Volume 24, Number 6,
March 15, 2016
, Pages 1362-1368
Author links open overlay panel, , , , , , , , , , , ,
https://doi.org/10.1016/j.bmc.2016.02.008Get rights and content
For the first time, we describe the chemical synthesis of tetramannoside, which contains botha(1→2) e b (1→2) see. dodecyltium (lauryl)glycosideswere prepared from odorless dodecyl thiols and used as donors forglycosylationsteps. This tetramannoside, linked to the mantle group, proved to be a perfect substrate for the β-mannosyltransferase Bmt3, confirming the proposed specificity and allowing the preparation of the pentamannoside sequence (β Man (1,2) β Man (1,2) α Man (1,2) α Man (1,2) α Man) which can be used as a new substrate for further elongation studies.
Candida albicans is a human pathogenic fungus involved in severe opportunistic infections. On the surface of its cell wall, it expresses β (1,2)-oligomannosides (β-Mans), which are crucial for yeast virulence mechanisms.1These β-Mans replace the terminal α-Man residues of various glycoconjugates, including polymannosylated N-linked glycans and the family of Candida-specific glycolipids, phospholipomannan (PLM). The structure of these mannoconjugates has been extensively studied in recent years, but their origin is still unclear. A family of nine genes (BMT) encoding β-mannosyltransferases (Bmt) was identified in this yeast using a mutant phenotypic approach.2Several of these Bmts are predicted to be involved in the biosynthesis of β-Mans from N-glycans and PLM.2,3 Recently, the activity and specificity of Bmt1 have been characterized using a recombinant form of the enzyme. It was shown that Bmt1 is responsible for the initiation of β-Mans in the acid-stable part of polymannosylated N-glycans. 4, 5 Now, the activity of Bmt3, an enzyme that is probably involved in the elongation of β-Mans N-glycans, needs to be investigated (Figure 1) .
In order to characterize the roles of β-mannosyl transferases, particularly their substrate requirements and their kinetics, fluorescently labeled synthetic oligomannosides are desirable. We recently published our work on Bmt14, 5 using coat-labeled α(1,2) oligomannosides as valuable substrates. In order to exploit the activity of the second enzyme Bmt3, an α(1,2) tetramannoside with a β unit at the non-reducing end was chosen as a suitable mimic of its natural substrate (Figure 2). No synthetic work on this mixture of α,β-oligomannosides has been reported in the literature.
Assembled1can also originate from enzymatic mannosylation of trimannosides using our recombinant Bmt1, chemical synthesis is preferred here as it offers larger amounts of a fully defined compound.
paragraphs of sections
Synthesis of1is based on two carbohydrate building blocks (Figure 3): an α-mannosyl donor (2), equipped with a thiolauryl leaving group, and a similar β-glucopyranosyl donor (3). Azidopropanol (5) was chosen as the ligand, and methyl isatoic anhydride (4) as the mantle fluorophore precursor. The mantle group is a 'discrete' fluorophore, it is small, polar and uncharged at pH 7 and should not interfere with the enzymatic process. It is proven to be effective in Bmt1 work. (Excitation 334 nm, emission
Synthetic mantle-mannonetetraose DP4 1 ([M+Na]+at m/z 879.4) was used as an acceptor substrate to study the enzymatic properties of Bmt3p. The presumed function of this enzyme is to add a β-Man residue to α-1,2 oligomannosides capped with a non-reducing terminal β-1,2 Man unit. Recombinant soluble Bmt3p enzyme was produced in Pichia pastoris11and the enzymatic evaluation was carried out using GDP-Man as the mannosyl unit donor. The reaction products were monitored by normal phase HPLC
For the first time, we describe the chemical synthesis of tetramannoside, which contains both α (1,2) and β (1,2) linkages. This tetramannoside proved to be a perfect substrate for the enzyme Bmt3,11confirming the proposed specificity of the enzyme. The resulting pentamannoside will be useful as a new substrate for studies of elongation by other transferases such as Bmt4.
Enzymatic assay of mannosyl transferase
A culture supernatant of an engineered strain of Pichia pastoris containing about 35 mgL−1Bmt3p, 0.05% Tween 20 and 0.5% Triton X-100 were used as the enzyme source. Details of the genetic construction and culture conditions are described elsewhere.11Assay mixture containing 2 μL supernatant, 0.06 mM mantyl-tetramannoside (1), 20 mM GDP-Man donor, 50 mM sodium citrate buffer pH 6.5, 20 mM CaCl2, 0.3% Triton X-100, in a total volume of 25 μL, was incubated for 30 minutes at 28°C (standard assay). ON
This work was supported by the Agency Nationale de la Recherche (ANR MIE CaBMT), with a grant fromEuropean UnionALLFUN (FP7/2007 2013, HEALTH-2010-260338) (Fungi in the Environment of Inflammation, Allergy and Autoimmune Diseases: Translating Basic Science into Clinical Practice “ALLFUN” and fromMinistry of Higher Education and Science(za G.S.-L. i T.H.).
