Templated Oligosaccharide Synthesis: Driving Forces and Mechanistic Aspects.

We previously communicated that high α-selectivity that can be achieved in intramolecular glycosylations using a rigid bisphenol A template supplemented with linkers of various lengths. Herein, we present our investigation of the mechanistic aspects of the templated synthesis that helped to design an improved template-linker combination. We demonstrate that bisphenol A as the template in combination with phthaloyl linker allows for superior stereoselectivity and yields in glycosylations. Several mechanistic studies explore origins of the enhanced stereoselectivity and yields achieved using the phthaloyl linker.

Synthesis of the Repeating Unit of Capsular Polysaccharide Staphylococcus aureus Type 5 To Study Chemical Activation and Conjugation of Native CP5.

The chemical synthesis of the repeating unit of S. aureus capsular polysaccharide type 5 equipped with capping methyl groups at the points of propagation of the polysaccharide sequence is described. This model compound was designed to study activation of the full length polysaccharide for conjugation to a carrier protein.

Templated Oligosaccharide Synthesis: The Linker Effect on the Stereoselectivity of Glycosylation.

A new method for intramolecular oligosaccharide synthesis that is conceptually related to the general molecular clamp approach is introduced. Exceptional α-selectivity has been achieved in a majority of applications. Unlike other related concepts, this approach is based on the bisphenol A template, which allows one to connect multiple building blocks to perform templated oligosaccharide synthesis with complete stereoselectivity. This principle was demonstrated by the synthesis of an α,α-linked trisaccharide.

2,3-Di-O-picolinyl building blocks as glycosyl donors with switchable stereoselectivity.

2-O-Picolinyl protected glycosyl donors lead to the formation of 1,2-trans glycosides with complete stereoselectivity. This is due to the participatory effect of the picolinyl nitrogen that is able to block the bottom face of the ring via a six-membered cyclic intermediate. Herein we demonstrate that if the nitrogen atom of the O-picolinyl moiety is temporarily blocked by coordination to the metal center (Pd), it becomes unavailable to participate in glycosylation and hence the stereoselectivity of 2-O-picolinyl-assisted glycosylations can be "switched".

A Concise Synthesis of the Repeating Unit of Capsular Polysaccharide Staphylococcus aureus Type 8.

The first synthesis of the repeating unit of S. aureus capsular polysaccharide type 8 is described. The repeating unit is an unusual trisaccharide sequence of three uncommon sugars, all connected via 1,2-cis linkages. The synthetic trisaccharide was equipped with capping methyl groups at the points of propagation of the polysaccharide sequence.

Square-wave voltammetry assays for glycoproteins on nanoporous gold.

Electrochemical enzyme-linked lectinsorbent assays (ELLA) were developed using nanoporous gold (NPG) as a solid support for protein immobilization and as an electrode for the electrochemical determination of the product of the reaction between alkaline phosphatase (ALP) and p-aminophenyl phosphate (p-APP), which is p-aminophenol (p-AP). Glycoproteins or concanavalin A (Con A) and ALP conjugates were covalently immobilized onto lipoic acid self-assembled monolayers on NPG. The binding of Con A - ALP (or soybean agglutinin - ALP) conjugate to glycoproteins covalently immobilized on NPG and subsequent incubation with p-APP substrate was found to result in square-wave voltammograms whose peak difference current varied with the identity of the glycoprotein. NPG presenting covalently bound glycoproteins was used as the basis for a competitive electrochemical assay for glycoproteins in solution (transferrin and IgG). A kinetic ELLA based on steric hindrance of the enzyme-substrate reaction and hence reduced enzymatic reaction rate after glycoprotein binding is demonstrated using immobilized Con A-ALP conjugates. Using the immobilized Con A-ALP conjugate, the binding affinity of immunoglobulin G (IgG) was found to be 105 nM, and that for transferrin was found to be 650 nM. Minimal interference was observed in the presence of 5 mg mL-1 BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl D-mannoside for the binding of TSF and IgG to Con A-ALP; IC50 values were found to be 90 µM and 286 µM, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay.

Superarming of glycosyl donors by combined neighboring and conformational effects.

A novel glycosyl donor that combines the concepts of both conformational and electronic superarming has been synthesized. The reactivity and selectivity of the donor have been tested in competition experiments.

O-Benzoxazolyl imidates as versatile glycosyl donors for chemical glycosylation.

Herein, we report a new class of glycosyl donors, benzoxazolyl imidates, for chemical glycosylation. The O-benzoxazolyl (OBox) leaving group was designed with an aim to compare the relative reactivity and stability of similarly structured S-benzoxazolyl (SBox) glycosides (thioimidates) developed in our lab and glycosyl trichloroacetimidates (TCAI, O-imidates) developed by Schmidt. Novel OBox donors can be activated under catalytic conditions and provided excellent yields in glycosylation. The OBox imidates were found to be more reactive than either SBox or TCAI donors. The high reactivity profile was confirmed in direct competitive experiments and was found beneficial for HPLC-assisted solid-phase synthesis.

Electrochemical characterization of globotriose-containing self-assembled monolayers on nanoporous gold and their binding of soybean agglutinin.

