De Novo Access to BODIPY C-Glycosides as Linker-Free Nonsymmetrical BODIPY-Carbohydrate Conjugates.

Recent years have witnessed an increasing interest in the synthesis and study of BODIPY-glycoconjugates. Most of the described synthetic methods toward these derivatives involve postfunctional modifications of the BODIPY core followed by the covalent attachment of the fluorophore and the carbohydrate through a "connector". Conversely, few de novo synthetic approaches to linker-free carbohydrate-BODIPY hybrids have been described. We have developed a reliable modular, de novo, synthetic strategy to linker-free BODIPY-sugar derivatives using the condensation of pyrrole C-glycosides with a pyrrole-carbaldehyde derivative mediated by POCl3. This methodology allows labeling of carbohydrate biomolecules with fluorescent-enough BODIPYs within the biological window, stable in aqueous media, and able to display singlet oxygen generation.

A Comparative Study of Ultrasmall Calcium Carbonate Nanoparticles for Targeting and Imaging Atherosclerotic Plaque.

Atherosclerosis is a complex disease that can lead to life-threatening events, such as myocardial infarction and ischemic stroke. Despite the severity of this disease, diagnosing plaque vulnerability remains challenging due to the lack of effective diagnostic tools. Conventional diagnostic protocols lack specificity and fail to predict the type of atherosclerotic lesion and the risk of plaque rupture. To address this issue, technologies are emerging, such as noninvasive medical imaging of atherosclerotic plaque with customized nanotechnological solutions. Modulating the biological interactions and contrast of nanoparticles in various imaging techniques, including magnetic resonance imaging, is possible through the careful design of their physicochemical properties. However, few examples of comparative studies between nanoparticles targeting different hallmarks of atherosclerosis exist to provide information about the plaque development stage. Our work demonstrates that Gd (III)-doped amorphous calcium carbonate nanoparticles are an effective tool for these comparative studies due to their high magnetic resonance contrast and physicochemical properties. In an animal model of atherosclerosis, we compare the imaging performance of three types of nanoparticles: bare amorphous calcium carbonate and those functionalized with the ligands alendronate (for microcalcification targeting) and trimannose (for inflammation targeting). Our study provides useful insights into ligand-mediated targeted imaging of atherosclerosis through a combination of in vivo imaging, ex vivo tissue analysis, and in vitro targeting experiments.

4,4'-Dicyano- versus 4,4'-Difluoro-BODIPYs in Chemoselective Postfunctionalization Reactions: Synthetic Advantages and Applications.

The presence of F or CN substituents at boron in BODIPYs causes a dramatic effect on their reactivity, which allows their chemoselective postfunctionalization. Thus, whereas 1,3,5,7-tetramethyl B(CN)2-BODIPYs displayed enhanced reactivity in Knoevenagel condensations with aldehydes, the corresponding BF2-BODIPYs can experience selective aromatic electrophilic substitution (SEAr) reactions in the presence of the former. These (selective) reactions have been employed in the preparation of BODIPY dimers and tetramers, with balanced fluorescence and singlet oxygen formation, and all-BODIPY trimers and heptamers, with potential application as light-harvesting systems.

Epidemiology of the Microsporidium Nosema ceranae in Four Mediterranean Countries.

Nosema ceranae is a highly prevalent intracellular parasite of honey bees' midgut worldwide. This Microsporidium was monitored during a long-term study to evaluate the infection at apiary and intra-colony levels in six apiaries in four Mediterranean countries (France, Israel, Portugal, and Spain). Parameters on colony strength, honey production, beekeeping management, and climate were also recorded. Except for São Miguel (Azores, Portugal), all apiaries were positive for N. ceranae, with the lowest prevalence in mainland France and the highest intra-colony infection in Israel. A negative correlation between intra-colony infection and colony strength was observed in Spain and mainland Portugal. In these two apiaries, the queen replacement also influenced the infection levels. The highest colony losses occurred in mainland France and Spain, although they did not correlate with the Nosema infection levels, as parasitism was low in France and high in Spain. These results suggest that both the effects and the level of N. ceranae infection depends on location and beekeeping conditions. Further studies on host-parasite coevolution, and perhaps the interactions with other pathogens and the role of honey bee genetics, could assist in understanding the difference between nosemosis disease and infection, to develop appropriate strategies for its control.