- C.mileand others
J. Biol. Chem.
- D.Crichand others
Jelly. chem. Society
- n.Shibataand others
Proc. Jpn. Agreement. Ser. B
- C.mileand others
- E.Fabreand others
There are more references available in the full text version of this article.
Synthesis of a biotechnologically obtained biotinylated β-D-mannopyranosyl-(1→2)-D-mannopyranose probe for the assessment of carbohydrate specificity of antibodies
2019, Carbohydrate Research
Excerpt from the quote:
Acidic hydrolysis of phosphomannan followed by enzymatic dephosphorylation efficiently yielded the disaccharide β-d-mannopyranosyl-(1 → 2)-d-mannopyranose 2 . The latter can be considered a precursor of the β-(1 → 2)-linked dimannoside block in the synthesis of oligosaccharide derivatives linked to mannan β-mannoside side chains of yeast  whose direct chemical synthesis is often a non-trivial task [3-5]. Here we describe the transformation of disaccharide 2 (Scheme 1) into the glycosyl donor 5 and subsequently into the biotinylated probe 9.
Disaharid β-d-manopiranosil-(1→2)-d-mannopyranose obtained by chemical cleavage and enzymatic dephosphorylation of biotechnologically available phosphomannan was transformed in six steps into a biotinylated probe suitable for assessing the carbohydrate specificity of antibodies induced by yeast cell wall preparations.
Synthesis of oligosaccharides related to polysaccharides of the cell wall of Candida and Aspergillus fungi
2017, Russian Chemical Reviews
Hot electron transport of PbSe quantum dots molecularly bonded to mesoporous films of tin and titanium oxide
Chemical Physics, Volume 471, 2016, pp. 54-58 (prikaz, ostalo).
The selective extraction of hot electrons to the appropriate energy contacts is a fundamental aspect of the development of heat-transferring solar cells. Here we use time-resolved THz spectroscopy (TRTS) to assess the extent to which hot electron transfer (HET) from 1Pe3nm PbSe colloidal quantum dot states molecularly linked by 3-mercaptopropionic acid to mesoporous SnO2i TiO2sensitized films. For PbSe–3MPA–SnO2samples, we have shown that the efficiency of hot electron transfer is negligibly small at room temperature, i.e. within the detection limit of approximately 10% of our measurements. The impact of spurious signals on TRTS data arising from carrier dynamics relative to QD aggregates - which can be misinterpreted as HETs - is discussed in detail. In contrast, according to previous reports, hot electron transfer was observed to occur from Pestates of colloidal PbSe QDs in PbSe-3MPA-TiO2system, with an efficiency of ⩾80%. These results are rationalized in terms of stronger donor-acceptor coupling between QDs and oxides for TiO2electrode compared to the SnO electrode2electrode, a factor that ultimately defines the kinetic competition between the rate of electron transfer to the oxide and the in-band cooling within the QD.
Resistance to heat transfer as a tool to quantify the efficiency of DNA hybridization on the surface of nanocrystalline diamond
Diamond and Related Materials, Volume 48, 2014, p. 32-36 (view, other).
In this paper, we report a label-free real-time method based on heat transfer resistance for thermally monitoring DNA denaturation and its potential for quantifying DNA fragments with a specific sequence of interest. The DNA probe, which consists of a 36-mer fragment, is covalently immobilized on the surface of a nanocrystalline diamond, created by chemical vapor deposition on a silicon substrate. Different concentrations of fully matched 29-mer target DNA fragments were hybridized with this DNA probe. We observed that the change in heat transfer resistance after denaturation depended on the target DNA concentration used during hybridization, which allowed us to determine a dose-response curve. Therefore, these results illustrate the potential of this technique to quantify the concentration of a specific DNA fragment and quantify the hybridization efficiency with its probe.
A short oligonucleotide prodrug with 5-fluoro and 5-iodouracil inhibits the proliferation of cancer cells in a photoreactive manner
Bioorganic & Medicinal Chemistry Letters, Volume 24, Number 16, 2014, p. 3736-3738
Photo-induced C1' hydrogen abstraction of 5-fluoro-2'-deoxyuridine is accepted as a key reaction for the release of the anticancer drug 5-fluorouracil (5-FU) from oligonucleotide chains. After photoirradiation after release of 5-FU, anticancer activity was expected. We show that the oligonucleotide tetramer, d(AFvasEUUA), can release 5-FU under physiological conditions in a photoreactive manner through photoinduced C1' hydrogen abstraction and that 5-FU released from d(AFvasEUUA) having a phosphorothioate skeleton clearly suppresses the proliferation of HeLa cells in a photoreactive manner.
Ca2+ dynamics correlate with phenotype and function in primary human neutrophils
Biophysical Chemistry, Volume 184, 2013, pp. 116-125 (prikaz, ostalo).
