene:hopene cyclase

[reaction: see text] Two indole-containing substrate analogues, in which a C20 isoprene unit is connected to indole (3-(geranylgeranyl)indole and 3-(farnesyldimethylallyl)indole), were synthesized and tested for enzymatic cyclization by squalene:hopene cyclase from Alicyclobacillus acidocaldarius. Interestingly, 3-(geranylgeranyl)indole was not a substrate for the bacterial squalene cyclase, while 3-(farnesyldimethylallyl)indole was efficiently converted to a 2:1 mixture of unnatural novel products.     

Preparation of linear oligosaccharides by a simple monoprotective chemo-enzymatic approach

A monoprotective approach, involving acetyl ester as unique protective group in oligosaccharides synthesis, has been developed. Starting from peracetylated monosaccharides and glycals, by using an efficient and selective chemo-enzymatic ‘one-pot’ strategy (a regioselective hydrolysis catalyzed by immobilized lipases followed by a chemical acyl migration), different carbohydrate acceptors, only protected with acetyl ester, can be achieved. If combined with the use of an acetylated glycosyl donor, the glycosylation reaction with these glycosyl acceptors leads to peracetylated oligosaccharides. These compounds can be directly used as intermediates for the synthesis of glycopeptides used as antitumoral vaccines and, at the end of the process, can be easily fully deprotected in only one step. Thus, these key building blocks have been successfully used in glycosylation reactions for an efficient construction of peracetylated disaccharides, such as the biological relevant lactosamine, in multigram scale. Subsequently, glycosylation with the 3OH-tetraacetyl-α-d-galactose, used as carbohydrate acceptor, allowed the synthesis of a peracetylated N-trisaccharidic precursor of the lacto-N-neo-tetraose antigen. Extending this strategy to a 3OH-di-acetyl galactal, one peracetylated precursor of the T tumor-associated carbohydrate antigen has been synthesized.This efficient approach, characterized by the use of the acetyl ester as only protecting group during all the synthetical steps expected, represents an easy and efficient alternative to the classical synthetic methods in carbohydrate chemistry that involve several protecting group manipulation.     

Synthesis of a highly hydrophobic cyclic decapeptide by solid-phase synthesis of linear peptide and cyclization in solution

<正>A general method was described to synthesize a highly hydrophobic cyclic peptide,cyclo[LWLWLWLWLQ]where underlines indicate D-configuration of the amino acid,by a two-step solid-phase/solution synthesis strategy.The linear decapeptide was assembled by standard Boc chemistry on solid-phase and subsequently cyclized in solution with high efficiency and reproducibility. In subsequent purification by semi-preparative HPLC,50%(v/v) DMF/H_2O was employed as the solvent to overcome the difficulty of solubilization for the hydrophobic cyclic decapeptide and achieved a total yield of 30-35%with a purity of over 98%.     

Amadori ketoses with calcium hydroxide and the Kiliani reaction on 1-deoxy ketoses: two approaches to the synthesis of saccharinic acids

Saccharinic acids (2-C-methyl aldonic acids) may be formed by treatment of Amadori ketoses with calcium hydroxide or by the Kiliani reaction of 1-deoxy ketoses with cyanide. Thus (i) N,N-dibenzyl or N,N-dimethyl-1-amino-1-deoxy-d-fructose with aqueous calcium hydroxide afforded 2-C-methyl-d-ribono-1,4-lactone under green conditions and (ii) reaction of methyl magnesium bromide with 2,3-O-isopropylidene-d-erythronolactone gave 1-deoxy-3,4-O-isopropylidene-d-ribulose, which on subsequent treatment with aqueous sodium cyanide and hydrolysis, formed 2-C-methyl-d-arabinono-1,4-lactone. Such branched sugar lactones are likely to be of value as chirons containing branched carbon chains.     