Effect of Nosema ceranae infection and season on the gut bacteriome composition of the European honeybee (Apis mellifera).

Nosema ceranae is an intracellular parasite that infects honeybees' gut altering the digestive functions; therefore, it has the potential of affecting the composition of the gut microbiome. In this work, individual bees of known age were sampled both in spring and autumn, and their digestive tracts were assessed for N. ceranae infection. Intestinal microbiome was assessed by sequencing the bacterial 16S rRNA gene in two different gut sections, the anterior section (AS; midgut and a half of ileum) and the posterior section (PS; second half of ileum and rectum). A preliminary analysis with a first batch of samples (n = 42) showed that AS samples had a higher potential to discriminate between infected and non-infected bees than PS samples. As a consequence, AS samples were selected for subsequent analyses. When analyzing the whole set of AS samples (n = 158) no changes in α- or ß-diversity were observed between infected and non-infected bees. However, significant changes in the relative abundance of Proteobacteria and Firmicutes appeared when a subgroup of highly infected bees was compared to the group of non-infected bees. Seasonality and bees' age had a significant impact in shaping the bacteriome structure and composition of the bees' gut. Further research is needed to elucidate possible associations between the microbiome and N. ceranae infection in order to find efficient strategies for prevention of infections through modulation of bees' microbiome.

Access to n-pentenyl tetra- and pentasaccharide analogues of the antitumor drug PI-88 based on 1,2-methyl orthoester glycosyl donors.

Convergent synthetic routes to PI-88 tetra- and pentasaccharide-component analogues, have been developed featuring regioselective glycosylations of mannose-polyol n-pentenyl glycosides (NPG) acceptors with 1,2-methyl orthoesters (MeOE) glycosyl donors.

Access to 2,6-Dipropargylated BODIPYs as "Clickable" Congeners of Pyrromethene-567 Dye: Photostability and Synthetic Versatility.

Hitherto unreported 2,6-dipropargyl-1,3,5,7-tetramethyl BODIPYs can be efficiently prepared by a Nicholas reaction/decomplexation protocol from 1,3,5,7-tetramethyl BODIPYs. The title compounds, which improve the BODIPY photostability by retaining their inherent photophysical and photochemical properties, can be engaged in efficient copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) "click-type" reactions with azido derivatives to provide all-BODIPY-triads or conjugated BODIPYs.

A SNP assay for assessing diversity in immune genes in the honey bee (Apis mellifera L.).

With a growing number of parasites and pathogens experiencing large-scale range expansions, monitoring diversity in immune genes of host populations has never been so important because it can inform on the adaptive potential to resist the invaders. Population surveys of immune genes are becoming common in many organisms, yet they are missing in the honey bee (Apis mellifera L.), a key managed pollinator species that has been severely affected by biological invasions. To fill the gap, here we identified single nucleotide polymorphisms (SNPs) in a wide range of honey bee immune genes and developed a medium-density assay targeting a subset of these genes. Using a discovery panel of 123 whole-genomes, representing seven A. mellifera subspecies and three evolutionary lineages, 180 immune genes were scanned for SNPs in exons, introns (< 4 bp from exons), 3' and 5´UTR, and < 1 kb upstream of the transcription start site. After application of multiple filtering criteria and validation, the final medium-density assay combines 91 quality-proved functional SNPs marking 89 innate immune genes and these can be readily typed using the high-sample-throughput iPLEX MassARRAY system. This medium-density-SNP assay was applied to 156 samples from four countries and the admixture analysis clustered the samples according to their lineage and subspecies, suggesting that honey bee ancestry can be delineated from functional variation. In addition to allowing analysis of immunogenetic variation, this newly-developed SNP assay can be used for inferring genetic structure and admixture in the honey bee.

