Protein Glycosylation through Sulfur Fluoride Exchange (SuFEx) Chemistry: The Key Role of a Fluorosulfate Thiolactoside.

Protein glycosylation is the most complex post-translational modification process. More than 50 % of human cells proteins are glycosylated, whereas bacteria such as E. coli do not have this modification machinery. Indeed, the carbohydrate residues in natural proteins affect their folding, immunogenicity, and stability toward proteases, besides controlling biological properties and activities. It is therefore important to introduce such structural modification in bioengineered proteins lacking the presence of carbohydrate residues. This is not trivial as it requires reagents and conditions compatible with the protein's stability and reactivity. This work reports on the introduction of lactose moieties in two natural proteins, namely ubiquitin (Ub) and l-asparaginase II (ANSII). The synthetic route employed is based on the sulfur(VI) fluoride exchange (SuFEx) coupling of a lactose tethered arylfluorosulfate (Lact-Ar-OSO2 F) with the ϵ-NH2 group of lysine residues of the proteins. This metal-free click SuFEx reaction relies on the properties of the fluorosulfate employed, which is easily prepared in multigram scale from available precursors and reacts chemoselectively with the ϵ-NH2 group of lysine residues under mild conditions. Thus, iterative couplings of Lact-Ar-OSO2 F to Ub and ANSII, afforded multiple glycosylations of these proteins so that up to three and four Lact-Ar-OSO2 groups were introduced in Ub and ANSII, respectively, via the formation of a sulfamoyl (OSO2 -NH) linkage.

SuFEx: a metal-free click ligation for multivalent biomolecules.

The Sulfur(vi) Fluoride Exchange (SuFEx), revived by Sharpless and co-workers from an unrecognized state, is an emerging new click reaction that is based on the high reactivity of sulfonyl fluorides and fluorosulfates with suitable nucleophiles such as silyl ethers and amines. Hence, we comment in this Perspective on the use of SuFEx for the synthesis of a new family of sugar containing sulfonamides from the reaction of a glycosylsulfonyl fluoride with aliphatic amines. We also highlight the applications of SuFEx to multivalent scaffolds such as tetraamino- and tetrafluorosulfonyl-calixarene leading to sulfonamide-linked sugar and iminosugar clusters. Finally, we report on the chemoselective functionalization of bovine serum albumin (BSA) which, owing to the amino group of lysine moieties, reacted with SO2F2 to give a multivalent BSA-SO2F system. The PEGylation of the same protein by coupling with a PEG-fluorosulfate is described as well.

Validating the alkene and alkyne hydrophosphonylation as an entry to organophosphonates.

The first paper on the hydrophosphonylation of terminal alkenes was published in 1958 by Stiles and coworkers. Afterwards various papers described organometal-catalyzed and free-radical reactions leading to linear anti-Markovnikov adducts and/or branched Markovnikov products. In 1996 Han and Tanaka reported the first example of alkyne hydrophosphonylation catalyzed by a palladium complex. Further studies using other metal catalysts registered poor selectivity as mixtures of adducts were obtained in most of the cases examined. The first example of alkyne hydrophosphonylation by H-phosphonates under free-radical conditions leading to Z- and E-vinylphosphonates in a 1 : 1 ratio was reported by our group. Nevertheless, Z- to E-isomerization took place upon irradiation in the presence of a thiol.

Tetravalent glycocyclopeptide with nanomolar affinity to wheat germ agglutinin.

A series of tetravalent glycocyclopeptides functionalized with GlcNAc was synthesized using copper(i)-catalysed alkyne-azide cycloaddition, oxime ligation and thiol-ene coupling. The binding ability of these compounds towards wheat germ agglutinin was studied by a competitive ELLA test and ITC experiments. While all compounds were able to inhibit WGA binding to GlcNAc-polymer coated surfaces at low concentrations, derivative 17 having an aliphatic spacer and thioether linkage was 4.9 × 10(6) times more potent on a per sugar basis than GlcNAc. This remarkably strong effect was confirmed by ITC experiments as these revealed an association constant of 9 nM for this compound, therefore presenting a gain of 200,000 times over GlcNAc. These results for compound 17 represent the highest binding properties reported for WGA.

Multivalent presentation of a hydrolytically stable GM(3) lactone mimetic as modulator of melanoma cells motility and adhesion.

A hydrolytically stable mimetic of the tumour antigen GM(3) lactone is used to decorate multivalent scaffolds. Two of them positively interfere on melanoma cell adhesion, migration and resistance to apoptosis (anoikis). Notably, their ability to hamper melanoma-cells adhesion and reduce the metastatic potential is enhanced when the two scaffolds, presenting a different shape, are used in combination.

Recent applications of thiol-ene coupling as a click process for glycoconjugation.

