One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities

Chemoenzymatic synthesis, which combines the flexibility of chemical synthesis and the high selectivity of enzymatic synthesis, is a powerful approach to obtain complex carbohydrates. It is a preferred method for synthesizing sialic acid-containing structures, including those with diverse naturally occurring and non-natural sialic acid forms, different sialyl linkages and different glycans that link to the sialic acid. Starting from N-acetylmannosamine, mannose or their chemically or enzymatically modified derivatives, sialic acid aldolase-catalyzed condensation reaction leads to the formation of sialic acids and their derivatives. These compounds are subsequently activated by a CMP-sialic acid synthetase and transferred to a wide range of suitable acceptors by a suitable sialyltransferase for the formation of sialosides containing natural and non-natural functionalities. The three-enzyme coupled synthesis of sialosides can be carried out in one pot without the isolation of intermediates. The time for synthesis is 4-18 h. Purification and characterization of the product can be completed within 2-3 d.     

One-pot multi-enzyme (OPME) chemoenzymatic synthesis of sialyl-Tn-MUC1 and sialyl-T-MUC1 glycopeptides containing natural or non-natural sialic acid

A series of STn-MUC1 and ST-MUC1 glycopeptides containing naturally occurring and non-natural sialic acids have been chemoenzymatically synthesized from Tn-MUC1 glycopeptide using one-pot multienzyme (OPME) approaches. In situ generation of the sialyltransferase donor cytidine 5′-monophosphate-sialic acid (CMP-Sia) using a CMP-sialic acid synthetase in the presence of an extra amount of cytidine 5′-triphosphate (CTP) and removal of CMP from the reaction mixture by flash C18 cartridge purification allow the complete consumption of Tn-MUC1 glycopeptide for quantitative synthesis of STn-MUC1. A Campylobacter jejuni β1–3GalT (CjCgtBΔ30-His₆) mutant has been found to catalyze the transfer of one or more galactose residues to Tn-MUC1 for the synthesis of T-MUC1 and galactosylated T-MUC1. Sialylation of T-MUC1 using Pasteurella multocida α2–3-sialyltransferase 3 (PmST3) with Neisseria meningitidis CMP-sialic acid synthetase (NmCSS) and Escherichia coli sialic acid aldolase in one pot produced ST-MUC1 efficiently. These glycopeptides are potential cancer vaccine candidates.     

The chemoenzymatic synthesis of usnic acid

Usnic acid, a highly functionalized dibenzofuran, is a polyketide secondary metabolite produced by several species of lichens. Synthesis of usnic acid from commercially available starting material was accomplished in two steps. The synthesis involves the methylation of phloracetophenone followed by oxidation with horseradish peroxidase. This work will lay the foundation for further biosynthetic studies on usnic acid.     

A chemoenzymatic synthesis of aromatic carboxylic acid vinyl esters

The practical synthesis of vinyl p-coumarate (vinyl 4-hydroxycinnamate) and vinyl ferulate (vinyl 4-hydroxy-3-methoxycinnamate) was accomplished via a transesterification to the corresponding aromatic acid using vinyl acetate and a catalytic amount of PdCl₂, followed by the lipase-catalyzed regioselective alcoholysis in EtOH.     

Chemo-enzymatic synthesis of C-9 acetylated sialosides

A chemo-enzymatic synthesis of [(5-acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2-->3)-O-(beta-D-galactopyranosyl)-(1-->3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)]-l-serine acetate (1) has been accomplished by a regioselective chemical acetylation of Neu5Ac (2) to give 9-O-acetylated sialic acid 3, which was enzymatically converted into CMP-Neu5,9Ac(2) (4) employing a recombinant CMP-sialic acid synthetase from Neisseria meningitis [EC 2.7.7.43]. The resulting compound was then employed for the enzymatic glycosylation of the C-3' hydroxyl of chemically prepared glycosylated amino acid 10 using recombinant rat alpha-(2-->3)-O-sialyltransferase expressed in Spodooptera frugiperda [EC 2.4.99.4] to give, after deprotection of the N(alpha)-benzyloxycarbonyl (CBz)-protecting group of serine, target compound 1. The N(alpha)-CBz-protected intermediate 11 can be employed for the synthesis of glycolipopeptides for immunization purposes.     

