How O-substitution of sialyl donors affects their stereoselectivity

The profound effect of substituents at C-5 of glycosyl sialosides on their stereoselectivity is well-known although the exact nature of this effect is somewhat less understood. Presented herein is a comparative study of a range of novel sialyl donors with various O-substituents. It is demonstrated that O-substituents at C-4 and C-7 may also have a significant effect on the reactivity of sialyl donors and on the stereoselectivity of chemical sialylation.     

A stereoselective approach for the synthesis of alpha-sialosides

A highly efficient synthesis of the human melanoma associated antigen GD(3) derivative has been described. A key feature of the synthetic approach was the use of sialyl donors that were protected with a C-5 trifluoroacetamide moiety. These sialyl donors gave high yields and excellent alpha-anomeric selectivities in direct glycosylations with a wide variety of glycosyl acceptors ranging from C-8 hydroxyls of sialic acids and C-3 hydroxyls of galactosides to reactive primary alcohols.     

N-trifluoroacetyl sialyl phosphite donors for the synthesis of alpha(2 --> 9) oligosialic acids

[reaction: see text] A new method for the synthesis of alpha(2 --> 9) oligosialic acids is developed using phosphite sialyl donors that are protected with a C-5 N-trifluoroacetyl (NHTFA) substituent. Compared with conventional donors, these donors gave a higher degree of alpha-anomeric selectivity during glycosidic bond formation and better yields during iterative sialylation in the synthesis of oligosialic acids.     

Thiomidoyl approach to the synthesis of α-sialosides

Novel sialosyl donors, S-benzoxazolyl (SBox) and S-thiazolyl (STaz) sialosides, have been synthesized and applied to the stereoselective synthesis of α-sialosides. It was also demonstrated that it is possible to selectively activate SBox sialyl donor over ethyl thioglycoside, allowing the direct synthesis of disaccharide donors that could be used in subsequent glycosylations without further manipulations.     

Highly Stereoselective Synthesis of Primary, Secondary, and Tertiary α-S-Sialosides under Lewis Acidic Conditions

N-Acetyl 4-O,5-N-oxazolidinone protected sialyl phosphates of either anomeric configuration are excellent donors for the formation of α-S-sialosides at -78 °C in dichloromethane with primary, secondary, and tertiary thiols including galactose 3-, 4-, and 6-thiols. The reactions, which proceed under typical Lewis acid promoted glycosylation conditions, are highly α-selective and do not suffer from competing elimination of the phosphate.     

Gold(I)‐Catalyzed Glycosylation with Glycosyl ortho‐Alkynylbenzoates as Donors: General Scope and Application in the Synthesis of a Cyclic Triterpene Saponin

Glycosyl ortho‐alkynylbenzoates have emerged as a new generation of donors for glycosidation under the catalysis of gold(I) complexes such as Ph₃PAuOTf and Ph₃PAuNTf₂ (Tf=trifluoromethanesulfonate). A wide variety of these donors, including 2‐deoxy sugar and sialyl donors, are easily prepared and shelf stable. The glycosidic coupling yields with alcohols are generally excellent; even direct coupling with the poorly nucleophilic amides gives satisfactory yields. Moreover, excellent α‐selective glycosylation with a 2‐deoxy sugar donor and β‐selective sialylation have been realized. Application of the present glycosylation protocol in the efficient synthesis of a cyclic triterpene tetrasaccharide have further demonstrated the versatility and efficacy of this new method, in that a novel chemoselective glycosylation of the carboxylic acid and a new one‐pot sequential glycosylation sequence have been implemented.     

alpha-selective sialylations at -78 degrees C in nitrile solvents with a 1-adamantanyl thiosialoside

Novel 1-adamantanylthio sialosides were synthesized and coupled to acceptors under NIS/TfOH promotion conditions. These donors showed higher reactivity than the phenylthio sialosides and could be activated by NIS/TfOH in nitrile solvents at -78 degrees C to afford improved alpha-sialylations. With the N-acetyl-5-N,4-O-oxazolidinone-protected 1-adamantanylthio sialyl donor high alpha-selectivities could be achieved in the sialylations of both primary and sterically hindered secondary acceptors, including the important galactose 3-OH acceptors.     

Total synthesis of the aminopropyl functionalized ganglioside GM1

GM1 is a common ganglioside pentasaccharide present on mammalian cell surface.It has been shown to play important roles in cellular communications and initiation of β-amyloid aggregation.In order to synthesize GM1,an efficient synthetic route was developed via a [3+2] strategy.The GM3 trisaccharide acceptor bearing an azido propyl group at the reducing end was prepared using the traditional acetamide protected sialyl thioglycosyl donor,which gave better stereoselectivity than sialyl donors protected with trichloroacetamide or oxazolidinone.The glycosylation of the axial 4-hydroxyl group of GM3 by the disaccharide donor was found to be highly dependent on donor protective groups.Donor bearing the more rigid benzylidene group gave low glycosylation yield.Replacing the benzylidene with acetates led to productive coupling and formation of the fully protected GM1 pentasaccharide.Deprotection of the pentasaccharide produced GM1 functionalized with the aminopropyl side chain,which will be a valuable probe for biological studies.     

