RESUMO
Reported herein is a new HPLC-based automated synthesizer (HPLC-A) capable of a temperature-controlled synthesis and purification of carbohydrates. The developed platform allows to perform various protecting group manipulations as well as the synthesis of O- and N-glycosides. A fully automated synthesis and purification was showcased in application to different carbohydrate derivatives including glycosides, oligosaccharides, glycopeptides, glycolipids, and nucleosides.
Assuntos
Carboidratos , Oligossacarídeos , Cromatografia Líquida de Alta Pressão , Carboidratos/química , Oligossacarídeos/síntese química , Oligossacarídeos/química , Glicosídeos/química , Glicosídeos/síntese química , Glicolipídeos/química , Glicolipídeos/síntese química , Glicolipídeos/isolamento & purificação , Glicopeptídeos/química , Glicopeptídeos/síntese química , Nucleosídeos/química , Nucleosídeos/síntese química , Automação , TemperaturaRESUMO
Superarmed glycosyl donors have higher reactivity compared to their perbenzylated armed counterparts. Generally, the 2-O- benzoyl-3,4,6-tri-O-benzyl protecting group pattern gives rise to increased reactivity due to an O-2/O-5 cooperative effect. Despite having a high reactivity profile and applicability in many expeditious strategies for glycan synthesis, regioselective introduction of the superarming protecting group pattern is tedious for most sugar series. Reported herein is a streamlined synthetic route to yield superarmed glycosyl donors of the d-gluco and d-galacto series equipped with an ethylthio, phenylthio, p-tolylthio, benzoxazol-2-ylthio, O-allyl, or O-pentenyl anomeric leaving group. This streamlined approach was made possible due to the refinement of the oxidative thioglycosylation reaction of the respective glucal and galactal precursors. The applicability of this approach to the direct formation of disaccharides is also showcased.
RESUMO
Reported herein is the development of a novel method for the activation of thioglycosides and thioimidates using benzyl trichloroacetimidate in the presence of catalytic triflic acid. Excellent yields have been achieved with reactive substrates, whereas efficiency of reactions with unreactive glycosyl donors and/or acceptors was modest.
Assuntos
Tioglicosídeos , CatáliseRESUMO
The reverse orthogonal strategy was invented in 2011 in an attempt to address drawbacks of other strategies for glycan assembly. Different from the classical orthogonal approach that relies on the orthogonality of leaving groups, the reverse strategy is based on orthogonal protecting groups that could be removed during the glycosylation step. This strategy remained largely unexplored due to only one combination of orthogonal protecting groups that would fit into this concept. Reported herein are new orthogonal combinations of leaving and protecting groups that help to streamline the glycan assembly. Also reported is further refinement of the previously reported reaction conditions.
Assuntos
Oligossacarídeos , Polissacarídeos , GlicosilaçãoRESUMO
Presented herein is an improved synthesis of a common 3-OH glycosyl acceptor. This compound is a building block that is routinely synthesized by many research groups to be used in glycosylation refinement studies. The only known direct synthesis by Koto lacks regioselectivity and relies on chromatography separation using hazardous solvents. Our improved synthetic approach relies on Koto's selective benzylation protocol, but it is followed by acylation-purification-deacylation sequence. Although this approach involves additional manipulations, it provides consistent results and is superior to other indirect strategies. Also obtained, albeit in minor quantities, is 4-OH acceptor, another common building block.
Assuntos
Oligossacarídeos , Sequência de Carboidratos , Glicosilação , Oligossacarídeos/químicaRESUMO
Hexasaccharide ß-D-Galp-(1ââ¯2)-[ß-D-Galp-(1â¯ââ¯3)]-ß-D-Galp-(1â¯ââ¯6)-[ß-D-Galp-(1â¯ââ¯2)-ß-D-Galf-(1â¯ââ¯4)]-D-GlcNAc (1) was found O-linked in mucins of Trypanosoma cruzi epimastigotes and metacyclic trypomatigotes. Studies on the biological pathways and functionalities of the mucin oligosaccharides are prompted in order to understand the interactions of these molecules with the insect host. Trisaccharide constituent ß-D-Galp-(1â¯ââ¯2)-ß-D-Galf-(1â¯ââ¯4)-D-GlcNAc was constructed from the reducing to the non-reducing end. We discuss the difficulties to introduce a Galp unit at the O-2 position of a partially protected galactofuranosyl unit which were overcome using an anchimerically superarmed donor. By this route and employing a [3 + 3] nitrilium convergent approach hexasaccharide 1 was synthesized in moderate yield.