A Concise Route to Water-Soluble 2,6-Disubstituted BODIPY-Carbohydrate Fluorophores by Direct Ferrier-Type C-Glycosylation.

Novel, linker-free, BODIPY-carbohydrate derivatives containing sugar residues at positions C2 and C6 are efficiently obtained by, hitherto unreported, Ferrier-type C-glycosylation of 8-aryl-1,3,5,7-tetramethyl BODIPYs with commercially available tri-O-acetyl-d-glucal followed by saponification. This transformation, which involves the electrophilic aromatic substitution (SEAr) of the dipyrrin framework with an allylic oxocarbenium ion, provides easy access to BODIPY-carbohydrate hybrids with excellent photophysical properties and a weaker tendency to aggregate in concentrated water solutions.

A Concise Synthesis of a BODIPY-Labeled Tetrasaccharide Related to the Antitumor PI-88.

A convergent synthetic route to a tetrasaccharide related to PI-88, which allows the incorporation of a fluorescent BODIPY-label at the reducing-end, has been developed. The strategy, which features the use of 1,2-methyl orthoesters (MeOEs) as glycosyl donors, illustrates the usefulness of suitably-designed BODIPY dyes as glycosyl labels in synthetic strategies towards fluorescently-tagged oligosaccharides.

Age and Season Effect the Timing of Adult Worker Honeybee Infection by Nosema ceranae.

The microsporidia Nosema ceranae is an intracellular parasite of honeybees' midgut, highly prevalent in Apis mellifera colonies for which important epidemiological information is still unknown. Our research aimed at understanding how age and season influence the onset of infection in honeybees and its development in the colony environment. Adult worker honeybees of less than 24h were marked and introduced into 6 different colonies in assays carried out in spring and autumn. Bees of known age were individually analyzed by PCR for Nosema spp. infection and those resulting positive were studied to determine the load by Real Time-qPCR. The age of onset and development of infection in each season was studied on a total of 2401 bees and the probability and the load of infection for both periods was established with two statistical models. First N. ceranae infected honeybees were detected at day 5 post emergence (p.e.; spring) and at day 4 p.e. (autumn) and in-hive prevalence increased from that point onwards, reaching the highest mean infection on day 18 p.e. (spring). The probability of infection increased significantly with age in both periods although the age variable better correlated in spring. The N. ceranae load tended to increase with age in both periods, although the age-load relationship was clearer in spring than in autumn. Therefore, age and season play an important role on the probability and the development of N. ceranae infection in honeybees, bringing important information to understand how it spreads within a colony.

Wide diversity of parasites in Bombus terrestris (Linnaeus, 1758) revealed by a high-throughput sequencing approach.

Assessing the extent of parasite diversity requires the application of appropriate molecular tools, especially given the growing evidence of multiple parasite co-occurrence. Here, we compared the performance of a next-generation sequencing technology (Ion PGM ™ System) in 12 Bombus terrestris specimens that were PCR-identified as positive for trypanosomatids (Leishmaniinae) in a previous study. These bumblebees were also screened for the occurrence of Nosematidae and Neogregarinorida parasites using both classical protocols (either specific PCR amplification or amplification with broad-range primers plus Sanger sequencing) and Ion PGM sequencing. The latter revealed higher parasite diversity within individuals, especially among Leishmaniinae (which were present as a combination of Lotmaria passim, Crithidia mellificae and Crithidia bombi), and the occurrence of taxa never reported in these hosts: Crithidia acanthocephali and a novel neogregarinorida species. Furthermore, the complementary results produced by the different sets of primers highlighted the convenience of using multiple markers to minimize the chance of some target organisms going unnoticed. Altogether, the deep sequencing methodology offered a more comprehensive way to investigate parasite diversity than the usual identification methods and provided new insights whose importance for bumblebee health should be further analysed.

In Vitro Antimicrobial Activity of Isopimarane-Type Diterpenoids.