The α‐Glycosidation of Partially Unprotected N‐Acetyl and N‐Glycolyl Sialyl Donors in the Absence of a Nitrile Solvent Effect

The synthesis of α‐sialosides is one of the most difficult reactions in carbohydrate chemistry and is considered to be both a thermodynamically and kinetically disfavored process. The use of acetonitrile as a solvent is an effective solution for the α‐selective glycosidation of N‐acetyl sialic acids. In this report, we report on the α‐glycosidation of partially unprotected N‐acetyl and N‐glycolyl donors in the absence of a nitrile solvent effect. The 9‐O‐benzyl‐N‐acetylthiosialoside underwent glycosidation in CH₂Cl₂ with a good α‐selectivity. On the other hand, the 4,7,8‐O‐triacetyl‐9‐O‐benzyl‐N‐acetylthiosialoside was converted to β‐sialoside as a major product under the same reaction conditions. The results indicate that the O‐acetyl protection of the sialyl donor was a major factor in reducing the α‐selectivity of sialylation. After tuning of the protecting groups of the hydroxy groups at the 4,7,8 position on the sialyl donor, we found that the 9‐O‐benzyl‐4‐O‐chloroacetyl‐N‐acetylthiosialoside underwent sialylation with excellent α‐selectivity in CH₂Cl₂. To demonstrate the utility of the method, straightforward synthesis of α(2,9) disialosides containing N‐acetyl and/or N‐glycolyl groups was achieved by using the two N‐acetyl and N‐glycolyl sialyl donors.     

Regioselective α(2→3)-Sialylation of LeX and Lea by Sialidase-Catalyzed Transglycosylation

Considerable effort has been devoted to the development of new methods for α-selective sialylation due to the growing importance of the synthetic sialoglycoconjugates in glycobiology³. The synthesis of α-sialoside has been establised by chemical routes,⁴ which often involve many steps and are complicated. The promising chemoenzymatic procedure through the use of sialyltransferases has already become a preparative technique.⁵ However, laborious isolation and the pronounced acceptor specificity of the transferases limit their synthetic potential. Recently, a novel procedure for α-sialylation has been reported, which uses sialosides of synthetic substrate as donors and is catalyzed by sialidase in place of sialyltransferase. Thiem et a1.⁶ have reported the enzymatic synthesis of α(2→6)-linked sialyl galactose, glucose, lactose and lactosamine in preference to the corresponding α(2→3)-linked derivatives employing sialidase from vibrio cholerae, while Ajisaka et al.⁷ have synthesized α(2→3)-linked sialyl lactose and lactosamine with sialidase from new castle disease virus.

     

Rapid and efficient conversion of sialyl thioglycosides to sialyl esters via NIS/BF3OEt2-promoted glycosylation

A rapid and efficient one-step conversion of sialyl thioglycosides to sialyl esters was disclosed. Under the promotion of NIS and BF₃OEt₂, the glycosylation of per-acetylated sialyl thioglycoside with a set of carboxylic acids provided β-sialyl esters as the major products in good to excellent yields within 5 min. Compared with the long-chain alkyl-, aryl- and α,β-unsaturated acids, complete β-selectivities were observed when the short-chain alkyl acids were selected as the coupling partners. The resultant β-selectivity for the glycosylation of the per-acetylated sialyl thioglycoside with acetic acid was compromised when the 5-N,4-O-oxazolidinone protected sialyl thioglycoside was employed as the coupling partner.     

Molecular recognition of sialyl Lewis(x) and related saccharides by two lectins.