Self-assembled monolayers (SAMs) of α-D-Gal-(1→4)-ß-D-Gal-(1→4)-ß-D-Glc-mercaptooctane (globotriose, Gb3-C8-SH) were prepared both as single-component SAMs and as mixed SAMs with either octanethiol (OCT) or 8-mercapto-3,6-dioxaoctanol (HO-PEG2-SH), on flat gold and on nanoporous gold (NPG) electrodes. The binding of soybean agglutinin (SBA) to the globotriose (Gb3) unit in the SAMs was then studied using electrochemical impedance spectroscopy (EIS), which is a label free method found to be quite sensitive to SAM composition and to the differences in SAM structure on NPG versus on flat Au. The affinity of SBA to the mixed SAM of HO-PEG2-SH and Gb3-C8-SH on NPG is found to be greater on NPG than on flat gold, and indicates a potential advantage for NPG as a substrate. The SAMs of HO-PEG2-SH were found to resist protein adsorption on either NPG or flat gold. The non-specific adsorption of SBA to OCT SAMs on flat Au was observed in EIS by the increase in charge transfer resistance; whereas, the increase seen on the NPG surface was smaller, and suggests that EIS measurements on NPG are less affected by non-specific protein adsorption. Atomic force microscopy (AFM) images of the SBA binding to mixed SAM of HO-PEG2-SH and Gb3-C8-SH on NPG showed a greater number of proteins on top of the OCT containing SAMs.

Lectin-carbohydrate interactions on nanoporous gold monoliths.

Monoliths of nanoporous gold (np-Au) were modified with self-assembled monolayers of octadecanethiol (C18-SH), 8-mercaptooctyl α-D-mannopyranoside (αMan-C8-SH), and 8-mercapto-3,6-dioxaoctanol (HO-PEG2-SH), and the loading was assessed using thermogravimetric analysis (TGA). Modification with mixed SAMs containing αMan-C8-SH (at a 0.20 mole fraction in the SAM forming solution) with either octanethiol or HO-PEG2-SH was also investigated. The np-Au monoliths modified with αMan-C8-SH bind the lectin Concanavalin A (Con A), and the additional mass due to bound protein was assessed using TGA analysis. A comparison of TGA traces measured before and after exposure of HO-PEG2-SH modified np-Au to Con A showed that the non-specific binding of Con A was minimal. In contrast, np-Au modified with octanethiol showed a significant mass loss due to non-specifically adsorbed Con A. A significant mass loss was also attributed to binding of Con A to bare np-Au monoliths. TGA revealed a mass loss due to the binding of Con A to np-Au monoliths modified with pure αMan-C8-SH. The use of mass losses determined by TGA to compare the binding of Con A to np-Au monoliths modified by mixed SAMs of αMan-C8-SH and either octanethiol or HO-PEG2-SH revealed that binding to mixed SAM modified surfaces is specific for the mixed SAMs with HO-PEG2-SH but shows a significant contribution from non-specific adsorption for the mixed SAMs with octanethiol. Minimal adsorption of immunoglobulin G (IgG) and peanut agglutinin (PNA) towards the mannoside modified np-Au monoliths was demonstrated. A greater mass loss was found for Con A bound onto the monolith than for either IgG or PNA, signifying that the mannose presenting SAMs in np-Au retain selectivity for Con A. TGA data also provide evidence that Con A bound to the αMan-C8-SH modified np-Au can be eluted by flowing a solution of methyl α-D-mannopyranoside through the structure. The presence of Con A proteins on the modified np-Au surface was also confirmed using atomic force microscopy (AFM). The results highlight the potential for application of carbohydrate modified np-Au monoliths to glycoscience and glycotechnology and demonstrate that they can be used for capture and release of carbohydrate binding proteins in significant quantities.

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.

Synthesis, characterization and reactivity of carbohydrate platinum(IV) complexes with thioglycoside ligands.