There has been over the past decades a resurgence of the free-radical thiol-ene coupling (TEC) as a method for assembling crosslinked networks and polymer functionalization. On the other hand the use of TEC in carbohydrate chemistry, a field of special importance due to the key role of carbohydrates in living organisms, is represented only by a handful of papers. Nevertheless it appears that TEC possesses many if not all the attributes of a click process proceeding with the assistance of the greenest catalyst such as visible light. This tutorial review focuses on the application of TEC on different topics, all related to glycochemistry, including: (a) carbohydrate modification, (b) oligosaccharide and glycosyl amino acid synthesis, (c) assembly of glycoclusters on rigid molecular platforms (calixarene, cyclodextrin, silsesquioxane, dendrimer), (d) peptide and protein glycosylation. Also the very recent development in peptide glycosylation by the closely related thiol-yne chemistry is described.

Glycoside and peptide clustering around the octasilsesquioxane scaffold via photoinduced free-radical thiol-ene coupling. The observation of a striking glycoside cluster effect.

Two series of multivalent octasilsesquioxane glyco- and peptido-conjugates were synthesized using the photoinduced free-radical thiol-ene coupling (TEC). The first series was obtained by coupling C-glycosylpropyl thiols and cysteine containing peptides with the known octavinyl octasilsesquioxane while the second series was obtained by reacting glycosyl thiols with a new octasilsesquioxane derivative displaying eight PEGylated chains functionalized with terminal allyl groups. The evaluation of the binding properties of mannoside and glucoside clusters toward Concanavalin A by Enzyme-Linked Lectin Assay (ELLA) revealed a modest glycoside cluster effect. On the other hand, the PEGylated POSS-based glycocluster featuring eight N-acetyl-glucosamine residues showed high affinity toward Wheat Germ Agglutinin to give a measured IC(50) at 3 nM. The calculated relative potency per number of sugar unit (rp/n) was superior to a value of 10(6), thus revealing the occurrence of a striking glycoside cluster effect.

Synthesis of sugar-based silica gels by copper-catalysed azide-alkyne cycloaddition via a single-step azido-activated silica intermediate and the use of the gels in hydrophilic interaction chromatography.

Novel sugar-based silica gels were prepared by exploiting the copper-catalysed azide-alkyne cycloaddition (CuAAC) of two different sugar alkynes, namely, ethynyl C-galactoside 1 and propargyl O-lactoside 2, with new single-step azido-activated silica gels. The fully characterised stationary phases were generally used for hydrophilic interaction chromatography (HILIC), with particular application in the stereoselective separation of monosaccharides. Dynamic HILIC (DHILIC) experiments were performed to evaluate the influence of mutarotation on the chromatographic peak shapes of two interconverting sugar anomers. The potential of such materials was shown in the separation of other highly polar compounds, including amino acids and flavonoids.

Photoinduced addition of glycosyl thiols to alkynyl peptides: use of free-radical thiol-yne coupling for post-translational double-glycosylation of peptides.

Double glycosylation of cysteine-containing peptides has been carried out by a one-pot two-step sequence comprising selective S-propargylation followed by photoinduced (lambda (max) 365 nm) free-radical hydrothiolation with glycosyl thiols. Conditions were established for the sequential introduction of two different thiol residues such as a glycosyl and a biotinyl derivative.

Modular approach to triazole-linked 1,6-α-D-oligomannosides to the discovery of inhibitors of Mycobacterium tuberculosis cell wall synthetase.

Aiming at developing inhibitors of mannosyltransferases, the enzymes that participate in the biosynthesis of the cell envelope of Mycobacterium tuberculosis, the synthesis of a range of designed triazole-linked 1,6-oligomannosides up to a hexadecamer has been accomplished by a modular approach centered on the Cu(I)-catalyzed azide-alkyne cycloaddition as key process. The efficiency and fidelity of the cycloaddition are substantiated by high yields (76-96%) and exclusive formation of the expected 1,4-disubstituted triazole ring in all oligomer assembling reactions. Key features of oligomers thus prepared are the anomeric carbon-carbon bond of all mannoside residues and the 6-deoxymannoside capping residue. Suitable bioassays with dimer, tetramer, hexamer, octamer, decamer, and hexadecamer showed variable inhibitor activity against mycobacterial α-(1,6)-mannosyltransferases, the highest activity (IC(50) = 0.14-0.22 mM) being registered with the hexamannoside and octamannoside.

Heterocycles in organic synthesis: thiazoles and triazoles as exemplar cases of synthetic auxiliaries.

