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N-linked glycosylation is a ubiquitous protein post-translational modification in which aberrant glycan biosynthesis has been linked to severe conditions like cancer. Accurate qualitative and quantitative analysis of N-glycans are crucial for investigating their physiological functions. Owing to the intrinsic absence of chromophores and high polarity of the glycans, current detection methods are restricted to liquid chromatography and mass spectrometry. Herein, we describe three new imidazolium-based glycan tags: 2'GITag, 3'GITag, and 4'GITag, that significantly improve both the limit of detection and limit of quantification of derivatized oligosaccharides, in terms of fluorescence intensity and ionisation efficiency. Our top-performing derivatisation agent, 4'GITag, shifted the detection sensitivity range from high femtomole to sub-femtomole levels in ESI-MS compared to traditional glycan label, 2AB, enabling the identification of 24 N-glycans in mouse serum, including those bearing sialic acids. Additionally, 4'GITag stabilized Na-salt forms of sialic acids, simplifying the simultaneous analysis of neutral and negative charged N-glycans significantly, avoiding the need for complex derivatisation procedures typically required for the detection of sialylated species. Overall, the favorable performance of imidazolium tags in the derivatisation and sensitive profiling of glycans has the potential for labeling tissue or live cells to explore disease biomarkers and for developing new targeted therapeutic strategies.
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Imidazóis , Polissacarídeos , Espectrometria de Massas por Ionização por Electrospray , Animais , Polissacarídeos/química , Polissacarídeos/sangue , Camundongos , Imidazóis/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Corantes Fluorescentes/química , Limite de Detecção , GlicosilaçãoRESUMO
Chagas disease is caused by the parasite Trypanosoma cruzi and affects over 7 million people worldwide. The two actual treatments, Benznidazole (Bzn) and Nifurtimox, cause serious side effects due to their high toxicity leading to treatment abandonment by the patients. In this work, we propose DNA G-quadruplexes (G4) as potential therapeutic targets for this infectious disease. We have found 174 PQS per 100,000 nucleotides in the genome of T. cruzi and confirmed G4 formation of three frequent motifs. We synthesized a family of 14 quadruplex ligands based in the dithienylethene (DTE) scaffold and demonstrated their binding to these identified G4 sequences. Several DTE derivatives exhibited micromolar activity against epimastigotes of four different strains of T. cruzi, in the same concentration range as Bzn. Compounds L3 and L4 presented remarkable activity against trypomastigotes, the active form in blood, of T. cruzi SOL strain (IC50 = 1.5-3.3 µM, SI = 25-40.9), being around 40 times more active than Bzn and displaying much better selectivity indexes.
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Doença de Chagas , Quadruplex G , Tripanossomicidas , Trypanosoma cruzi , Trypanosoma cruzi/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Ligantes , Doença de Chagas/tratamento farmacológico , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Tripanossomicidas/síntese química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Relação Dose-Resposta a Droga , Testes de Sensibilidade Parasitária , Antiparasitários/farmacologia , Antiparasitários/química , Antiparasitários/síntese químicaAssuntos
Neoplasias da Mama , Ossificação Heterotópica , Humanos , Feminino , Neoplasias da Mama/patologia , Neoplasias da Mama/diagnóstico por imagem , Ossificação Heterotópica/diagnóstico por imagem , Dura-Máter/diagnóstico por imagem , Dura-Máter/patologia , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/radioterapiaRESUMO
This mini review article provides an overview on the use of hypervalent iodine compounds (HICs) in carbohydrate synthesis, focusing on their chemistry and recent applications. HICs are similar to transition metals in their reactivity but have the added benefit of being environmentally benign, and are therefore commonly used as selective oxidants and eco-friendly reagents in organic synthesis. Herein, we summarize various synthetic uses of hypervalent iodine reagents in reactions such as glycosylation, oxidations, functionalization, and C-C bond-forming reactions. The goal of this review is to illustrate the advantages and versatility of using HICs as an environmentally sustainable alternative to heavy metals in carbohydrate chemistry.