The interaction of sialyl Lewis(x), Lewis(x), and alpha-L-Fuc-(1-->3)-beta-D-GlcNAc with isolectin A from Lotus tetragonolobus (LTL-A), and with Aleuria aurantia agglutinin (AAA) was studied using NMR experiments and surface plasmon resonance. Both lectins are specific for fucose residues. From NMR experiments it was concluded that alpha-L-Fuc-(1-->3)-beta-D-GlcNAc and Lewis(x) bound to both lectins, whereas sialyl Lewis(x) only bound to AAA. Increased line broadening of 1H NMR signals of the carbohydrate ligands upon binding to AAA and LTL-A suggested that AAA bound to the ligands more tightly. Further comparison of line widths showed that for both lectins binding strengths decreased from alpha-L-Fuc-(1-->3)-beta-D-GlcNAc to Lewis(x) and were lowest for sialyl Lewis(x). Surface plasmon resonance measurements were then employed to yield accurate dissociation constants. TrNOESY, QUIET-trNOESY, and trROESY experiments delivered bioactive conformations of the carbohydrate ligands, and STD NMR experiments allowed a precise epitope mapping of the carbohydrates bound to the lectins. The bioactive conformation of Lewis(x) bound to LTL-A, or AAA revealed an unusual orientation of the fucose residue, with negative values for both dihedral angles, phi and psi, at the alpha(1-->3)-glycosidic linkage. A similar distortion of the fucose orientation was also observed for sialyl Lewis(x) bound to AAA. From STD NMR experiments it followed that only the L-fucose residues are in intimate contact with the protein. Presumably steric interactions are responsible for locking the sialic acid residue of sialyl Lewis(x) in one out of many orientations that are present in aqueous solution. The sialic acid residue of sialyl Lewis(x) bound to AAA adopts an orientation similar to that in the corresponding sialyl Lewis(x)/E-selectin complex.     

Stabilization of sialyl cation in axial conformation assisted by remote acyl groups

Stereoselective synthesis of α-sialosides by the glycosylation reaction (sialylation) is an important task in carbohydrate chemistry. Using quantum chemical calculations, the conformations of the sialyl cation formed from the sialyl donor under conditions of sialylation reaction were studied. Although the "axial conformation" of sialyl cation itself is energetically unfavorable, it is possible to stabilize it through the participation of O- and N-acyl protective groups. The obtained results open the possibility to modulate the stereoselectivity of sialylation by directed variation of the nature of protective groups in the sialyl donor molecule. 2     

Exhaustive Conformational Search for Sialyl Cation Reveals Possibility of Remote Participation of Acyl Groups

Finding convenient ways for the stereoselective α-sialylation is important due to the high practical significance of α-sialic acid-containing glycans and neoglycoconjugates. It was proposed that sialylation stereoselectivity is determined by the structure of the sialyl cation (also known in biochemistry as “sialosyl cation”), a supposed intermediate in this reaction. Here we design a new approach for studying the conformational space of highly flexible sialyl cation and find 1625 unique conformers including those stabilized by covalent remote participation (also known as long-range participation) of 4-O-acetyl (4-OAc), 5-N-trifluoroacetyl (5-NTFA), as well as 7,8,9-OAc from both α and β sides. The most energetically stable sialyl cation conformers are featured by 4-OAc participation, closely followed by 5-NTFA- and 7-OAc-stabilized conformers; unstabilized sialyl cation conformers are ∼10 kcal mol⁻¹ less stable than the 4-OAc-stabilized ones. Analysis of all the obtained conformers by means of substituents positions, side chain conformations and ring puckering led us to a new “eight-conformer hypothesis” which describes interconversions among the most important sialyl cation conformers and predicts that stronger remote participation of acyl groups favors β-anomers. Thus, selective synthesis of the desired α-sialosides requires minimization of acyl groups participation.     

Fluorescent Mimetics of CMP‐Neu5Ac Are Highly Potent,Cell‐Permeable Polarization Probes of Eukaryotic and Bacterial Sialyltransferases and Inhibit Cellular Sialylation

Oligosaccharides of the glycolipids and glycoproteins at the outer membranes of human cells carry terminal neuraminic acids, which are responsible for recognition events and adhesion of cells, bacteria, and virus particles. The synthesis of neuraminic acid containing glycosides is accomplished by intracellular sialyl transferases. Therefore, the chemical manipulation of cellular sialylation could be very important to interfere with cancer development, inflammations, and infections. The development and applications of the first nanomolar fluorescent inhibitors of sialyl transferases are described herein. The obtained carbohydrate‐nucleotide mimetics were found to bind all four commercially available and tested eukaryotic and bacterial sialyl transferases in a fluorescence polarization assay. Moreover, it was observed that the anionic mimetics intruded rapidly and efficiently into cells in vesicles and translocated to cellular organelles surrounding the nucleus of CHO cells. The new compounds inhibit cellular sialylation in two cell lines and open new perspectives for investigations of cellular sialylation.     

Synthetic study on α(2→8)-linked oligosialic acid employing 1,5-lactamization as a key step

An attempt to synthesize α(2→8)-linked oligosialic acid utilizing a 1,5-lactamized sialyl acceptor is described. 1,5-Lactamization was experimentally proven to proceed only for α-sialoside, which was integrated into the synthetic cycle of oligosialic acid as a chemical sorting step to collect the desired α-sialoside and as a transformation step to produce a reactive sialyl acceptor for the next sialylation. Lactamized oligosialyl acceptors served as favorable coupling partners for sialylation, providing high stereoselectivities and high yields.     

