Protecting‐Group‐Controlled Enzymatic Glycosylation of Oligo‐N‐Acetyllactosamine Derivatives

We describe a chemoenzymatic strategy that can give a library of differentially fucosylated and sialylated oligosaccharides starting from a single chemically synthesized tri‐N‐acetyllactosamine derivative. The common precursor could easily be converted into 6 different hexasaccharides in which the glucosamine moieties are either acetylated (GlcNAc) or modified as a free amine (GlcNH₂) or Boc (GlcNHBoc). Fucosylation of the resulting compounds by a recombinant fucosyl transferase resulted in only modification of the natural GlcNAc moieties, providing access to 6 selectively mono‐ and bis‐fucosylated oligosaccharides. Conversion of the GlcNH₂ or GlcNHBoc moieties into the natural GlcNAc, followed by sialylation by sialyl transferases gave 12 differently fucosylated and sialylated compounds. The oligosaccharides were printed as a microarray that was probed by several glycan‐binding proteins, demonstrating that complex patterns of fucosylation can modulate glycan recognition.     

1‐Picolinyl‐5‐azido Thiosialosides: Versatile Donors for the Stereoselective Construction of Sialyl Linkages

With the picolinyl (Pic) group as a C‐1 located directing group and N₃ as versatile precursor for C5‐NH₂, a novel 1‐Pic‐5‐N₃ thiosialyl donor was designed and synthesized, based on which a new sialylation protocol was established. In comparison to conventional sialylation methods, the new protocol exhibited obvious advantages, including excellent α‐stereoselectivity in the absence of a solvent effect, broad substrate scope encompassing the challenging sialyl 8‐ and 9‐hydroxy groups of sialic acid acceptors, flexibility in sialoside derivative synthesis, high temperature tolerance and easy scalability. In particular, the applicability to the synthesis of complex and bioactive N‐glycan antennae when combined with the MPEP glycosylation protocol via the “latent‐active” strategy has been shown. Mechanistically, the excellent α‐stereoselectivity of the novel sialylation protocol could be attributed to the dramatic electron‐withdrawing effect of the protonated Pic groups, which was supported by control reactions and DFT calculations.     

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.

     

Effect of Anomeric Linkage on the Sialylation of Glycosides by Cells

The synthesis of sialylated glycosides using saccharide primers and cells was investigated. α · and β · Saccharide primers were chemically synthesized and introduced into B16 melanoma cells to prime oligosaccharide synthesis. Incorporation of α‐ and β‐dodecyl lactosides into B16 cells resulted in the sialylation of the galactose residue to give GM3‐type oligosaccharides. The β‐dodecyl galactoside primer was sialylated but the α‐dodecyl galactoside primer was not. Both the α‐ and β‐dodecyl glucoside primers were not elongated. In the glycosylation of primers by cells, this research confirmed that sialyl transferases tolerate acceptor modifications and are permissive to primer elongation regardless of the α‐ or β‐linkage to the aglycon unit. However, the presence of the terminal galactose residue that is β‐linked to the adjacent saccharide or aglycon unit is essential for sialylation by cellular enzymes to occur.     

Dehydrative sialylation with C2-hemiketal sialyl donors

[reaction: see text] A new method for sialylation involving the dehydrative coupling of sialyl donors with the reagent combination of (p-nitrophenyl)(phenyl) sulfoxide and triflic anhydride is reported. This process establishes sialyl C2-hemiketals as viable sialyl donors for complex carbohydrate synthesis.     

Divergent Chemoenzymatic Synthesis of Asymmetrical‐Core‐Fucosylated and Core‐Unmodified N‐Glycans

A divergent chemoenzymaytic approach for the preparation of core‐fucosylated and core‐unmodified asymmetrical N‐glycans from a common advances precursor is described. An undecasaccharide was synthesized by sequential chemical glycosylations of an orthogonally protected core fucosylated hexasaccharide that is common to all mammalian core fucosylated N‐glycans. Antennae‐selective enzymatic extension of the undecasaccharide using a panel of glycosyl transferases afforded core fucosylated asymmetrical triantennary N‐glycan isomers, which are potential biomarkers for breast cancer. A unique aspect of our approach is that a fucosidase (FucA1) has been identified that selectively can cleave a core‐fucoside without affecting the fucoside of a sialyl Lewis^X epitope to give easy access to core‐unmodified compounds.     

Selective Engineering of Linkage‐Specific α2,6‐N‐Linked Sialoproteins Using Sydnone‐Modified Sialic Acid Bioorthogonal Reporters

The metabolic oligosaccharide engineering (MOE) strategy using unnatural sialic acids has recently enabled the visualization of the sialome in living systems. However, MOE only reports on global sialylation and dissected information regarding subsets of sialosides is missing. Described here is the synthesis and utilization of sialic acids modified with a sydnone reporter for the metabolic labeling of sialoconjugates. The positioning of the reporter on the sugar significantly altered its metabolic fate. Further in vitro enzymatic assays revealed that the 9‐modified neuraminic acid is preferentially accepted by the sialyltransferase ST6Gal‐I over ST3Gal‐IV, leading to the favored incorporation of the reporter into linkage‐specific α2,6‐N‐linked sialoproteins. This sydnone sugar presents the possibility of investigating the roles of specific sialosides.     