The antimicrobial evaluation of twelve natural and hemisynthetic isopimarane diterpenes are reported. The compounds were evaluated against a panel of Gram-positive bacteria, including two methicillin-resistant Staphylococcus aureus (MRSA) strains and one vancomycin-resistant Enterococcus (VRE) strain. Only natural compounds 7,15-isopimaradien-19-ol (1) and 19-acetoxy-7,15-isopimaradien-3ß-ol (6) showed promising results. Isopimarane (1) was the most active, showing MIC values between 6.76 µM against S. aureus (ATCC 43866) and 216.62 µM against E. faecalis (FFHB 427483) and E. flavescens (ATCC 49996). Compound (6) showed moderated activity against all tested microorganisms (MIC between value 22.54 and 45.07 µM). These compounds were found to be active against the methicillin-sensitive strains of S. aureus (CIP 106760 and FFHB 29593), showing MIC values of 13.55 (1) and 22.54 (6) µM. Both compounds were also active against vancomycin-resistant E. faecalis (ATCC 51299) (MIC values of 54.14 and 45.07 µM, respectively). In addition, the cytotoxicity of nine compounds 7,15-isopimaradien-3ß,19-diol (2); mixture: 15-isopimarene-8ß-isobutyryloxy-19-ol and 15-isopimarene-8ß-butyryloxy-19-ol (3); 3ß-acetoxy-7,15-isopimaradiene-19-ol (5); 19-acetoxy-7,15-isopimaradiene-3ß-ol (6); 3ß,19-diacetoxy-7,15-isopimaradiene (8); 15-isopimarene-8ß,19-diol (9); 19-O-ß-d-glucopyranoside-7,15-isopimaradiene (10); lagascatriol-16-O-ß-d-glucopyranoside (11) and lagascatriol-16-O-α-d-mannopyranoside (12) was evaluated in the human breast cancer cell line MDA-MB-231. Isopimarane (2) was the only compound showing some cytotoxicity. The IC50 value of compound (2) was 15 µM, suggesting a mild antiproliferative activity against these breast cancer cells.

BODIPYs as Chemically Stable Fluorescent Tags for Synthetic Glycosylation Strategies towards Fluorescently Labeled Saccharides.

A series of fluorescent boron-dipyrromethene (BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes have been designed to participate, as aglycons, in synthetic oligosaccharide protocols. As such, they served a dual purpose: first, by being incorporated at the beginning of the process (at the reducing-end of the growing saccharide moiety), they can function as fluorescent glycosyl tags, facilitating the detection and purification of the desired glycosidic intermediates, and secondly, the presence of these chromophores on the ensuing compounds grants access to fluorescently labeled saccharides. In this context, a sought-after feature of the fluorescent dyes has been their chemical robustness. Accordingly, some BODIPY derivatives described in this work can withstand the reaction conditions commonly employed in the chemical synthesis of saccharides; namely, glycosylation and protecting-group manipulations. Regarding their photophysical properties, the BODIPY-labeled saccharides obtained in this work display remarkable fluorescence efficiency in water, reaching quantum yield values up to 82 %, as well as notable lasing efficiencies and photostabilities.

Tuning the Photonic Behavior of Symmetrical bis-BODIPY Architectures: The Key Role of the Spacer Moiety.

Herein we describe the synthesis, computationally assisted spectroscopy, and lasing properties of a new library of symmetric bridged bis-BODIPYs that differ in the nature of the spacer. Access to a series of BODIPY dimers is straightforward through synthetic modifications of the pending ortho-hydroxymethyl group of readily available C-8 (meso) ortho-hydroxymethyl phenyl BODIPYs. In this way, we have carried out the first systematic study of the photonic behavior of symmetric bridged bis-BODIPYs, which is effectively modulated by the length and/or stereoelectronic properties of the spacer unit. The designed bis-BODIPYs display bright fluorescence and laser emission in non-polar media. The fluorescence response is governed by the induction of a non-emissive intramolecular charge transfer (ICT) process, which is significantly enhanced in polar media. The effectiveness of the fluorescence quenching and also the prevailing charge transfer mechanism (from the spacer itself or between the BODIPY units) rely directly on the electron-releasing ability of the spacer. Moreover, the linker moiety can also promote intramolecular excitonic interactions, leading to excimer-like emission characterized by new spectral bands and the lengthening of lifetimes. The substantial influence of the bridging moiety on the emission behavior of these BODIPY dyads and their solvent-sensitivity highlight the intricate molecular dynamics upon excitation in multichromophoric systems. In this regard, the present work represents a breakthrough in the complex relationship between the molecular structure of the chromophores and their photophysical signatures, thus providing key guidelines for rationalizing the design of tailored bis-BODIPYs with potential advanced applications.