New stabilized FastPrep-CLEAs for sialic acid synthesis

N-acetyl-d-neuraminic acid aldolase, a key enzyme in the biotechnological production of N-acetyl-d-neuraminic acid (sialic acid) from N-acetyl-d-mannosamine and pyruvate, was immobilized as cross-linked enzyme aggregates (CLEAs) by precipitation with 90% ammonium sulfate and crosslinking with 1% glutaraldehyde. Because dispersion in a reciprocating disruptor (FastPrep) was only able to recover 40% of the activity, improved CLEAs were then prepared by co-aggregation of the enzyme with 10 mg/mL bovine serum albumin followed by a sodium borohydride treatment and final disruption by FastPrep (FastPrep-CLEAs). This produced a twofold increase in activity up to 86%, which is a 30% more than that reported for this aldolase in cross-linked inclusion bodies (CLIBs). In addition, these FastPrep-CLEAs presented remarkable biotechnological features for Neu5Ac synthesis, including, good activity and stability at alkaline pHs, a high K_M for ManNAc (lower for pyruvate) and good operational stability. These results reinforce the practicability of using FastPrep-CLEAs in biocatalysis, thus reducing production costs and favoring reusability.     

Engineering of a sialic acid synthesis pathway in transgenic plants by expression of bacterial Neu5Ac-synthesizing enzymes

Plants are a low-cost and contamination-free factory for the production of recombinant pharmaceutical proteins. However, plant-made pharmaceuticals differ from their mammalian homologues by the structure of their N -linked glycans. For instance, most mammalian glycoproteins harbour terminal sialic acids that control their half-life in the bloodstream. The absence of the whole sialylation machinery in plants is of major concern as non-sialylated plant-made pharmaceuticals may not perform at their full potential in humans, because of their removal from the circulation through the involvement of hepatic cell receptors. In this context, we have investigated the synthesis of N -acetylneuraminic acid (Neu5Ac) in the cytosol of plants by either the re-routing of the endogenous 3-deoxy- d - manno -2-octulosonic acid (Kdo) biosynthetic pathway or the expression of microbial Neu5Ac-synthesizing enzymes. In this paper, we demonstrate that the plant Kdo-8P synthase is not able to use N -acetyl d -mannosamine as a substrate, and thus re-routing of the Kdo pathway for the synthesis of Neu5Ac is not possible. Consequently, we expressed genes encoding Neu5Ac lyase from Escherichia coli and Neu5Ac synthase ( neuB2 ) from Campylobacter jejuni in plants. These resulted in the production of functional enzymes in the cytosol, which in turn can catalyse the synthesis of Neu5Ac in vitro . Experiments were carried out on two models, Bright Yellow 2 (BY2) tobacco cells and Medicago sativa (alfalfa), the perennial legume crop.     

Concise chemoenzymatic synthesis of epi-inositol

epi-Inositol was synthesized in six steps in 40% overall yield from a bacterial bromobenzene metabolite. The chemoenzymatic route involved toluene dioxygenase oxidation, substrate-directed catalytic osmylation, m-CPBA epoxidation, radical debromination, and Amberlite-catalized hydrolysis. The route described is amenable to scaleup and could allow access to cis-inositol, and deoxy derivatives of epi-inositol.     

Biosynthesis and metabolic degradation of sphingolipids not containing sialic acid

Interest in sphingolipid metabolism has increased rapidly during the past decade, and many of the steps involved in the biosynthesis and metabolic degradation of sphingolipids are now known. In this review these studies are critically examined. Emphasis has been placed on the in vitro studies with cell-free systems, since these represent the groundwork for further purification and characterization of the enzyme systems involved. Experimental problems specific to this field of study, and the manner in which these may affect interpretation of experimental results, are discussed.     

