N-acetylneuraminic Acid Analogues Research Articles

Development and evaluation of an immunoassay for the quantification of N-acetylneuraminic acid (Neu5Ac) in foods and biosamples

N-acetylneuraminic acid is an active ingredient in tonic foods and an important additive in foods and biopharmaceuticals. To address the limitations of existing methods of N-acetylneuraminic acid quantification, we developed an immunoassay based on antibodies induced in hens using artificial antigen, showing high sensitivity and specificity with no cross-reactivity with eight N-acetylneuraminic acid analogues. An IgY-based indirect competitive enzyme-linked immunosorbent assay showed a detection range of 1.14 to 70.08 ng/mL and a limit of detection of 0.57 ng/mL. In spiked samples, recoveries by the indirect competitive enzyme-linked immunosorbent assay ranged from 74.05% to 110.87% compared with HPLC (73.01% to 108.8%). Consistency between the indirect competitive enzyme-linked immunosorbent assay and HPLC was satisfactory (R2 = 0.9736), demonstrating this established immunoassay as a rapid and reliable approach for N-acetylneuraminic acid analysis. The assay described in this study provides an important method for the screening of N-acetylneuraminic acid in biological samples and foodstuffs.

Synthesis of 4-O-Alkylated N-Acetylneuraminic Acid Derivatives.

The synthesis of 4-O-alkyl analogues of N-acetylneuraminic acid (Neu5Ac) and the scope of the reaction are described. Activated alkyl halides and sulfonates and primary alkyl iodides give products in useful yields. The utility of the methodology is exemplified using a thiophenyl Neu5Ac building block to synthesize a 4-O-alkyl DANA analogue. These results expand the toolbox of Neu5Ac chemistry with value in drug discovery and for the design of novel tools to study the biology of Neu5Ac lectins.

Syntheses of 2-deoxy-2,3-didehydro- N-acetylneuraminic acid analogues modified by N-sulfonylamidino groups at the C-4 position and biological evaluation as inhibitors of human parainfluenza virus type 1

Eleven novel sialidase inhibitors 9 and 10 with an N-sulfonylamidino group at the C-4 position of Neu5Ac2en 1 against human parainfluenza virus type 1 (hPIV-1) were synthesized using copper-catalyzed three-component coupling reactions, and their inhibitory activities against hPIV-1 sialidase were studied.

Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution

The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P21 at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme–substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.

A first synthesis of sulfonic acid analogues of N-acetylneuraminic acid

Sulfonic acid analogues of N-acetylneuraminic are synthesized from 1-thio- l-fucoside derivatives with the introduction of an azido group at C-4 of the fucose moiety and carbanionic addition onto fully protected lactones. The analogues in the form of methyl glycosides are subjected to a neuraminidase inhibition assay.

Structure-guided saturation mutagenesis of N-acetylneuraminic acid lyase for the synthesis of sialic acid mimetics

Analogues of N-acetylneuraminic acid (sialic acid, NANA, Neu5Ac), including 6-dipropylcarboxamides, have been found to be selective and potent inhibitors of influenza sialidases. Sialic acid analogues are, however, difficult to synthesize by traditional chemical methods and the enzyme N-acetylneuraminic acid lyase (NAL) has previously been used for the synthesis of a number of analogues. The activity of this enzyme towards 6-dipropylcarboxamides is, however, low. Here, we used structure-guided saturation mutagenesis to produce variants of NAL with improved activity and specificity towards 6-dipropylcarboxamides. Three residues were targeted for mutagenesis, Asp191, Glu192 and Ser208. Only substitution at position 192 produced significant improvements in activity towards the dipropylamide. One variant, E192N, showed a 49-fold improvement in catalytic efficiency towards the target analogue and a 690-fold shift in specificity from sialic acid towards the analogue. These engineering efforts provide a scaffold for the further tailoring of NAL for the synthesis of sialic acid mimetics.

Synthesis of 2-deoxy-2,3-didehydro- N-acetylneuraminic acid analogues modified at the C-4 and C-9 positions and their behaviour towards sialidase from influenza virus and pig liver membrane

The synthesis of novel 2-deoxy-2,3-didehydro- N-acetylneuraminic acid analogues structurally varied at C-4 and C-9 by transformation from versatile key intermediates and their inhibitory activity against sialidase from influenza virus A and pig liver membrane are described.