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ß-Mannosides are ubiquitous in nature, with diverse roles in many biological processes. Notably, Manß1,4GlcNAc a constituent of the core N-glycan in eukaryotes was recently identified as an immune activator, highlighting its potential for use in immunotherapy. Despite their biological significance, the synthesis of ß-mannosidic linkages remains one of the major challenges in glycoscience. Here we present a chemoenzymatic strategy that affords a series of novel unnatural Manß1,4GlcNAc analogues using the ß-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase, BT1033. We show that the presence of fluorine in the GlcNAc acceptor facilitates the formation of longer ß-mannan-like glycans. We also pioneer a "reverse thiophosphorylase" enzymatic activity, favouring the synthesis of longer glycans by catalysing the formation of a phosphorolysis-stable thioglycoside linkage, an approach that may be generally applicable to other phosphorylases.
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Dual-emission fluorescence probes that provide high sensitivity are key for biomedical diagnostic applications. Nontoxic carbon dots (CDs) are an emerging alternative to traditional fluorescent probes; however, robust and reproducible synthetic strategies are still needed to access materials with controlled emission profiles and improved fluorescence quantum yields (FQYs). Herein, we report a practical and general synthetic strategy to access dual-emission CDs with FQYs as high as 0.67 and green/blue, yellow/blue, or red/blue excitation-dependent emission profiles using common starting materials such as citric acid, cysteine, and co-dopants to bias the synthetic pathway. Structural and physicochemical analysis using nuclear magnetic resonance, absorbance and fluorescence spectroscopy, Fourier-transform infrared spectroscopy, and X-ray photoelectron spectroscopy in addition to transmission electron and atomic force microscopy (TEM and AFM) is used to elucidate the material's composition which is responsible for the unique observed photoluminescence properties. Moreover, the utility of the probes is demonstrated in the clinical setting by the synthesis of green/blue emitting antibody-CD conjugates which are used for the immunohistochemical staining of human brain tissues of glioblastoma patients, showing detection under two different emission channels.
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Pontos Quânticos , Humanos , Pontos Quânticos/química , Carbono/química , Espectroscopia Fotoeletrônica , Corantes Fluorescentes/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Babesia microti is a tick-transmitted protozoan parasite of wildlife that can also cause serious disease in humans. It is now well established that B. microti represents an assemblage of different strains or species, only some of which are important zoonotic pathogens. Therefore, in order to assess the potential public health risk associated with B. microti in any given location, it is important to determine the strains that are present. This is the first study on the presence and identity of B. microti in Ireland. Overall, 314 wood mice (Apodemus sylvaticus), 243 bank voles (Myodes glareolus) and 634 questing Ixodes ricinus nymphs collected in various locations across Ireland were screened for the presence of B. microti by metabarcoding and nested PCR, respectively. Overall 8 rodent spleen samples (1.4%) were positive for B. microti, while all tick samples tested negative. Rodent isolates were identified as the 'Munich' strain which rarely causes human disease and is chiefly transmitted by the mouse tick, Ixodes trianguliceps. Together with reports from the UK these results suggest that B. microti does not represent a significant public health risk in Britain or Ireland.
