Preparation and use of microarrays containing synthetic heparin oligosaccharides for the rapid analysis of heparin-protein interactions

Heparin is a highly sulfated, linear polymer that participates in a plethora of biological processes by interaction with many proteins. The chemical complexity and heterogeneity of this polysaccharide can explain the fact that, despite its widespread medical use as an anticoagulant drug, the structure-function relationship of defined heparin sequences is still poorly understood. Here, we present the chemical synthesis of a library containing heparin oligosaccharides ranging from di- to hexamers of different sequences and sulfation patterns. An amine-terminated linker was placed at the reducing end of the synthetic structures to allow for immobilization onto N-hydroxysuccinimide activated glass slides and creation of heparin microarrays. Key features of this modular synthesis, such as the influence of the amine linker on the glycosidation efficiency, the use of 2-azidoglucose as glycosylating agents for oligosaccharide assembly, and the compatibility of the protecting group strategy with the sulfation-deprotection steps, are discussed. Heparin microarrays containing this oligosaccharide library were constructed using a robotic printer and employed to characterize the carbohydrate binding affinities of three heparin-binding growth factors. FGF-1, FGF-2 and FGF-4 that are implicated in angiogenesis, cell growth and differentiation were studied. These heparin chips aided in the discovery of novel, sulfated sequences that bind FGF, and in the determination of the structural requirements needed for recognition by using picomoles of protein on a single slide. The results presented here highlight the potential of combining oligosaccharide synthesis and carbohydrate microarray technology to establish a structure-activity relationship in biological processes.     

Microarrays of synthetic heparin oligosaccharides

We present the first preparation of microarrays containing synthetic heparin oligosaccharides in order to elucidate the heparin-protein interactions involved in a variety of biological processes. For this purpose, we have developed a novel linker strategy that is compatible with the protecting-group manipulations required for the synthesis of the highly sulfated oligosaccharides and can also be extended to an automated solid phase approach. Strategic placement of the orthogonally protected amine linker was key to the success of the array construction. These heparin chips allow for the high-throughput screening of oligosaccharides by using approximately picomoles of protein. The potential of the new method was demonstrated by probing the carbohydrate affinity of two heparin-binding growth factors, FGF-1 and FGF-2, that are implicated in the development and differentiation of several tumors.     

Synthesis of a Potential 10E4 Tetrasaccharide Antigen Involved in Scrapie Pathogenesis

To test the hypothesis that tetrasaccharide 3 is involved in scrapie pathogenesis, tetrasaccharide derivative 32 functionalized with an amine linker at the reducing end was synthesized. A (2 + 2) glycosylation approach was chosen to furnish the target compound in fully protected form. To investigate its biological role, tetrasaccharide 32 was further functionalized to the corresponding thiol 33 using Traut's reagent. During the course of the synthesis, the N,N‐diacetyl protecting group proved surprisingly labile to radical and acidic conditions.     

Total synthesis of a unique tetrasaccharide present in the human clotting factor IX and mammalian Notch 1 receptor

The synthesis of a unique tetrasaccharide linked to the serine 61 of human clotting factor IX through an α-l-fucose residue has been achieved for the first time in excellent yield. All glycosylation and protecting group manipulation steps are high yielding and reproducible for a scale-up preparation. A sequential glycosylation strategy has been used to assemble suitably protected monosaccharide synthons for the preparation of the target tetrasaccharide.     

Anomeric reactivity-based one-pot synthesis of heparin-like oligosaccharides

A highly efficient one-pot methodology is described for the synthesis of heparin and heparan sulfate oligosaccharides utilizing thioglycosides with well-defined reactivity as building blocks. L-Idopyranosyl and D-glucopyranosyl thioglycosides 5 and 10 were used as donors due to low reactivity of uronic acids as the glycosyl donors in the one-pot synthesis. The formation of uronic acids by a selective oxidation at C-6 was performed after assembly of the oligosaccharides. The efficiency of this programmable strategy with the flexibility for sulfate incorporation was demonstrated in the representative synthesis of disaccharides 17, 18, tetrasaccharide 23, and pentasaccharide 26.     