This Perspective article illustrates the key role of thiazole and triazole in the work carried out in the author's laboratory over three decades and deals with the synthesis of carbohydrate-based bioactive molecules. The first part reports on the development of synthetic strategies exploiting the use of various thiazole-based reagents and the ready conversion of thiazole into the formyl group. After describing the chain elongation of monosaccharides into higher-carbon homologues, the synthesis of target natural and non-natural carbohydrates, or their ultimate precursors, is presented. These include some sphingoids, neuraminic and destomic acids, lincosamine, various 3-deoxy-2-ulosonic acids (KDO, KDN, iso-Neu4Ac), iminosugars (nojirimycin, mannojirimycin, galactostatin) and homoazasugars. Also prepared were the disaccharide subunit of bleomycin A(2) and the side-chain of taxol and taxotere.((R)) The use of 1,2,3-triazole is discussed in the second part of the paper. The service of this heterocycle that is easily formed by the Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) is considered in light of its use as a robust linker (a sort of keystone) of complex and diverse molecular architectures. Thus, the assembly of triazole-linked glycosyl amino acids, non-natural nucleotides, 1,6-oligomannosides, sialoside clusters on calixarene platfom via CuAAC is described and the biological relevance of these compounds is discussed in brief.

Fucosylated ubiquitin and orthogonally glycosylated mutant A28C: conceptually new ligands for Burkholderia ambifaria lectin (BambL).

Two orthogonal, metal free click reactions, enabled to glycosylate ubiquitin and its mutant A28C forming two protein scaffolds with high affinity for BambL, a lectin from the human pathogen Burkholderia ambifaria. A new fucoside analogue, with high affinity with BambL, firstly synthetized and co-crystallized with the protein target, provided the insights for sugar determinants grafting onto ubiquitin. Three ubiquitin-based glycosides were thus assembled. Fuc-Ub, presented several copies of the fucoside analogue, with proper geometry for multivalent effect; Rha-A28C, displayed one thio-rhamnose, known for its ability to tuning the immunological response; finally, Fuc-Rha-A28C, included both multiple fucoside analogs and the rhamnose residue. Fuc-Ub and Fuc-Rha-A28C ligands proved high affinity for BambL and unprecedented immune modulatory properties towards macrophages activation.

Design of triazole-tethered glycoclusters exhibiting three different spatial arrangements and comparative study of their affinities towards PA-IL and RCA 120 by using a dna-based glycoarray.

Sugar-coated chips: Glycoside clusters are valuable tools for carbohydrate-lectin recognition studies. However, the spatial arrangement of the sugar residues is a key issue in the design of high-affinity glycoclusters. Here the affinities of linear and antenna- and calixarene-based galactoside clusters towards two lectins derived from Pseudomonas aeruginosa and Ricinus communis were compared by means of glycoarrays.Interactions between proteins and carbohydrates are involved in a large number of crucial biological events. Many efforts have been devoted to the design and synthesis of unnatural saccharides displaying high affinities towards targeted lectins. Among others, glycoside clusters have proven to be valuable tools for these recognition studies. However, the spatial arrangements of the sugar residues are a key issue in the design of high-affinity glycoclusters. Here, the affinities of linear and antenna- and calixarene-based galactoside clusters against two lectins, derived from Pseudomonas aeruginosa and Ricinus communis, have been compared by means of glycoarrays.

Photoinduced thiol-ene coupling as a click ligation tool for thiodisaccharide synthesis.

The high efficiency and selectivity of the thiol-ene radical reaction has been validated by the photoinduced coupling of anomeric sugar thiols with sugar alkenes to give 1,6-linked S-disaccharides in good to excellent yields (76-92%) and high diastereoselectivities (up to 99%). The reaction appears to be well-qualified as an exemplar click process.

Synthesis of thiourea-tethered C-glycosyl amino acids via isothiocyanate-amine coupling.

A new class of C-glycosyl amino acids displaying a thiourea segment as a linker has been designed and synthesized by addition of peracetylated glycosylmethyl isothiocyanates to an amine-functionalized amino acid (N(alpha)-Fmoc-beta-amino-l-alanine). Three pairs of compounds with alpha- and beta-galacto, alpha- and beta-gluco, and alpha- and beta-manno configuration have been prepared with yields ranging between 70 and 75%. The orthogonal set of protective groups (O-acetyl in the carbohydrate moiety and N-Fmoc in the amino acid residue) makes these compounds suitable substrates for the co-translational modification of natural peptides. The couplings of model hydroxy-free and perbenzylated glycosylmethyl isothiocyanates with the above N(alpha)-Fmoc-beta-amino-l-alanine and the N(alpha)-Boc-protected analogue have been carried out as well, thus broadening the scope of the coupling reaction. Nevertheless, there are limitations of this isothiocyanate-amine coupling in complex systems, and these are briefly discussed.

Single and dual glycoside clustering around calix[4]arene scaffolds via click thiol-ene coupling and azide-alkyne cycloaddition.