Formation of lactones from sialylated MUC1 glycopeptides

The tumor-associated carbohydrate antigens TN, T, sialyl TN and sialyl T are expressed on mucins in several epithelial cancers. This has stimulated studies directed towards development of glycopeptide-based anticancer vaccines. Formation of intramolecular lactones involving sialic acid residues and suitably positioned hydroxyl groups in neighboring saccharide moieties is known to occur for glycolipids such as gangliosides. It has been suggested that these lactones are more immunogenic and tumor-specific than their native counterparts and that they might find use as cancer vaccines. We have now investigated if lactonization also occurs for the sialyl TN and T antigens of mucins. It was found that the model compound sialyl T benzyl glycoside , and the glycopeptide Ala-Pro-Asp-Thr-Arg-Pro-Ala from the tandem repeat of the mucin MUC1, in which Thr stands for the 2,3-sialyl-T antigen, lactonized during treatment with glacial acetic acid. Compound gave the 1'--> 2' lactone as the major product and the corresponding 1'--> 4' lactone as the minor product. For glycopeptide the 1'--> 4' lactone constitued the major product, whereas the 1'--> 2' lactone was the minor one. When lactonized was dissolved in water the 1'--> 4' lactone underwent slow hydrolysis, whereas the 1'--> 2' remained stable even after a 30 days incubation. In contrast the corresponding 2,6-sialyl-TN glycopeptide did not lactonize in glacial acetic acid.     

Regio/Stereoselective Glycosylation of Diol and Polyol Acceptors in Efficient Synthesis of Neu5Ac‐α‐2,3‐LacNPhth Trisaccharide

A concise approach to a Neu5Ac‐α‐2,3‐LacNPhth trisaccharide derivative was developed. First, the regio/stereoselective glycosylation between glycoside donors and glucoNPhth diol acceptors was investigated. It was found that the regioselectivity depends not only on the steric hindrance of the C2‐NPhth group and the C6‐OH protecting group of the glucosamine acceptors, but also on the leaving group and protecting group of the glycoside donors. Under optimized conditions, LacNPhth derivatives were synthesized in up to 92 % yield through a regio/stereoselective glycosylation between peracetylated‐α‐galactopyranosyl trichloroacetimidate and p‐methoxyphenyl 6‐O‐tert‐butyldiphenylsilyl‐2‐deoxy‐2‐phthalimido‐β‐d ‐glucopyranoside, avoiding the formation of glycosylated orthoesters and anomeric aglycon transfer. Then, the LacNPhth derivative was deacylated and then protected on the primary position by TBDPS to form a LacNPhth polyol acceptor. Finally, the Neu5Ac‐α‐2,3‐LacNPhth derivative was synthesized in 48 % yield through the regio/stereoselective glycosylation between the LacNPhth polyol acceptor and a sialyl phosphite donor. Starting from d ‐glucosamine hydrochloride, the target Neu5Ac‐α‐2,3‐LacNPhth derivative was synthesized in a total yield of 18.5 % over only 10 steps.     

Three-dimensional mapping of N-linked oligosaccharides using anion-exchange, hydrophobic and hydrophilic interaction modes of high-performance liquid chromatography

To determine the structure of N-linked oligosaccharides, a three-dimensional (3-D) sugar mapping technique for pyridylaminated neutral and sialyl oligosaccharides is proposed. The pyridylaminated oligosaccharide mixture is first separated by HPLC on a diethylaminoethyl anion-exchange column and the elution data are placed on the Z-axis. Neutral and mono-, di-, tri- and tetrasialyloligosaccharides are then individually separated on both a hydrophobic octadecylsilylsilica column and a hydrophilic amide-silica column under the same conditions as described previously for neutral oligosaccharides. The validity of the 3-D mapping technique was tested using sialyl pyridylaminated oligosaccharides from human serum glycoproteins.     

Synthesis of 1,1-linked galactosyl mannosides carrying a thiazine ring as mimetics of sialyl Lewis X antigen: investigation of the effect of carboxyl group orientation on P-selectin inhibition

This paper describes the synthesis of 1,1-linked galactosyl mannosides as sialyl Lewis X mimetics that contain a spiro-ring to position the carboxylate group in a well-defined orientation. It was found that compound 4 is more active as a P-selectin inhibitor (IC50 = 19 microM) than the parent disaccharide 2, which contains a flexible carboxyl group (IC50 = 193 microM). This result is consistent with that observed in the previous NMR study of sialyl Lewis X bound to P-selectin. The chemistry described here should be useful for the development of selective inhibitors of E-, P-, and L-selectins.