The synthesis and evaluation of novel sialic acid analogues bound to matrices for the purification of sialic acid-recognising proteins

A novel N-acetylneuraminic acid analogue, 2- S-(5′-aminopentyl) 5-acetamido-3,5-dideoxy-2-thio- d- glycero-α- d- galacto-2-nonulopyranosidonic acid, as well as the thiosialoside 2- S-(2′-aminoethyl) 5-acetamido-3,5-dideoxy-2-thio- d- glycero-α- d- galacto-2-nonulopyranosidonic acid, have been synthesised and successfully coupled to CNBr-activated Sepharose 4B through the terminal amino group. The resultant affinity resins have proved efficient in purifying a number of sialic acid-recognising proteins such as Vibrio cholerae sialidase, sialidase-L from leech, trans-sialidase from Trypanosoma cruzi, and sialyltransferases from rat liver, all in high yield.

ChemInform Abstract: N‐Monoalkylation of Tetra‐O‐benzyl‐D‐arabinonamide: Synthesis of Some Open‐Chain Analogues of N‐Acetylneuraminic Acid and Their Evaluation as Sialidase Inhibitors.

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Chemoenzymatic synthesis of an N-acetylneuraminic acid analogue having a carbamoylmethyl group at C-4 as an inhibitor of sialidase from influenza virus

5,9-Diacetamido-2,6-anhydro- O-4-carbamoylmethyl-3,5,9-trideoxy- d- glycero- d- galacto-non-2-enonic acid ( 1) was synthesized via a key intermediate 2 through the Neu5Ac aldolase [E.C.4.1.3.3]-catalyzed aldol reaction of 2-acetamido-2,6-dideoxy-6-azido- d-glucose with sodium pyruvate operating under alkaline conditions (pH 10.5) in order to accelerate epimerization C-2 of N-acetyl- d-glucosamine ( d-GlcNAc) derivatives. Compound 1 showed inhibitory activity against sialidase.

N-Monoalkylation of Tetra-O-benzyl-D-arabinonamide: Synthesis of Some Open-Chain Analogues ofN-Acetylneuraminic Acid and Their Evaluation as Sialidase Inhibitors

N-Monoalkylation of Tetra-O-benzyl-D-arabinonamide: Synthesis of Some Open-Chain Analogues ofN-Acetylneuraminic Acid and Their Evaluation as Sialidase Inhibitors

A palladium(>0>)-mediated approach to some novel C-4-substituted Neu5Ac2en derivatives

The preparation of a range of novel C-4-modified 2,3-unsaturated N-acetylneuraminic acid analogues is reported. These compounds have been prepared from the 4-chloro-4-deoxy-N-acetylneuraminic acid derivatives 10 and 12 by using a palladium(>0>)-mediated coupling with organostannanes.

Diastereoselective routes to side-chain truncated analogues of N-acetylneuraminic acid†

Vitamin C 3 has been converted, in a completely stereocontrolled manner, into the side-chain truncated analogues 4 and 5 of N-acetylneuraminic acid 1.

Synthesis of Phosphonic Acid Analogues of Sialic Acids (Neu5Ac and KDN) as Potential Sialidase Inhibitors

Five phosphonic acid analogues of N-acetylneuraminic acid (Neu5Ac) and 3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (KDN) have been synthesized. The synthesis was accomplished using an indium-mediated coupling of N-acetylmannosamine or mannose with dimethyl (3-bromopropen-2-yl)phosphonate in aqueous media. The potential of these phosphonate acid analogues of sialic acids as sialidase inhibitors was evaluated and found to show moderate biological acitivities.

Synthesis of C-4-disubstituted analogues of N-acetylneuraminic acid

The synthesis of some novel C-4-disubstituted analogues of N-acetylneuraminic acid has been achieved by nucleophilic opening of the epoxide ring in the acetyl-4,4′-anhydroneuraminic acid derivatives 6 and 7. This has provided the azide 8, the methyl ether 9, the nitrile 10 and the chloride 11 of the 8,9-isopropylidenated β-methyl ketoside of methyl N-acetylneuraminate in moderate to good yields.

Synthesis of C-5 Analogs of N-Acetylneuraminic Acid via Indium-Mediated Allylation of N-Substituted 2-Amino-2-deoxymannoses.