Capillary zone electrophoresis of linear and branched oligosaccharides

The electrophoretic behavior of derivatized linear and branched oligosaccharides from various sources was examined in capillary zone electrophoresis with polyether-coated fused-silica capillaries. Two UV-absorbing (also fluorescent) derivatizing agents (2-amino-pyridine and 6-aminoquinoline) were utilized for the electrophoresis and sensitive dtection of neutral oligosaccharides, e.g., N-acetylchitooligosaccharides, high-mannose glycans and xyloglucan oligosaccharides. The oligosaccharides labelled with 6-aminoquinoline yielded eight times higher signal than those tagged with 2-aminopyridine. Plots of logarithmic electrophoretic mobilities of labelled N-acetylchitooligosaccharides with 6-aminoquinoline or 2-aminopyridine versus the number of sugar residues in the homologous series yielded straight lines in the size range studied, the slopes of which were independent of the tagging functions. The slopes of these lines are referred to as the N-acetylglucosaminyl group mobility decrement. The oligosaccharides were better resolved in the presence of tetrabutylammonium bromide in the running electrolyte. Furthermore, the electrophoretic mobilities of branched oligosaccharides were indexed with respect to linear homooligosaccharides, an approach that may prove valuable in correlating and predicting the mobilities of complex oligosaccharides.     

Enzymatic synthesis of prebiotic oligosaccharides

Prebiotic oligosaccharides are nondigestible carbohydrates that can be obtained by enzymatic synthesis. Glucosyltransferases can be used to produce these carbohydrates through an acceptor reaction synthesis. When maltose is the acceptor a trisaccharide composed of one maltose unit and one glucose unit linked by an alpha-1,6-glycosidic bond (panose) is obtained as the primer product of the dextransucrase acceptor reaction. In this work, panose enzymatic synthesis was evaluated by a central composite experimental design in which maltose and sucrose concentration were varied in a wide range of maltose/sucrose ratios in a batch reactor system. A partially purified enzyme was used in order to reduce the process costs, because enzyme purification is one of the most expensive steps in enzymatic synthesis. Even using high maltose/sucrose ratios, dextran and higher-oligosaccharide formation were not avoided. The results showed that intermediate concentrations of sucrose and high maltose concentration resulted in high panose productivity with low dextran and higher-oligosaccharide productivity.     

生物催化在非天然寡糖合成中的应用

酶已经成为现代有机合成中一种不可或缺的工具。近年来人们在克隆技术，微生物学以及蛋白纯化方面的研究成果为我们提供了越来越多的生物催化剂，酶最初用于天然产物的合成，而今其在非天然化合物中的应用更引起了生物化学家的广泛关注。酶催化反应可以使不加保护的高官能团化底物进行条件温和，环境友好地转化，得到具有高度化学、区域和立体选择性的产物。本文对近年来糖基转移酶在糖生物学中的应用，特别是其在利用Leloir方法合成非天然寡糖中的应用作一简要回顾。     

Modular synthesis of heparin oligosaccharides

A general, modular strategy for the first completely stereoselective synthesis of defined heparin oligosaccharides is described. Six monosaccharide building blocks (four differentially protected glucosamines, one glucuronic and one iduronic acid) were utilized to prepare di- and trisaccharide modules in a fully selective fashion. Installation of the alpha-glucosamine linkage was controlled by placing a conformational constraint on the uronic acid glycosyl acceptors thereby establishing a new concept for stereochemical control. Combination of disaccharide modules to form trans-uronic acid linkages was completely selective by virtue of C2 participating groups. Coupling reactions between disaccharide modules exhibited sequence dependence. While the union of many glucosamine uronic acid disaccharide modules did not meet any problems, certain sequences proved not accessible. Elaboration of glucosamine uronic acid disaccharide building blocks to trisaccharide modules by addition of either one additional glucosamine or uronic acid allowed for stereoselective access to oligosaccharides as demonstrated on the example of a hexasaccharide resembling the ATIII-binding sequence. Final deprotection and sulfation yielded the fully synthetic heparin oligosaccharides.