Reactions of fac-[PtMe3(4,4'-R2bpy)(Me2CO)][BF4] (R = H, 1a; tBu, 1b) and fac-[PtMe3(OAc-kappa2O,O')(Me2CO)] (2), respectively, with thioglycosides containing thioethyl (ch-SEt) and thioimidate (ch-STaz, Taz = thiazoline-2-yl) anomeric groups led to the formation of the carbohydrate platinum(IV) complexes fac-[PtMe3(4,4-R2bpy)(ch*)][BF4] (ch* = ch-SEt, 8-14; ch-STaz, 15-23) and fac-[PtMe3(OAc-kappa2O,O')(ch*)] (ch* = ch-SEt, 24-28; ch-STaz = 29-35), respectively. NMR (1H, 13C, 195Pt) spectroscopic investigations and a single-crystal X-ray diffraction analysis of 19 (ch-STaz = 2-thiazolinyl 2,3,4,6-tetra-O-benzoyl-1-thio-beta-D-galactopyranose) revealed the S coordination of the ch-SEt glycosides and the N coordination of the ch-STaz glycosides. Furthermore, X-ray structure analyses of the two decomposition products fac-[PtMe3(bpy)(STazH-kappaS)][BF4] (21a) and 1,6-anhydro-2,3,4-tri-O-benzoyl-beta-D-glucopyranose (23a), where a cleavage of the anomeric C-S bond had occurred in both cases, gave rise to the assumption that this decomposition was mediated due to coordination of the thioglycosides to the high electrophilic platinum(IV) atom, in non-strictly dried solutions. Reactions of fac-[PtMe3(Me2CO)3][BF4] (3) with ch-SEt as well as with ch-SPT and ch-Sbpy thioglycosides (PT = 4-(pyridine-2-yl)-thiazole-2-yl; bpy = 2,2'-bipyridine-6-yl), having N,S and N,N heteroaryl anomeric groups, respectively, led to the formation of platinum(IV) complexes of the type fac-[PtMe3(ch*)][BF4] (ch* = ch-SEt, 36-40, ch-SPT 42-44, ch-Sbpy 45, 46). The thioglycosides were found to be coordinated in a tridentate kappaS,kappa2O,O, kappaS,kappaN,kappaO and kappaS,kappa2N,N coordination mode, respectively. Analogous reactions with ch-STaz ligands succeeded for 2-thiazolinyl 2,3,4-tri-O-benzyl-6-O-(2,2'-bipyridine-6-yl)-1-thio-beta-D-glucopyranoside (5h) resulting in fac-[PtMe3(ch-STaz)][BF4] (41, ch-STaz = 5h), having a kappa3N,N',N''coordinated thioglycoside ligand.

Coordination chemistry approach to the long-standing challenge of stereocontrolled chemical glycosylation.

This study clearly demonstrates that a multi-dentate metal coordination to the leaving group, along with O-5 and/or a protecting group at O-6, has a strong effect on the stereoselectivity of chemical glycosylation.

STICS: surface-tethered iterative carbohydrate synthesis.

A new surface-tethered iterative carbohydrate synthesis (STICS) technology is presented in which a surface functionalized 'stick' made of chemically stable high surface area porous gold allows one to perform cost efficient and simple synthesis of oligosaccharide chains; at the end of the synthesis, the oligosaccharide can be cleaved off and the stick reused for subsequent syntheses.

Unexpected orthogonality of S-benzoxazolyl and S-thiazolinyl glycosides: application to expeditious oligosaccharide assembly.

Thorough mechanistic studies of the alkylation pathway for the activation of glycosyl thioimidates have led to the development of the "thioimidate-only orthogonal strategy". Discrimination among S-thiazolinyl (STaz) and S-benzoxazolyl (SBox) anomeric leaving groups was achieved by fine-tuning of the activation conditions. Preferential glycosidation of a certain thioimidate is not simply determined by the strength of activating reagents; instead, the type of activation--direct vs indirect--comes to the fore and plays the key role.

4-(pyridin-2-yl)thiazol-2-yl thioglycosides as bidentate ligands for oligosaccharide synthesis via temporary deactivation.

This study focusses on a new concept for oligosaccharide synthesis based on 4-(pyridin-2-yl)thiazol-2-yl thioglycosides that can either act as effective glycosyl donors or can be deactivated by stable bidentate complexation with palladium(II) bromide.

Silver(I) tetrafluoroborate as a potent promoter for chemical glycosylation.

We have identified silver tetrafluoroborate (AgBF(4)) as an excellent promoter for the activation of various glycosyl donors including glycosyl halides, trichloroacetimidates, thioimidates, etc. Easy handling and no requirement for azeotropic dehydration prior to application makes AgBF(4) especially beneficial in comparison to the commonly used AgOTf. Selective activation of glycosyl halides or thioimidates over thioglycosides or n-pentenyl glycosides, including simple sequential one-pot syntheses, has been also demonstrated. Versatility of glycosyl thioimidates was further explored by converting these intermediates into a variety of other classes of glycosyl donors.

S-thiazolinyl (STaz) glycosides as versatile building blocks for convergent selective, chemoselective, and orthogonal oligosaccharide synthesis.

In the aim of developing new procedures for efficient oligosaccharide assembly, a range of S-thiazolinyl (STaz) glycosides have been synthesized. These novel derivatives were evaluated against a variety of reaction conditions and were shown to be capable of being chemoselectively activated in the armed-disarmed fashion. Moreover, the S-thiazolinyl moiety exhibited a remarkable propensity for selective activation over other common leaving groups. Conversely, a variety of leaving groups could be selectively activated over the STaz moiety, which, in turn, allowed STaz/S-ethyl and STaz/S-phenyl orthogonal approaches. To demonstrate versatility of novel STaz derivatives, a number of oligosaccharide targets have been synthesized in a convergent selective, orthogonal, and chemoselective fashion.

Synthesis of cancer-associated glycoantigens: stage-specific embryonic antigen 3 (SSEA-3).

The synthesis of the tumor-associated carbohydrate antigens SSEA-3 and Gb3 in a semi-convergent fashion using building blocks bearing a S-thiazolinyl (STaz) moiety is reported. Complete stereoselective control of a difficult alpha-(1-->4)-galactosylation and high overall yields were achieved.