A crosslinked inclusion body process for sialic acid synthesis

The propensity of a recombinant protein produced in bacteria to aggregate has been assumed to be unpredictable, and inclusion bodies have been thought of as wasted cell material. However, a target protein can be purposely driven to inclusion bodies, which demonstrate full cell tolerable activity. Sialic acid aldolase, N-terminally fused with the cellulose-binding module of Clostridium cellulovorans, was almost quantitatively physiologically aggregated into active inclusion bodies. These inclusion bodies were entrapped in alginate beads and crosslinked by glutaraldehyde. The immobilized biocatalyst generated by this crosslinked inclusion bodies (CLIB) technology was used in a repetitive batch protocol for sialic acid production that was monitored on-line by flow calorimetry. The required substrate, N-acetyl-D-mannosamine, was obtained by partially improved alkaline epimerization.     

Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers

The length of the hyaluronan (HA) polysaccharide chain dictates its biological effects in many cellular and tissue systems. Long and short HA polymers often appear to have antagonistic or inverse effects. However, no source of very defined, uniform HA polymers with sizes greater than 10 kDa is currently available. We present a method to produce synthetic HA with very narrow size distributions in the range of approximately 16 kDa to approximately 2 MDa. The Pasteurella HA synthase enzyme, pmHAS, catalyzes the synthesis of HA polymer utilizing monosaccharides from UDP-sugar precursors. Recombinant pmHAS will also elongate exogenously supplied HA oligosaccharide acceptors in vitro in a nonprocessive fashion. As a result of bypassing the slow initiation step in vitro, the elongation process is synchronized in the presence of acceptor; thus all of polymer products are very similar in length. In contrast, without the use of an acceptor, the final polymer size range is difficult to predict and the products are more polydisperse. HA polymers of a desired size are constructed by controlling the reaction stoichiometry (i.e. molar ratio of precursors and acceptor molecules). The use of modified acceptors allows the synthesis of HA polymers containing tags (e.g. fluorescent, radioactive). In this scheme, each molecule has a single foreign moiety at the reducing terminus. Alternatively, the use of radioactive UDP-sugar precursors allows the synthesis of uniformly labeled native HA polymers. Overall, synthetic HA reagents with monodisperse size distributions and defined structures should assist in the elucidation of the numerous roles of HA in health and disease.     

A new facile chemoenzymatic synthesis of levamisole

An efficient and facile chemoenzymatic synthesis of levamisole by employing lipase-mediated resolution of 3-hydroxy-3-phenylpropanenitrile followed by its conversion to beta-amino alcohol as the key intermediate is described.     

C群流脑多糖菌苗唾液酸含量测定试验

Ｃ群脑膜炎球菌多糖菌苗是我们近年开发的一种新制品,其中唾液酸（ＳＡ）是该制品的有效成份之一。为了严格控制该制品的质量,经反复试验,对比各种试验条件,选择出较理想的测定ＳＡ方法－改良法,该法与Ｓｖｅｎｎｅｒｈｏｌｍ氏法比较,其标准曲线变异系数（ｃｖ％）为０．０３％,低于Ｓｖｅｎｎｅｒｈｏｌｍ氏法（０．３１％）;其回收率平均为１００．６２％。与中检所方法比较,无显著差异（Ｐ〉０．０５）,对批样品进行６     

Metabolism of 4-O-methyl-N-acetylneuraminic acid a synthetic sialic acid

1. 4-O-Methyl-N-acetylneuraminic acid shows a strong positive periodate-thiobarbiturate reaction. The mechanism of dye formation in this test for sialic acids is discussed in view of the studies already published. 2. An efficient preparation of a tritium-labelled 4-O-methyl-N-acetylneuraminic acid, with high specific radioactivity, by an oxymercuration-demercuration procedure is presented. 3. Sialytransferase activities in microsomal fractions of equine liver using desialylated fetuin are studied. The enzyme activity, assayed in a radioactive procedure, shows an apparent Km value for CMP-N-acetylneuraminic acid of 0.7 mM, whereas this value is 3.4 mM for CMP-4-O-methyl-N-acetylneuraminic acid. Differences are also observed in the maximal velocity for the two substrates. 4. The equine liver system can be used to prepare substantial amounts of fetuin containing radioactive N-acetylneuraminic acid or 4-O-methyl-N-acetylneuraminic acid. The isolated reaction products show similar sialic acid release by treatments with acid or fowl-plague virus neuraminidase. In contrast, 4-O-methyl-N-acetylneuraminic acid-fetuin displays a marked resistance to desialylation by Vibrio cholerae neuraminidase. 5. Free 4-O-methyl-N-acetylneuraminic acid is completely resistant to the action of acylneuraminate pyruvate-lyase. It does not inhibit the enzymic cleavage reaction of N-acetylneuraminic acid. 6. The influence of a substitution at C-4 neuraminic acid on the enzymatic reaction mechanisms is discussed.     