Synthetic directions towards capsular polysaccharide of Streptococcus pneumoniae serotype 18C

An efficient synthetic strategy has been developed for the synthesis of the tri, tetrasaccharide block and pentasaccharide corresponding to the capsular polysaccharide of Streptococcus pneumonia serotype 18C as their 2-aminoethyl glycosides. A one-pot glycosylation-deprotection, sequential glycosylations strategy has been adopted for the construction of the fragments and pentasaccharide derivative, which were then transformed into target compound after a series of functional group transformations. The synthetic method relies on the use of p-methoxybenzyl ether as an in situ-removable protecting group to reduce the number of reaction steps significantly. Here H₂SO₄-silica has been used successfully as a promoter for all glycosylation reaction. In addition, the synthetic target also contained a free amino group at its reducing end, facilitating its conjugation with other molecules for various biological studies and applications.     

Zwitterionic‐Type Molten Salt‐Catalyzed Multicomponent Reactions: One‐Pot Synthesis of Substituted Imidazoles Under Solvent‐Free Conditions

4‐(1‐Imidazolium) butane sulfonate is an excellent catalyst for the synthesis of 2,4,5‐trisubstituted and 1,2,4,5‐tetrasubstituted imidazoles through the condensation of 1,2‐dicarbonyl compounds, aldehydes, and ammonium acetate or amine via multicomponent condensation strategy under solvent‐free conditions. The key advantages of this process are high yields, reusability of catalyst, environmental friendliness, easy work‐up and purification of products by nonchromatographic methods.     

Solution and solid-support synthesis of a potential leishmaniasis carbohydrate vaccine

The synthesis of a potential carbohydrate vaccine for the parasitic disease leishmaniasis is described. New solution- and solid-phase synthetic strategies were explored for the assembly of a unique tetrasaccharide antigen found on the Leishmania lipophosphoglycan. An initial solution-phase synthesis relied on thioglycosides as building blocks and the establishment of the central disaccharide from lactal via an oxidation-reduction sequence. A second approach was completed both in solution and on solid support. The solid-phase synthesis relied on assembly from monosaccharide units and was used to evaluate different glycosylating agents in the efficient installation of the galactose beta-(1-->4) mannoside. Glycosyl phosphates proved most successful in this endeavor. This first solid-phase synthesis of the Leishmania cap provided rapid access to the tetrasaccharide in 18% overall yield while requiring only a single purification step. The synthetic cap tetrasaccharide was conjugated to the immunostimulator Pam3Cys to create fully synthetic carbohydrate vaccine 1 and to the carrier protein KLH to form semisynthetic vaccine 2. Currently, both constructs have entered initial immunological experiments in mice targeted at the development of a vaccine against the parasitic disease leishmaniasis.     

Structural elucidation of the tetrasaccharide pool in enoxaparin sodium

Low-molecular-weight heparins (LMWHs) are produced from heparin by various depolymerization strategies, which result in a reduction of the average molecular weight of the polysaccharide chains, a reduction of the anti-factor IIa activity (and a concomitant increase in the anti-factor Xa/anti-factor IIa ratio), and introduction of process-related structural signatures. Numerous techniques have been developed to characterize LMWHs and to measure the type and extent of structural modifications that are introduced as a function of the depolymerization process. We present here an analysis of the tetrasaccharide pool of enoxaparin sodium, a LMWH produced by chemical β-elimination of heparin benzyl ester. We identify the predominant sequences present within the tetrasaccharide pool and demonstrate that this pool provides a sensitive, specific readout of the physicochemical process conditions used to generate enoxaparin sodium.     

Synthesis of a 2,3,4-triglycosylated rhamnoside fragment of rhamnogalacturonan-II side chain A using a late stage oxidation approach

Pectic polysaccharide RG-II, a key component of plant primary cell walls, is known to exist as a dimer formed by means of borate diester cross-links between apiosyl residues of one of its constituent side-chain oligosaccharides. Described herein is the strategy for the synthesis of the branched tetrasaccharide alpha-d-GalA-(1-->2)-[beta-D-GalA-(1-->3)]-[alpha-L-Fuc-(1-->4)]-alpha-L-Rha-OMe, an RG-II fragment that is linked to the apiosyl residue that is thought to be responsible for the borate complexation in RG-II dimer. Iterative glycosylation of the rhamnoside acceptors derived from the key 2,3-orthoacetate of methyl 4-O-methoxybenzyl-alpha-d-rhamnopyranoside afforded the protected tetrasaccharide. The target dicarboxylic acid saccharide was subsequently prepared by removal of protecting groups followed by TEMPO-mediated oxidation of galactopyranosyl residues to galactopyranosyluronic acids.     