Towards Efficient and Photostable Red-Emitting Photonic Materials Based on Symmetric All-BODIPY-Triads, -Pentads, and -Hexads.

The development of efficient and stable red and near-IR emitting materials under hard radiation doses and/or prolonged times is a sought-after task due to their widespread applications in optoelectronics and biophotonics. To this aim, novel symmetric all-BODIPY-triads, -pentads, and -hexads have been designed and synthesized as light-harvesting arrays. These photonic materials are spectrally active in the 655-730 nm region and display high molar absorption across UV-visible region. Furthermore, they provide, to the best of our knowledge, the highest lasing efficiency (up to 68 %) and the highest photostability (tolerance >1300 GJ mol-1 ) in the near-IR spectral region ever recorded under drastic pumping conditions. Additionally, the modular synthetic strategy to access the cassettes allows the systematic study of their photonic behavior related to structural factors. Collectively, the outstanding behavior of these multichromophoric photonic materials provides the keystone for engineering multifunctional systems to expedite the next generation of effective red optical materials.

A Malonyl-Based Scaffold for Conjugatable Multivalent Carbohydrate-BODIPY Presentations.

A concise synthetic route from methylmalonate to a tetravalent aliphatic scaffold has been developed. The ensuing tetra-tethered derivative is equipped with two hydroxyl groups, as well as orthogonal alkene and alkyne functionalities. The usefulness of the scaffold has been demonstrated with the preparation of two representative multivalent derivatives: (i) a tetravalent compound containing two D-mannose units, one fluorescent boron-dipyrromethene (BODIPY) dye and a suitably functionalized amino acid and (ii) by way of dimerization and saponification, a water-soluble tetramannan derivative containing two fluorescent BODIPY units. Additionally, photophysical measurements conducted on these derivatives support the viability of the herein designed single and double BODIPY-labeled carbohydrate-based clusters as fluorescent markers.

Ready access to a branched Man5 oligosaccharide based on regioselective glycosylations of a mannose-tetraol with n-pentenyl orthoesters.

A branched Man(5) oligosaccharide has been synthesized by sequential regioselective glycosylations on a mannose-tetraol with n-pentenyl orthoester glycosyl-donors promoted by NIS/BF(3)·Et(2)O, in CH(2)Cl(2). An extended n-pentenyl chain was incorporated into the tetraol acceptor to facilitate (a) the solubility of the starting tetraol in CH(2)Cl(2), and (b) future manipulations at the reducing end of the Man(5) oligosaccharide.

Unexpected stereocontrolled access to 1α,1'ß-disaccharides from methyl 1,2-ortho esters.

Mannopyranose-derived methyl 1,2-orthoacetates (R = Me) and 1,2-orthobenzoates (R = Ph) undergo stereoselective formation of 1α,1'ß-disaccharides, upon treatment with BF(3)·Et(2)O in CH(2)Cl(2), rather than the expected acid-catalyzed reaction leading to methyl glycosides by way of a rearrangement-glycosylation process of the liberated methanol.

Reaction of 1,2-orthoesters with HF-pyridine: a method for the preparation of partly unprotected glycosyl fluorides and their use in saccharide synthesis.

Glycosyl fluorides can be prepared in an efficient manner by treatment of pyranose- or furanose-derived 1,2-orthoesters, with hydrogen fluoride pyridine (HF-py). The method is compatible with the presence of a variety of protecting groups, including tert-butyldiphenyl silyl ethers, and can be applied to sugar derivatives with free hydroxyl groups, thus avoiding the need for the protection-deprotection steps.