Assuntos
Galactose/química , Mucinas/química , Oligossacarídeos/química , Oligossacarídeos/síntese química , Trypanosoma cruzi/química , Técnicas de Química Sintética , Glicosilação , EstereoisomerismoRESUMO
BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, is a protozoan parasite transmitted to humans by blood-sucking triatomine vectors. However, and despite its utmost biological and epidemiological relevance, T. cruzi development inside the digestive tract of the insect remains a poorly understood process. METHODS/PRINCIPLE FINDINGS: Here we showed that Gp35/50 kDa mucins, the major surface glycoproteins from T. cruzi insect-dwelling forms, are involved in parasite attachment to the internal cuticle of the triatomine rectal ampoule, a critical step leading to its differentiation into mammal-infective forms. Experimental evidence supporting this conclusion could be summarized as follows: i) native and recombinant Gp35/50 kDa mucins directly interacted with hindgut tissues from Triatoma infestans, as assessed by indirect immunofluorescence assays; ii) transgenic epimastigotes over-expressing Gp35/50 kDa mucins on their surface coat exhibited improved attachment rates (~2-3 fold) to such tissues as compared to appropriate transgenic controls and/or wild-type counterparts; and iii) certain chemically synthesized compounds derived from Gp35/50 kDa mucins were able to specifically interfere with epimastigote attachment to the inner lining of T. infestans rectal ampoules in ex vivo binding assays, most likely by competing with or directly blocking insect receptor(s). A solvent-exposed peptide (smugS peptide) from the Gp35/50 kDa mucins protein scaffolds and a branched, Galf-containing trisaccharide (Galfß1-4[Galpß1-6]GlcNAcα) from their O-linked glycans were identified as main adhesion determinants for these molecules. Interestingly, exogenous addition of a synthetic Galfß1-4[Galpß1-6]GlcNAcα derivative or of oligosaccharides containing this structure impaired the attachment of Dm28c but not of CL Brener epimastigotes to triatomine hindgut tissues; which correlates with the presence of Galf residues on the Gp35/50 kDa mucins' O-glycans on the former but not the latter parasite clone. CONCLUSION/SIGNIFICANCE: These results provide novel insights into the mechanisms underlying T. cruzi-triatomine interplay, and indicate that inter-strain variations in the O-glycosylation of Gp35/50 kDa mucins may lead to differences in parasite differentiation and hence, in parasite transmissibility to the mammalian host. Most importantly, our findings point to Gp35/50 kDa mucins and/or the Galf biosynthetic pathway, which is absent in mammals and insects, as appealing targets for the development of T. cruzi transmission-blocking strategies.
Assuntos
Mucinas/metabolismo , Proteínas de Protozoários/metabolismo , Triatoma/parasitologia , Trypanosoma cruzi/metabolismo , Animais , Doença de Chagas/parasitologia , Doença de Chagas/transmissão , Humanos , Mucinas/genética , Proteínas de Protozoários/genética , Reto/parasitologia , Trypanosoma cruzi/genéticaRESUMO
The hexasaccharide ß-D-Galp-(1â2)-[ß-D-Galp-(1â3)]-ß-D-Galp-(1â6)-[ß-D-Galp(1â2)-ß-D-Galf(1â4)]-D-GlcNAc (10) and its ß-D-Galf-(1â2)-ß-D-Galf containing isomer (7) are the largest carbohydrates in mucins of some strains of Trypanosoma cruzi. The terminal ß-D-Galp units are sites of sialylation by the parasite trans-sialidase. Hexasaccharide 10 was chemically synthesized for the first time by a [3+3] nitrilium based convergent approach, using the trichloroacetimidate method of glycosylation. The (1)H NMR spectrum of its alditol was identical to the spectrum of the product released by ß-elimination from the parasite mucin. The trans-sialylation reaction studied on the benzyl glycoside of 10 showed two monosialylated products whose relative abundance changed with time. On the other hand, only one product was produced by sialylation of the benzyl glycoside of 7. A preparative synthesis of the latter and spectroscopic analysis of the product unequivocally established the sialylation site at the less hindered (1â3)-linked galactopyranose.
Assuntos
Glicoproteínas/metabolismo , Mucinas/química , Mucinas/metabolismo , Neuraminidase/metabolismo , Oligossacarídeos/síntese química , Trypanosoma cruzi/química , Configuração de Carboidratos , Sequência de Carboidratos , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismoRESUMO
The hexasaccharide ß-D-Galp-(1â2)-[ß-D-Galp-(1â3)]-ß-D-Galp-(1â6)-[ß-D-Galf(1â2)-ß-D-Galf(1â4)]-D-GlcNAc (1) is the largest carbohydrate structure released as alditol by reductive ß-elimination from mucins of some strains of T. cruzi. The terminal ß-D-Galp units are sites of sialylation by trans-sialidase which transfers sialic acid from the host to the parasite. Hexasaccharide 1 was synthesized by a [3 + 3]-convergent strategy based on a nitrile assisted glycosylation, using the trichloroacetimidate method. The ß-D-Galf-(1â2)-ß-D-Galf-D-GlcNAc synthon was sequentially constructed from the reducing end to the non-reducing end employing benzyl α-D-galactofuranoside as starting material for the internal Galf unit. The choice of this novel precursor, obtained in one-reaction step from galactose, allowed the introduction of an orthogonal and participating levulinoyl group at O-2. Thus, the diastereoselective construction of the Galf-ß(1â4)-GlcNAc linkage by the trichloroacetimidate method of glycosylation was achieved. The (1)H NMR spectrum of alditol 2 was identical to the product released by ß-elimination from the parasite mucin.
Assuntos
Mucinas/química , Oligossacarídeos/síntese química , Proteínas de Protozoários/química , Trypanosoma cruzi/química , Sequência de Carboidratos , Galactose/síntese química , Galactose/química , Glicosilação , Dados de Sequência Molecular , Oligossacarídeos/química , Álcoois Açúcares/síntese química , Álcoois Açúcares/químicaRESUMO
The trisaccharides beta-D-Galf-(1-->2)-beta-D-Galf-(1-->4)-D-GlcNAc (5) and beta-D-Galp-(1-->2)-beta-D-Galf-(1-->4)-D-GlcNAc (6) constitute novel structures isolated as alditols when released by reductive beta-elimination from mucins of Trypanosoma cruzi (Tulahuen strain). Trisaccharides 5 and 6 were synthesized employing the aldonolactone approach. Thus, a convenient D-galactono-1,4-lactone derivative was used for the introduction of the internal galactofuranose and the trichloroacetimidate method was employed for glycosylation reactions. Due to the lack of anchimeric assistance on O-2 of the galactofuranosyl precursor, glycosylation studies were performed under different conditions. The nature of the solvent strongly determined the stereochemical course of the glycosylation reactions when the galactofuranosyl donor was substituted either by 2-O-Galp or 2-O-Galf.