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.     

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.     

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     

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.     

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.     

Oligonucleotide‐Based Mimetics of Hepatocyte Growth Factor

Oligonucleotide‐based hepatocyte growth factor (HGF) mimetics are described. A DNA aptamer to Met, a cognate receptor for HGF, was shown to induce Met activation when used in dimer form. The most potent aptamer dimer, ss‐0, which was composed solely of 100‐mer single‐stranded DNA, exhibited nanomolar potency. Aptamer ss‐0 reproduced HGF‐induced cellular behaviors, including migration and proliferation. The present work sheds light on oligonucleotides as a novel chemical entity for the design of growth factor mimetics.     

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.     

Efficient synthesis of MUC4 sialylglycopeptide through the new sialylation using 5-acetamido-neuraminamide donors

Sialylation reactions using a new sialyl donor, diethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-O-beta-D-glycero-D-galacto-2-nonulopyranosylonamide phosphite (Neu5Ac-1-amide-2-phosphite) derivatives, and the synthesis of the sialyl-T N-MUC4 glycopeptide are described. The sialylation was performed in CH2Cl2 solvent toward the 6-hydroxyl group of several monosugar acceptors and generated alpha-sialoside in good yield under low temperature and TMSOTf activation system. Amide derivatives of sialoside were easily converted into naturally occurring sialoside after hydrolysis of the amide group. Sialyl-alpha(2,6)-GalN3 was also prepared by this new sialylation protocol, and then this sialoside was further converted into a Fmoc-protected sialyl-TN serine derivative for solid-phase glycopeptides synthesis. The solid-phase glycopeptide synthesis using this sialyl-TN serine derivative in which the sugar hydroxyl group was free afforded the target sialyl-TN-MUC4 glycopeptide.     

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.     

Parallel Synthesis of Sialyl Lewis X Mimetics on a Solid Phase: Access to a Library of Fucopeptides

A novel anchoring group p-(acyloxymethyl)benzylidene acetal (p-AMBA) enables the bidirectional functionalization of glycosylated amino acid derivatives and thus the rapid parallel synthesis of fucopeptides as sialyl Lewis X mimetics on a solid phase [Eq. (a), PEG-PS=poly(ethylene glycol) graft copolymer]. This led to the discovery of new mimetics against P-selectin with IC₅₀ values in the low μM range.     

Environment‐sensitive Fluorescent Turn‐on Chemical Probe for the Specific Detection of O‐Methylguanine‐DNA Methyltransferase (MGMT) in Living Cells

MGMT protein, which has been associated with resistance to antitumor alkylation drugs for many patients, is a very useful prognostic marker to provide a guide for therapeutic decisions. Considering the large number of cellular samples that have to be handled daily at the hospitals, it is thus important to develop a rapid and simple analytical method to distinguish MGMT activity in different types of cells. In this paper, we describe a MGMT‐activated fluorescence turn‐on probe for the rapid no‐wash imaging of MGMT in living cells. The probe consists of a specific MGMT suicide pseudosubstrate, O⁶‐benzyl‐guanine and an environment‐sensitive fluorophore, SBD. In the presence of MGMT, the enzyme transfers SBD to the protein active site where the hydrophobic surrounding causes the fluorophore to exhibit more than 50‐fold fluorescence enhancement. With this probe, bright fluorescence was observed for MGMT‐positive, Hela S3 and MCF‐7 cells, while MGMT‐deficient CHO cells displayed no fluorescence. We believe that this fluorescence activation probe design can also be extended to detect other transferases, for which there are still no effective methods to image them in living cells.     

Conformationally constrained dipeptides. Obtention of enantiomerically pure 6‐acetamido‐5‐oxo‐1,2,3,5,6,7‐hexahydro‐3‐indolizine carboxylic acid

The separation of a stereoisomeric mixture of esters 6, and the synthesis of the new enantiomerically pure unsaturated 6,5‐fused bicyclic lactam 2 are described. The key‐step involves trimethylsilyl iodide cleavage, without racemization, of a vinylogous carbamate. The cis N‐ acetylamino acid 2 is a new scaffold for the synthesis of rigid β‐turn mimetics.     

An approach towards the synthesis of sialyl nucleoside mimetics

An approach towards the synthesis of novel sialyl nucleoside mimetics based on d-fructose is described. The synthesis of these mimetics is achieved in good overall yield in seven steps. The key synthetic step is the coupling reaction of pyrimidine bases (uracil, 5-fluorouracil and cytosine) to the C-1 position of the modified d-tagatofructofuranoside.