The central function of neutrophil immune defense is sensing, movement and killing. Neutrophils acquire different cellular states necessary to fulfill these functions, each associated with a specific phenotype. The cells that make up the neutrophil population are probably not synchronized with respect to their actual state, eg by expiration or preactivation. They are also likely to exhibit varying degrees of phenotypic plasticity (ie, the ability to switch to a particular state). Calcium is known to play a key role in neutrophils such as cell motility. This study focuses on characterizing cell-to-cell variability at the morphological level as well as at the level of calcium dynamics by studying individual primary human neutrophils. We applied long-term multivariate live cell imaging to (i) characterize the phenotypes of neutrophils of different functional states, (ii) analyze the distribution of cells in these states, and (iii) study the individual intracellular calcium response simultaneously with shape changes. We are able to distinguish five different subpopulations of neutrophils based on quantitative parameters of cell morphology and motility. As the main result, we showed that calcium dynamics in individual cells is correlated with their respective functional status. Finally, we see a series of cells undergoing spontaneous phenotypic changes from one cellular state to another. These events are preceded by the display of calcium dynamics in the future state or switching to the appropriate calcium dynamics parallel to the change in morphology. Based on our results, we conclude that calcium-specific dynamics carry key information for neutrophil function and phenotype.
Synthesis and evaluation of (S)-[18F]fesetron in rat brain as a potential agent for PET imaging of serotonin 5-HT3 receptors
Bioorganic & Medicinal Chemistry Letters, Volume 26, Number 8, 2016, p. 1919-1924
Serotonin 5-HT3receptors are involved in several brain functions, including targeting vomiting during cancer chemotherapy. Here we report the development of (S)-2,3-dimethoxy-5-(3'-[18F]fluoropropyl)-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide ([18F]fesetron) as a potential PET agent for recording serotonin 5-HT3receivers. By radioactive labeling of ((S)-2,3-dimethoxy-5-(3'-tosyloxypropyl)-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide) with fluoro-18, (S)- [18F]fesetron was obtained in decay-corrected yields of 5 to 10% and with specific activities >74GBq/μmol at the end of the radiosynthesis. PET images in rats showed low uptake [18F]fesetron in the brain with retention of binding in the striatal and cerebellar regions. Using the colliculus as a reference region, the ratios were 3.4 for the striatum and 2.5 for the cerebellum. Ex vivo PET analysis of the brain showed binding [18F]fezetron in hippocampus, striatum and cerebellum regions. Cerebellar regions corresponded to the area postrema and the nucleus of the solitary tract known to contain 5-HT3receivers. The dorsal hippocampus showed the highest uptake with a ratio >17 compared to the colliculus, while the area postrema and striatum had ratios >10. Therefore, [18F]fesetron showed a unique binding profile to regions of the rat brain known to contain significant amounts of serotonin 5-HT3receivers. However, very low brain uptake limits its utility as a PET radiopharmaceutical in this animal model.
Anticancer efficacy of p-dodecylaminophenol against refractory and high-risk neuroblastoma cells in vitro and in vivo
Bioorganic & Medicinal Chemistry Letters, Volume 27, Number 20, 2017, p. 4664-4672
Neuroblastoma is an aggressive, drug-resistant cancer. The high-risk human neuroblastoma cell line SK-N-AS (non-amplified N-myc) is derived from stromal cells and is resistant to retinoic acid treatment.1, RA), which is a chemotherapeutic agent used to induce the differentiation of neuroblastoma neuronal cells. We developed p-dodecylaminophenol (3, p-DDAP), based on N-(4-hydroxyphenyl)retinamide (2, 4-HPR), synthetic amide1, from1e2are associated with the side effect of nyctalopia. To assess the effects3in high-risk neuroblastomas we use SK-N-AS cells as well as another high-risk human neuroblastoma cell line, IMR-32, which is derived from neuronal cells (amplified N-myc, drug-sensitive). Connection3suppressed the growth of SK-N-AS cells and IMR-32 cells more effectively than1,2, p-decilaminofenol (4, p-DAP), N-(4-hydroxyphenyl)dodecananamide (5, 4-HPDD) or N-(4-hydroxyphenyl)decananamide (6, 4-HPD). In SK-N-AS cells,3G induced0/G1arrest and apoptosis to a greater extent than1e2. In IMR-32 cells,3induced apoptosis to a similar extent1e2, potentially inhibiting N-myc expression. Also, i.p. administration of3suppressed tumor growth in mice implanted with SK-N-AS in vivo. From3showed no effect on blood retinol concentrations, as opposed to a decrease after administration2, showed excellent anticancer efficacy against high-risk neuroblastoma SK-N-AS and IMR-32 expressing different levels of N-myc. Compound3may have potential for clinical use in the treatment of refractory neuroblastoma with reduced side effects.
Copyright © 2016 Elsevier Ltd. All rights reserved.