Synthesis of polyamino nitriles, en route to acylpolyamine neurotoxins, via the regioselective michael cyanoethylation of unprotected polyamines. Unusual behaviour of 1-(2-aminoethyl)piperazine

The Michael cyanoethylation of 1-(2-aminoethyl)piperazine, 4,4′-methylenebis(cyclohexylamine) and bis(3-aminopropylamine)amine, leading to acrylonitrile free (<100 ppm) polyamino nitriles, as a key step in the synthesis of higher polyamines useful in the synthesis of acylpolyamine neurotoxins, was carried out regioselectively on a multigram scale by careful tuning of reaction conditions, without a necessity to protect nitrogen atoms. The higher reactivity of primary amino groups in aliphatic diamines and triamines [as in bis(3-aminopropylamine)amine] was also observed in the cyclic amine, 4,4′-methylenebis(cyclohexylamine), but reversed in 1-(2-aminoethyl)piperazine. The compounds with a dicyanoethylated nitrogen atom were thermally less stable than the monocyanoethylated ones.     

Synthesis of a tetrasaccharide corresponding to the teichoic acid from the cell wall of Streptomyces sp. VKM Ac-2275

A concise chemical synthesis of a tetrasaccharide found in the teichoic acid from the cell wall of Streptomyces sp. VKM Ac-2275 was achieved in high yield. A [2+2] block synthetic strategy has been adopted for the construction of the target tetrasaccharide by exploiting the orthogonal property of a thioglycoside. For the first time, the 2-(4-methoxyphenoxy) ethyl group has been used as the anomeric protecting group to make the glycone moiety with a readily available linker for its conjugation to a protein without destroying the cyclic structure at the reducing end. Yields were high in all of the intermediate steps.     

Metal complexes of 1-oxa-4,7,10-triazacyclododecane-N,N',N''-triacetic acid

The macrocyclic complexone 1-oxa-4,7,10-triazacyclododecane-N,N',N'-triacetic acid (cODTA) has been synthesized and its protona constants, stability constants of metal complexes and enthalpy changes for the formation of alkaline-earth complexes have been determined. Although it is not so powerful a complexing agent as the N-acetate derivative of the corresponding tetra-aza macrocycle, cDOTA, this is still one of the strongest complexones known, particularly towards the alkaline-earth metals. The complexes of the transition metals are also very stable and there is an inversion of the Irving-Williams order of stability for the complexes of cobalt and nickel.     

Synthesis and anticoagulant activity of bioisosteric sulfonic-Acid analogues of the antithrombin-binding pentasaccharide domain of heparin

Two pentasaccharide sulfonic acids that were related to the antithrombin-binding domain of heparin were prepared, in which two or three primary sulfate esters were replaced by sodium-sulfonatomethyl moieties. The sulfonic-acid groups were formed on a monosaccharide level and the obtained carbohydrate sulfonic-acid esters were found to be excellent donors and acceptors in the glycosylation reactions. Throughout the synthesis, the hydroxy groups to be methylated were masked in the form of acetates and the hydroxy groups to be sulfated were masked with benzyl groups. The disulfonic-acid analogue was prepared in a [2+3] block synthesis by using a trisaccharide disulfonic acid as an acceptor and a glucuronide disaccharide as a donor. For the synthesis of the pentasaccharide trisulfonic acid, a more-efficient approach, which involved elongation of the trisaccharide acceptor with a non-oxidized precursor of the glucuronic acid followed by post-glycosidation oxidation at the tetrasaccharide level and a subsequent [1+4] coupling reaction, was elaborated. In vitro evaluation of the anticoagulant activity of these new sulfonic-acid derivatives revealed that the disulfonate analogue inhibited the blood-coagulation-proteinase factor?Xa with outstanding efficacy; however, the introduction of the third sulfonic-acid moiety resulted in a notable decrease in the anti-Xa activity. The difference in the biological activity of the disulfonic- and trisulfonic-acid counterparts could be explained by the different conformation of their L-iduronic-acid residues.     

Synthesis of phosphorylated Neisseria meningitidis inner core lipopolysaccharide structures

A phosphoethanolamine-substituted tetrasaccharide structure, 2-aminoethyl 2-acetamido-2-deoxy-α-d-glucopyranosyl-(1→2)-6-O-[2-(tert-butyloxycarbonylaminoethyl)-phosphono]-l-glycero-α-d-manno-heptopyranosyl-(1→3)-[β-d-glucopyranosyl-(1→4)]-l-glycero-α-d-manno-heptopyranoside, corresponding to the non-reducing part of the conserved part of Neisseria meningitidis lipopolysaccharides has been synthesized. Orthogonal protection of the phosphoethanolamino group in combination with the presence of a free amino-containing anomeric spacer allows conjugation to proteins to construct conjugate vaccine candidates. The tetrasaccharide is built up using a linear strategy, where the introduction of the terminal α-GlcNAc moiety is performed using a 2-azido-thioglucoside as a donor and NIS/AgOTf as a promoter. The synthetic pathway includes tetrasaccharide intermediates appropriately designed to permit other phosphorylation patterns as well as elongation at the reducing end.     