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Babesia microti , Ixodes , Animais , Humanos , Camundongos , Babesia microti/genética , Irlanda/epidemiologia , Ixodes/parasitologia , Animais Selvagens , Murinae , ArvicolinaeRESUMO
The ability to append targeting biomolecules to chelators that efficiently coordinate to the diagnostic imaging radionuclide, 99mTc, and the therapeutic radionuclide, 188Re, can potentially enable receptor-targeted "theranostic" treatment of disease. Here we show that Pt(0)-catalyzed hydrophosphination reactions are well-suited to the derivatization of diphosphines with biomolecular moieties enabling the efficient synthesis of ligands of the type Ph2PCH2CH2P(CH2CH2-Glc)2 (L, where Glc = a glucose moiety) using the readily accessible Ph2PCH2CH2PH2 and acryl derivatives. It is shown that hydrophosphination of an acrylate derivative of a deprotected glucose can be carried out in aqueous media. Furthermore, the resulting glucose-chelator conjugates can be radiolabeled with either 99mTc(V) or 188Re(V) in high radiochemical yields (>95%), to furnish separable mixtures of cis- and trans-[M(O)2L2]+ (M = Tc, Re). Single photon emission computed tomography (SPECT) imaging and ex vivo biodistribution in healthy mice show that each isomer possesses favorable pharmacokinetic properties, with rapid clearance from blood circulation via a renal pathway. Both cis-[99mTc(O)2L2]+ and trans-[99mTc(O)2L2]+ exhibit high stability in serum. This new class of functionalized diphosphine chelators has the potential to provide access to receptor-targeted dual diagnostic/therapeutic pairs of radiopharmaceutical agents, for molecular 99mTc SPECT imaging and 188Re systemic radiotherapy.
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Rênio , Tecnécio , Camundongos , Animais , Tecnécio/química , Quelantes/química , Distribuição Tecidual , Radioisótopos/química , Rênio/química , Compostos Radiofarmacêuticos/química , Glucose , Catálise , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
PURPOSE: Molecular analysis of peritoneal fluid in staging laparoscopy of gastric cancer is performed to improve the detection of free intraperitoneal tumor cells. Nevertheless, its significance is controversial, especially in patients with negative cytology but positive molecular analysis. The aim of this study was to analyze the sensitivity of molecular analysis and its prognostic value. METHODS: A retrospective analysis from April 2011 to October 2019 was performed. Cytology (Cyt) and molecular analysis were analyzed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) of the carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) tumor makers. RESULTS: During the study period, 138 staging laparoscopies were performed. Macroscopic carcinomatosis was found in 12.3%. Of the remaining 87.7%, 9.9% were Cyt + and 11.6% were Cyt- RT-PCR + . Of the latter, 9 responded to chemotherapy and underwent radical surgery. The sensitivity of cytology and molecular analysis was 0.70 and 0.76, respectively (p = 0.67). The 2-year overall survival (OS) of Cyt- RT-PCR + vs. Cyt + was similar (p = 0.1). The 2-year OS of Cyt-RT-PCR + subgroup who underwent radical surgery vs. Cyt-RT-PCR- patients was similar (p = 0.69), but disease-free survival was shorter in the first group (p = 0.005). CONCLUSION: Our results show that the sensitivity of molecular analysis is similar to that of cytology. The prognostic value of positive molecular analysis was similar to positive cytology in terms of 2-year overall survival, except in the subgroup of operated patients in whom the overall survival was similar to that of those with a negative molecular analysis, albeit with a shorter disease-free survival.
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Laparoscopia , Neoplasias Gástricas , Humanos , Líquido Ascítico/química , Líquido Ascítico/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/cirurgia , Estudos Retrospectivos , Terapia Neoadjuvante , Antígeno Carcinoembrionário , Prognóstico , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cell surface carbohydrates mediate a wide range of carbohydrate-protein interactions key to healthy and disease mechanisms. Many of such interactions are multivalent in nature and in order to study these processes at a molecular level, many glycan-presenting platforms have been developed over the years. Among those, carbon nanoforms such as graphene and their derivatives, carbon nanotubes, carbon dots and fullerenes, have become very attractive as biocompatible platforms that can mimic the multivalent presentation of biologically relevant glycosides. The most recent examples of carbon-based nanoplatforms and their applications developed over the last few years to study carbohydrate-mediate interactions in the context of cancer, bacterial and viral infections, among others, are highlighted in this review.