We present the first synthesis of calix[4]arene-based S-glycoclusters via photoinduced multiple thiol-ene coupling of tetra- and octa-allyl calix[4]arenes with peracetylated glucosyl thiol (67-88% yields). Moreover we describe the dual clustering at the upper and lower rim of a calix[4]arene with two different sugars (galactose and glucose) via sequential copper(i)-catalyzed azide-alkyne cycloaddition and photoinduced thiol-ene coupling.

Production of carbohydrate building blocks from red seaweed polysaccharides. Efficient conversion of galactans into C-glycosyl aldehydes.

Agarans and carrageenans are abundant natural polysaccharides which are obtained on a large scale by water extraction from a variety of red seaweeds. These galactans, in addition to being valuable products for the pharmaceutical and food industries, are low cost starting materials for the preparation of useful and rare carbohydrate-based building blocks whose access by total synthesis is difficult and expensive. The semisynthesis of two sets of C-glycosyl aldehydes with l- and d-configuration from agarose and kappa-carrageenan respectively is described. Succinctly, the partial acid-catalyzed mercaptolysis of the two galactans under mild conditions afforded agarobiose and carrabiose (beta-d-Galp-(1-->4)-3,6-anhydro-aldehydo-l- and d-galactose, respectively) derivatives. Complete depolymerization of agarose and kappa-carrageenan under harsher conditions produced 3,6-anhydro l- and d-galactose aldehyde derivatives. Chain shortening of these products via alditol formation and oxidative carbon-carbon bond cleavage furnished C-formyl alpha-l- and alpha-d-threofuranosides. The above C-glycosyl aldehydes were all prepared on a meaningful preparative scale starting from gram quantities of galactans. Finally, a new procedure for the preparation of the 2,3-O-benzyl l-threofuranose was established by Baeyer-Villiger oxidation of the benzylated C-formyl alpha-l-threofuranoside here prepared from agarose.

Dihydropyridine C-glycoconjugates by organocatalytic Hantzsch cyclocondensation. Stereoselective synthesis of alpha-threofuranose C-nucleoside enantiomers.

The Hantzsch reaction of C-glycosyl aldehyde/enamino ester/beta-ketoester systems under l-proline catalysis to give dihydropyridine C-glycoconjugates is reported. Asymmetric cyclocondensations of differentially substituted enamine and beta-dicarbonyl components with formyl alpha-L-C-threofuranoside and with the alpha-D-isomer were also carried out. Each reaction occurred with high yet opposite stereoselectivity (de >95%) so that the pair of alpha-threofuranose C-nucleoside enantiomers was prepared.

Click azide-nitrile cycloaddition as a new ligation tool for the synthesis of tetrazole-tethered C-glycosyl alpha-amino acids.

Glycoproteins play a key role in a multitude of biological events in living organisms. Hence, neoglycopeptides obtained from unnatural C-glycosyl alpha-amino acids can be used as synthetic probes in studies aiming at clarifying the role of the carbohydrate domain in glycoprotein biological activity. A new class of C-glycosyl alpha-amino acids featuring a nitrogenated heterocycle ring holding the carbohydrate and glycinyl moiety was designed in our laboratory. Having previously prepared isoxazole-, 1,2,3-triazole-, and pyridine-tethered compounds, the family has now been enlarged by a group of newcomers represented by tetrazole derivatives. Two sets of compounds have been prepared, one being constituted of C-galactosyl and C-ribosyl O-tetrazolyl serines while the other contains S-tetrazolyl cysteine derivatives. In both cases, the synthetic scheme involved a two-step route, the first one being the thermal cycloaddition of a sugar azide with p-toluensulfonyl cyanide (TsCN) to give a 1-substituted 5-sulfonyl tetrazole and the second the replacement of the tosyl group with a serine or cysteine residue. For the high efficiency and operational simplicity, the azide-TsCN cycloaddition appears to be a true click process. Finally, one of the amino acids prepared was incorporated into a tripeptide.

Microwave-enhanced ionothermal CuAAC for the synthesis of glycoclusters on a calix[4]arene platform.

A tetra-azido calix[4]arene derivative was allowed to react with ethynyl tetra- O-benzyl- C-galactoside in the presence of CuI and i-Pr 2EtN in three different ionic liquids, that is, [C 8dabco][N(CN) 2], [C 8dabco][Br], and Ammoeng 110. Reactions were performed at 80 degrees C by thermal and MW dielectric heating. In all cases, multiple cycloadditions took place to give a triazole-linked tetra- C-galactosyl-calix[4]arene in up to 90% yield. The [C 8dabco][N(CN) 2] ionic liquid was also used to perform the multiclick reactions with propargyl O-lactoside and S-sialoside.