Stereoselective Synthesis of α‐Linked 2‐Deoxy Glycosides Enabled by Visible‐Light‐Mediated Reductive Deiodination

2‐Deoxy sugars and their derivatives occur abundantly in many pharmaceutically important natural products. However, the construction of specific 2‐deoxy‐glycosidic bonds remains as a challenge. Herein, we report an efficient way to prepare 2‐deoxy‐α‐glycosides by glycosylation of 2‐iodo‐glycosyl acetate and subsequent visible‐light‐mediated tin‐free reductive deiodination. We have successfully applied the postglycosylational‐deiodination strategy in the synthesis of more than 30 mono‐, di‐, tri‐, tetra‐ and pentadeoxysaccharides with excellent stereoselectivity and efficiency. This method has also been applied to the synthesis of a 2‐deoxy‐tetrasaccharide containing four α‐linkages.     

Convergent synthesis of a pentasaccharide repeating unit corresponding to the cell wall O-antigen of Salmonella enterica O44

A convergent synthetic strategy has been developed for the synthesis of a pentasaccharide fragment corresponding to the O-antigen of Salmonella enterica O44 strain. An intermediate tetrasaccharide derivative was prepared by a [2+2] block glycosylation of two disaccharide derivatives. The p-methoxybenzyl (PMB) group has been used as the in situ temporary protecting group minimizing the number of functional group manipulation steps. The application of the armed–disarmed glycosylation concept reduced the number of steps in the synthetic strategy. The glycosylation steps were highly stereoselective and high yielding.     

Synthesis and Preclinical Evaluation of QS-21 Variants Leading to Simplified Vaccine Adjuvants and Mechanistic Probes

QS-21 is a potent immunostimulatory saponin that is currently under clinical investigation as an adjuvant in various vaccines to treat infectious diseases, cancers, and cognitive disorders. Herein, we report the design, synthesis, and preclinical evaluation of simplified QS-21 congeners to define key structural features that are critical for adjuvant activity. Truncation of the linear tetrasaccharide domain revealed that a trisaccharide variant is equipotent to QS-21, while the corresponding disaccharide and monosaccharide congeners are more toxic and less potent, respectively. Modification of the acyl chain domain in the trisaccharide series revealed that a terminal carboxylic acid is well-tolerated while a terminal amine results in reduced adjuvant activity. Acylation of the terminal amine can, in some cases, restore adjuvant activity and enables the synthesis of fluorescently labeled QS-21 variants. Cellular studies with these probes revealed that, contrary to conventional wisdom, the most highly adjuvant active of these fluorescently labeled saponins does not simply associate with the plasma membrane, but rather is internalized by dendritic cells.     

Synthetic study and structural analysis of the antifreeze agent xylomannan from Upis ceramboides

The novel antifreeze factor, xylomannan, first isolated from the freeze-tolerant Alaskan beetle Upis ceramboides , demonstrates a high degree of thermal hysteresis, comparable to that of the most active insect antifreeze proteins. Although the presence of a lipid component in this factor has not yet been verified, it has been proposed that the glycan backbone consists of a β-D-mannopyranosyl-(1→4)-β-D-xylopyranose-disaccharide-repeating structure according to MS and NMR analyses. In this contribution, we report the stereoselective synthesis of the tetrasaccharide β-D-mannopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-mannopyranosyl-(1→4)-D-xylopyranoside, a structural component of xylomannan. Our synthesis features the use of 2-naphthylmethyl (NAP)-ether-mediated intramolecular aglycon delivery (IAD) as the key reaction in obtaining β-mannopyranoside stereoselectively. Various donors for NAP-IAD were tested to determine the most suitable for the purposes of this synthesis. Fragment coupling between a disaccharyl fluoride and a disaccharide acceptor obtained from a common β-D-mannopyranosyl-(1→4)-β-D-xylopyranoside derivative was successfully carried out to afford the desired tetrasaccharide in the presence of Cp(2)HfCl(2)-AgClO(4). Structural analysis of the resulting synthetic tetrasaccharide using NMR techniques and molecular modeling was performed in order to demonstrate the presence of the proposed xylomannan linkages in this molecule.