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Nanotubos de Carbono , Polissacarídeos , Carboidratos , GlicosídeosRESUMO
The functionalization of glycosides with ionic compounds such as ionic liquids provides enhanced polarity for the labelled glycans thanks to the presence of a permanent positive charge. The chemical derivatisation of glycans with ionic liquids constitutes an emerging strategy to boost the detection sensitivity in MS applications. This allows the straightforward monitoring and detection of the presence of labelled glycans in complex matrices and in those cases where very limited amounts of material were available such as in biological samples and chemoenzymatic reactions. The use of ionic liquid based derivatisation agents can be further exploited for the labelling of live cells via metabolic oligosaccharide engineering for the detection of cancer biomarkers and for the tuning of live cells-surface properties with implications in cancer prognosis and progression. In this mini-review we summarise the latest development of the ionic liquid based derivatisation agents in glycoscience focussing on their use for sensitive MS applications.
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Líquidos Iônicos , Líquidos Iônicos/química , ÍonsRESUMO
A new practical, catalytic, and highly stereoselective method for directly accessing 1,1-α,α'-linked 2-deoxy trehalose analogues via AuCl3-catalyzed dehydrative glycosylation using hemiacetal glycosyl donors and acceptors is described. The method relies on the chemoselective Brønsted acid-type activation of tribenzylated 2-deoxy hemiacetals in the presence of other less reactive hemiacetals.
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Trealose , Catálise , Glicosilação , EstereoisomerismoRESUMO
Naphthalene diimide (NDI) is a central scaffold that has been commonly used in the design of G-quadruplex (G4) ligands. Previous work revealed notable anticancer activity of a disubstituted N-methylpiperazine propyl NDI G4 ligand. Here, we explored structure-activity relationship studies around ligand bis-N,N-2,7-(3-(4-methylpiperazin-1-yl)propyl)-1,4,5,8-naphthalenetetracarboxylic diimide, maintaining the central NDI core whilst modifying the spacer and the nature of the cationic groups. We prepared new disubstituted NDI derivatives of the original compound and examined their in vitro antiproliferative and antiparasitic activity. Several N-methylpiperazine propyl NDIs showed sub-micromolar activity against Trypanosoma brucei and Leishmania major parasites with up to 30 fold selectivity versus MRC-5 cells. The best compound was a dimorpholino NDI with an IC50 of 0.17 µM against T.brucei and 40 fold selectivity versus MRC-5 cells. However, no clear correlation between G4 binding of the new NDI derivatives and antiproliferative or antiparasitic activity was observed, indicating that other mechanisms of action may be responsible for the observed biological activity.
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Antiparasitários , Quadruplex G , Antiparasitários/química , Antiparasitários/farmacologia , Imidas/química , Imidas/farmacologia , Ligantes , Naftalenos , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: Percutaneous endoscopic gastrostomy (PEG) is a useful intervention for patients with impaired swallowing and a functional gastrointestinal system. Neurological diseases that cause neuromotor dysphagia, brain tumors, and cerebrovascular disease are the most frequent indications; complications are rare, and morbidity and mortality rates are low. OBJECTIVE: To describe the usefulness of PEG in patients with neurological diseases, and its impact on care, survival, and costs and benefits. MATERIAL AND METHODS: We performed a retrospective observational study, reviewing clinical files of patients hospitalised at the National Institute of Neurology and Neurosurgery (years 2015-2017) who underwent PEG placement. RESULTS: The sample included 51 patients: 62.7% were women and the mean (SD) age was 54.4 (18.6) years (range, 18-86). Diagnosis was tumor in 37.3% of cases and cerebrovascular disease in 33.3%. Sixteen patients (33.3%) died and 11 presented minor complications. The PEG tube remained in place for a mean of 9.14 months; in 52.9% of patients it was removed due to lack of improvement and/or tolerated oral intake, with removal occurring after a mean of 5.1 (4.4) months. Among patients' family members, 78.4% reported a great benefit, 43.1% reported difficulty caring for the PEG, and 45.1% reported complicated care in general. The monthly cost of maintaining the PEG was 175.78 on average (range, 38.38-293.45). DISCUSSION AND CONCLUSIONS: This preliminary study reveals that PEG was well indicated in patients with neurological diseases, with survival rates similar to those reported in other studies with long follow-up periods. In patients with cerebrovascular disease, the PEG tube remained in place a mean of 9.14 months, during recovery of swallowing function; however, the cost is high for our population.