Efficient synthesis of a sialyl T-antigen-linked glycopeptide by the chemoenzymatic method

A sialyl T-antigen-linked tetrapeptide was prepared by the combined method of chemical synthesis and enzymatic synthesis. The GalNAc-linked peptide was first obtained by using a commercial peptide synthesizer, and then a galactose residue was attached with beta-(1-->3)-linkage by transglycosylating with a recombinant beta-galactosidase from Bacillus circulans. The sialic acid residue was then combined by alpha-(2-->3)-linkage with sialytransferase from rat liver.     

NeuD plays a role in the synthesis of sialic acid in Escherichia coli K1

The polysialic acid capsule of Escherichia coli K1 is an essential virulence determinant. The kps gene cluster, which encodes the proteins necessary for polymer synthesis and transport, is divided into three functional regions. In this report, we present evidence that the neuD gene from region 2 is involved in sialic acid synthesis. A non-polar chromosomal deletion in neuD was constructed. The defect was complemented by neuD in trans or by the addition of exogenous sialic acid. A NeuD homologue, Neu(III)D, from serotype III Streptococcus agalactiae (GBS) also restored capsule expression to the neuD deletion strain. These data confirm the role of neuD in E. coli sialic acid capsule synthesis and demonstrate that the neu(III)D homologue from GBS shares a similar enzymatic function.     

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsinwere analysed by sequential exoglycosidase digestion and gelfiltration chromatography, following reductive tritiation. Inaddition, selected tryptic glycopeptides obtained from frogretinal rod outer segment membranes were examined by electrospraymass spectrometry (ES-MS), fast atom bombardment mass spectrometry(FAB-MS), amino acid sequence and composition analysis, andcarbohydrate composition analysis. The amino acid sequence datademonstrated that the glycopeptides were derived from rhodopsinand confirmed the presence of twoN-glycosylation sites, at residuesAsn² and Asn¹⁵. The predominant glycan (60% of total) had thestructure GlcNAcß1–2Man1–3(Man1–6)Manß1–4GlcNAcß1–4GlcNAc-(Asn),with the remaining structures containing 1–3 additionalhexose residues, as reported previously for bovine rhodopsin.Unlike bovine rhodopsin, however, a sizable fraction of thetotal giycans of frog rhodopsin also contained sialic acid (NeuAc),with the sialylated oligosaccharides being present exclusivelyat the Asn² site. FAB-MS analysis of oligosaccharides releasedfrom the Asn² site gave, among other signals, an abundant quasimolecularion corresponding to a glycan of composition NeuAc₁Hex₆HexNAc₃(where Hex is hexose and HexNAc is N-acetylhexosamine), consistentwith a hybrid structure. The potential biological implicationsof these results are discussed in the context of rod outer segmentmembrane renewal. glycoforms oligosaccharide structure rhodopsin     

Characterization of Neisseria meningitidis capsular polysaccharides containing sialic acid by pyrolysis mass spectrometry

The group B, C, W-135, and Y capsular polysaccharides of Neisseria meningitidis which contain sialic acid were differentiated by Curie-point pyrolysis low-voltage mass spectrometry. A large series of partially purified group B polysaccharide preparations obtained from pathogenic as well as nonpathogenic strains were analyzed by the same technique. It was shown that the carbohydrate structure of these group B polysaccharides appears to be the same throughout the whole series. Slight immunogenicity of some of the group B polysaccharide preparations tested is probably due to protein impurities. Automated pyrolysis mass spectrometry coupled with multivariate analysis of the spectral data by computer turns out to be a rapid method of characterizing microgram samples of large series of polysaccharide preparations.