This paper presents a short synthesis of new analogs of N-acetylneuraminic acid (Neu5Ac) varied structurally at C-5. The synthetic strategy includes indium-mediated coupling reactions between ethyl 2-(bromomethyl)acrylate and N-derivatized mannosamines, and the ozonolysis of the resulting enoates. The main advantage of this indium-mediated allylation for the synthesis of neuraminic acids comes from the efficient, stereoselective C-C bond formation, which affords predominantly the correct diastereomer having a threo relationship between the newly generated hydroxyl group and the C-2 amide group of mannosamine. By this approach, Neu5Boc (4a), Neu5Gly (4b), Neu5(6-NHCbz)hexanoyl (4c), and Neu5(1-naphthyl)acetyl (4d) were prepared in three steps (overall approximately 50%). In addition, several N-substituted neuraminic acids were synthesized by N-acylation of the amino functionality of neuraminic acid (5b), which was obtained by deprotecting the N-Boc group of Neu5Boc (4a). These analogs include Neu5BrAc (6a), Neu5acryloyl (6b), Neu5benzoyl (6c) and Neu5benzoyl-4-benzoyl (6d). The N-acylation method is especially suited for synthesis of neuraminic acids bearing substituents that can not tolerate ozonolysis or that are unstable (photo)chemically. Finally, we illustrate the utility of synthetic neuraminic acids by converting 4c to a derivative of 2-deoxy-2,3-didehydroneuraminic acid (8c), a precursor to inhibitors of neuraminidases.

Synthesis of C11 chain-extended analogues of N-acetylneuraminic acid

The synthesis of novel C11 chain-extended analogues of N-acetylneuraminic acid has been achieved from the N-acetyl-9-hydroxyneuraminic acid derivative 7 via a Swern oxidation/Wittig olefination sequence. In this way the novel α,β-unsaturated ester 12, the saturated ester 13 and the α,β-unsaturated aldehyde 14 of N-acetylneuraminic acid have been prepared in good overall yield.

The Synthesis and N.M.R. Investigation of a Methyloxime Derivative of N-Acetylneuraminic Acid

The synthesis of a novel 4-modified N-acetylneuraminic acid analogue and its structural characterization by n.m.r. spectroscopy are reported.

Synthesis of potential inhibitors of hemagglutination by influenza virus: chemoenzymic preparation of N-5 analogs of N-acetylneuraminic acid.

A chemoenzymic route to neuraminic acid 2 is described. This method is based on conversion of N-carbobenzoxymannosamine (ManCBz) 3 to N-carbobenzoxyneuraminic acid (Neu5CBz) 4, catalyzed by Neu5Ac aldolase. The Neu5CBz 4 was converted to the α-methyl glycoside 8 and deprotected to afford the free amine 2. This procedure has been scaled up to generate 10-gram quantities of 2. N-Acylation of 2 produced several new N-acyl neuraminic acid analogs; these have been evaluated as inhibitors of adhesion of influenza virus to chicken erythrocytes in a hemagglutination inhibition assay (HAI). This preparation of neuraminic acid 2 is compared with other literature procedures.

Fluorescent CMP‐sialic acids as a tool to study the specificity of the CMP‐sialic acid carrier and the glycoconjugate sialylation in permeabilized cells

The specificity of the Golgi carrier for CMP-sialic-acids and the lumenal sialylation of glycoconjugates in mechanically permeabilized cells (semi-intact CHO 15B cells) was studied with CMP-activated fluorescent sialic acids as sensitive markers. Semi-intact cells represent a well-established cellular model for studies on the constitutive secretion pathway because the perforated plasma membrane allows membrane-impermeable CMP-sialic-acids to gain access to cellular organelles. The subcellular structures of semi-intact cells remain morphologically intact and hence synthetic CMP-sialic-acids can be assayed as substrates for the corresponding Golgi sugar-nucleotide transporter. The results prove that the CMP-sialic-acid carrier is able to translocate fluorescent CMP-glycosides, despite the bulky fluoresceinyl residue located at position C5 or C9 of the sialic-acid moiety; the data suggest a slightly higher affinity of the carrier for the C9-substituted CMP-glycoside, whereas the affinity of cellular sialyltransferases is fourfold higher for CMP-5-N-fluoresceinylaminoacetylneuraminic acid (5-FTIUNeuAc; 5-N-fluoresceinylaminoneuraminic acid). Using CMP-9-fluoresceinylthioureido-N-acetylneuraminic acid (CMP-9-FTIUNeuAc), an easy and sensitive fluorometric assay was established for the lumenal sialylation in semi-intact cells. Cellular proteins and gangliosides are both labelled by covalent incorporation of the fluorescent N-acetylneuraminic acid analogue. The assay allows rapid screening for small biomolecules or proteins that influence cellular sialyl transport and sialyl transfer; the lumenal fluorescence incorporation does not require ATP or cytosolic compounds. The suitability of fluorescent CMP-glycosides as markers for intracellular sialylation, proven in this paper, introduces the use of synthetic sialic acids for visualisation of cellular sialic acid pathways by fluorescence microscopy. Based on the data presented here, specific CMP-N-acetylneuraminic-acid analogues can be produced and used for the characterization of the Golgi CMP-sialic-acid carrier.