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Neoplasias Encefálicas , Transtornos Cerebrovasculares , Transtornos de Deglutição , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Transtornos de Deglutição/etiologia , Nutrição Enteral/efeitos adversos , Feminino , Gastrostomia/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Direct measurements to determine the degree of surface coverage of nanoparticles by functional moieties are rare, with current strategies requiring a high level of expertise and expensive equipment. Here, a practical method to determine the ratio of the volume of the functionalisation layer to the particle volume based on measuring the refractive index of nanoparticles in suspension is proposed. As a proof of concept, this technique is applied to poly(methyl methacrylate) (PMMA) nanoparticles and semicrystalline carbon dots functionalised with different surface moieties, yielding refractive indices that are commensurate to those from previous literature and Mie theory. In doing so, it is demonstrated that this technique is able to optically detect differences in surface functionalisation or composition of nanometre-sized particles. This non-destructive and rapid method is well-suited for in situ industrial particle characterisation and biological applications.
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The development of efficient and sensitive tools for the detection of brain cancer in patients is of the utmost importance particularly because many of these tumours go undiagnosed until the disease has advanced and when treatment is less effective. Current strategies employ antibodies (Abs) to detect Glial Fibrillary Acid Protein (GFAP) in tissue samples, since GFAP is unique to the brain and not present in normal peripheral blood, and it relies on fluorescent reporters. Herein we describe a low cost, practical and general method for the labelling of proteins and antibodies with fluorescent carbon dots (CD) to generate diagnostic probes that are robust, photostable and applicable to the clinical setting. The two-step protocol relies on the conjugation of a dibenzocyclooctyne (DBCO)-functionalised CD with azide functionalised proteins by combining amide conjugation and strain promoted alkyne-azide cycloaddition (SPAAC) ligation chemistry. The new class of Ab-CD conjugates developed using this strategy was successfully used for the immunohistochemical staining of human brain tissues of patients with glioblastoma (GBM) validating the approach. Overall, these novel fluorescent probes offer a promising and versatile strategy in terms of costs, photostability and applicability which can be extended to other Abs and protein systems.
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The development of robust and reproducible synthetic strategies for the production of carbon dots (CDs) with improved fluorescence quantum yields and distinct emission profiles is of great relevance given the vast range of applications of CDs. The fundamental understanding at a molecular level of their formation mechanism, chemical structure and how these parameters are correlated to their photoluminescence (PL) properties is thus essential. In this study, we describe the synthesis and structural characterization of a range of CDs with distinct physico-chemical properties. The materials were prepared under three minutes of microwave irradiation using the same common starting materials (D-glucosamine hydrochloride 1 and ethylenediamine 2) but modifying the stoichiometry of the reagents. We show that small variation in reaction conditions leads to changes in the fluorescent behaviour of the CDs, especially in the selective enhancement of overlapped fluorescence bands. Structural analysis of the different CD samples suggested different reaction pathways during the CD formation and surface passivation, with the latter step being key to the observed differences. Moreover, we demonstrate that these materials have distinct reversible response to pH changes, which we can be attribute to different behaviour towards protonation/deprotonation events of distinct emission domains present within each nanomaterial. Our results highlight the importance of understanding the reaction pathways that lead to the formation of this carbon-based nanomaterials and how this can be exploited to develop tailored materials towards specific applications.
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The ability to modulate the cell surface structure provides a powerful tool to understand fundamental processes and also to elicit desired cellular responses. Here we report the development of a new class of 'clickable labels' to reengineer the cell surface charges of live cells. The method relies on the use of metabolic oligosaccharide engineering (MOE) combined with chemo selective labeling of cell surface azido-containing sialic acids with dibenzocyclooctyne (DBCO) ionic-probes. Using this strategy, we demonstrate that reducing the negative charge induced by the overexpression of cell surface sialic acids in cancer cells leads to a reduction in cell migration without affecting drug supceptibility.