Carbohydrate-appended photocytotoxic (imidazophenanthroline)-oxovanadium(IV) complexes for cellular targeting and imaging

Oxovanadium(IV) complexes VO(aip)(L)](ClO4)(2) (L = phtpy, 1; stpy, 2) and VO(pyip)(L)](ClO4)(2) (L = phtpy, 3; stpy, 4), where aip is 2-(9-anthryl)-1H-imidazo4,5-f]1,10] phenanthroline, pyip is 2-(1-pyrenyl)-1Himidazo4,5-f]1,10] phenanthroline, phtpy is (4'-phenyl)-2,2': 6',2 `'-terpyridine and stpy is (2,2': 6', 2 `'-terpyridin-4'-oxy) ethyl-beta-D-glucopyranoside, were prepared, characterized and their DNA binding and photocleavage activity, cellular uptake and photocytotoxicity in visible light were studied. The complexes are avid binders to calf thymus DNA (K-b similar to 10(5) mol(-1)). They efficiently cleave pUC19 DNA in red light of 705 nm via the formation of HO center dot species. The glucose appended complexes 2 and 4 showed higher photocytotoxicity in HeLa and Hep G2 cells over the normal HEK 293T cells. No such preference was observed for the phtpy complexes 1 and 3. No significant difference in IC50 values was observed for the HEK 293T cells. Cell cycle analysis showed that the glucose appended complexes 2 and 4 are more photocytotoxic in cancer cells than in normal cells. Fluorescence microscopy was done to study the cellular localization of complex 4 having a pendant pyrenyl group.

Carbohydrate-Appended Tumor Targeting Iron(III) Complexes Showing Photocytotoxicity in Red Light

Glucose-appended photocytotoxic iron(III) complexes of a tridentate Schiff base phenolate ligand Fe(bpyag) (L)] (NO3) (1-3), where bpyag is N,N-bis(2- pyridylmethyl)-2-aminoethyl-beta-D-glucopyranoside and H2L is 3-(2-hydroxyphenylimino)-1-phenylbutan-1-one (H(2)phap) in 1, 3-(2-hydroxyphenylimino)-9-anthrylbutan-1-one (H(2)anap) 2, and 3- (2-hydroxyphenylimino)-1-pyrenylbutan-1-one (H(2)pyap) in 3, were synthesized and characterized. The complex Fe(dpma)(anapn(NO3) (4), having bis-(2-pyridylmethyl)benzylamine (dpma), in which the glucose moiety of bpyag is substituted by a phenyl group, was used as a control, and the complex Fe(dpma)(anap)](PF6) (4a) was structurally characterized by X-ray crystallography. The structure shows a FeN4O2 core in a distorted octahedral geometry. The high-spin iron(III) complexes with magnetic moment value of similar to 5.9 mu(B) showed a low-energy phenolate-to-Fe(III) charge-transfer (CT) absorption band as a shoulder near 500 nm with a tail extending to 700 nm and an irreversible Fe(III)-Fe(II) redox couple near -0.6 V versus saturated calomel electrode. The complexes are avid binders to calf thymus DNA and showed photocleavage of supercoiled pUC19 DNA in red (647 nm) and green (532 nm) light. Complexes 2 and 3 displayed significant photocytotoxicity in red light, with an IC50 value of similar to 20 mu M in HeLa and HaCaT cells, and no significant toxicity in dark. The cell death is via an apoptotic pathway, by generation of reactive oxygen species. Preferential internalization of the carbohydrate-appended complexes 2 and 3 was evidenced in HeLa cells as compared to the control complex 4. A 5-fold increase in the cellular uptake was observed for the active complexes in HeLa cells. The photophysical properties of the complexes are rationalized from the density functional theory calculations.

Novel synthesis of carbohydrate fused alpha-amino gamma-lactams and glycopeptides by NIS mediated ring opening of donor-acceptor substituted cyclopropanes

alpha-Amino gamma-lactams have been synthesized from carbohydrate derived cyclopropanecarboxylates using N-iodosuccinimide (NIS) and NaN3. Cyclopropane ring opening with NIS and NaN3 in different solvents has been studied. Reductive cyclization of the intermediate di-azides leads to the carbohydrate fused alpha-amino gamma-lactam and gamma-lactams. Additionally, the methodology has been successfully extended to the synthesis of a glycopeptide. (C) 2014 Elsevier Ltd. All rights reserved.

sigma-Ferrier rearrangement of carbohydrate derived vinylcyclopropanes: a facile approach to oxepane analogs

This article presents our work on the sigma-Ferrier ring-expansion of carbohydrate derived vinylcyclopropanes (VCPs) under electrophilic conditions mediated by chloramine-T and a phase-transfer catalyst. The present work serves as the first example on the studies of the reactivity of carbohydrate VCPs towards the synthesis of densely functionalized oxepane analogues. The work elaborates on a reasonable mechanism for the product formation and our observations on the diastereoselectivity based on control experiments and gas-phase calculations. (C) 2014 Elsevier Ltd. All rights reserved.

Jacalin-carbohydrate interactions: distortion of the ligand molecule as a determinant of affinity

Jacalin is among the most thoroughly studied lectins. Its carbohydrate-binding site has also been well characterized. It has been postulated that the lower affinity of beta-galactosides for jacalin compared with beta-galactosides is caused by steric interactions of the substituents in the former with the protein. This issue has been explored energetically and structurally using different appropriate carbohydrate complexes of jacalin. It turns out that the earlier postulation is not correct. The interactions of the substituent with the binding site remain essentially the same irrespective of the anomeric nature of the substitution. This is achieved through a distortion of the sugar ring in beta-galactosides. The difference in energy, and therefore in affinity, is caused by a distortion of the sugar ring in beta-galactosides. The elucidation of this unprecedented distortion of the ligand as a strategy for modulating affinity is of general interest. The crystal structures also provide a rationale for the relative affinities of the different carbohydrate ligands for jacalin.

Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry

A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W-102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W-102) in the activity of the lectin. (c) 2015 IUBMB Life, 67(12):943-953, 2015

Kinetic measurements of cell surface E-selectin/carbohydrate ligand interactions

Selectin/ligand interactions initiate the multistep adhesion and signaling cascades in the recruitment of leukocytes from circulation to inflamed tissues and may also play a role in tumor metastasis. Kinetic properties of these interactions are essential determinants governing blood-borne cells' tethering to and rolling on the vessel wall. Extending our recently developed micropipette method, we have measured the kinetic rates of E-selectin/ligand interactions. Red cells coated with an E-selectin construct were allowed to bind HL-60 or Colo-205 cells bearing carbohydrate ligands. Specific adhesions were observed to occur at isolated points, the frequency of which followed a Poisson distribution. These point attachments were formed at the same rate with both the HL-60 and Colo-205 cells (0.14 +/- 0.04 and 0.13 +/- 0.03 mum(2) s(-1) per unit density of E-selectin, respectively) but dissociated from the former at a rate twice as fast as did from the latter (0.92 +/- 0.23 and 0.44 +/- 0.10 s(-1), respectively). The reverse rates agree well with those measured by the flow chamber. The forward rates are orders of magnitude higher than those of Fc gamma receptors interacting with IgG measured under similar conditions, consistent with the rapid kinetics requirement for the function of E-selectin/ligand binding, which is to capture leukocytes on endothelial surfaces from flow.

Seasonal Biomass and Carbohydrate Allocation Patterns in Southern Minnesota Curlyleaf Pondweed Populations

Four southern Minnesota populations of curlyleaf pondweed ( Potamogeton crispus L.) were sampled monthly from January 2001 to November 2002 to determine seasonal phenological, biomass, and carbohydrate allocation patterns. Low periods of carbohydrate storage in the seasonal phenological cycle indicate potentially vulnerable periods in the plant’s life cycle and may be the ideal time to initiate management and control efforts.

Nutrient digestibility and growth response of rainbow trout (Salmo gairdneri ) fed different carbohydrate types

Seven groups of fingerling rainbow trout (S. gairdneri ) were fed for 10 weeks on 0%, 10%, 20% and 30% of cassava or rice in isonitrogenous diets. Optimum growth and food utilization was at 20% dietary cassava. High fiber content of the control diet did not suppress protein digestibility in this group. Rather, at all levels, protein digestibility was good and remained between 84.4% and 90.1%. However, in the control group, carbohydrate digestibility was very poor. The cassava diets which had the highest digestible energy as carbohydrate produced the best growth performance, food utilization and protein sparing. At the levels studied, the dietary carbohydrates produced no hyperglycamic effect on the fish

Dietary carbohydrate requirement of Heterobranchus longifilis

The dietary carbohydrate requirement of Heterobranchus longifilis was evaluated in two separate experiments.In the first experiment, varying levels of carbohydrate ranging from 28, 24 to58 72% were fed to the fish of mean weight 1.83~c0.02g. Results revealed that the polynomial regression curve for the mean weight gain and the carbohydrate levels did not present a point where Y-max is equal to X-max and so the requirement was not obtained. The second experiment was therefore, conducted with lower levels of carbohydrate ranging from 17.00 to 20.86% and fed to fish with mean weight 0.49~c0.02g. Based on growth and feed efficiency data the carbohydrate requirement was determined to be 19.5%

Molecular mechanism of sulfated carbohydrate recognition: structural and biochemical studies of the cysteine-rich domain of mannose receptor

Mannose receptor (MR) is widely expressed on macrophages, immature dendritic cells, and a variety of epithelial and endothelial cells. It is a 180 kD type I transmembrane receptor whose extracellular region consists of three parts: the amino-terminal cysteine-rich domain (Cys-MR); a fibronectin type II-like domain; and a series of eight tandem C-type lectin carbohydrate recognition domains (CRDs). Two portions of MR have distinct carbohydrate recognition properties: Cys-MR recognizes sulfated carbohydrates and the tandem CRD region binds terminal mannose, fucose, and N-acetyl-glucosamine (GlcNAc). The dual carbohydrate binding specificity allows MR to interact with sulfated and nonsulfated polysaccharide chains, and thereby facilitating the involvement of MR in immunological and physiological processes. The immunological functions of MR include antigen capturing (through binding non-sulfated carbohydrates) and antigen targeting (through binding sulfated carbohydrates), and the physiological roles include rapid clearance of circulatory luteinizing hormone (LH), which bears polysaccharide chains terminating with sulfated and non-sulfated carbohydrates.

We have crystallized and determined the X-ray structures of unliganded Cys-MR (2.0 Å) and Cys-MR complexed with different ligands, including Hepes (1.7 Å), 4SO_4-N-Acetylgalactosamine (4SO_4-GalNAc; 2.2 Å), 3SO_4-Lewis^x (2.2 Å), 3S04-Lewis^a (1.9 Å), and 6SO_4-GalNAc (2.5 Å). The overall structure of Cys-MR consists of 12 anti-parallel β-strands arranged in three lobes with approximate three fold internal symmetry. The structure contains three disulfide bonds, formed by the six cysteines in the Cys-MR sequence. The ligand-binding site is located in a neutral pocket within the third lobe, in which the sulfate group of ligand is buried. Our results show that optimal binding is achieved by a carbohydrate ligand with a sulfate group that anchors the ligand by forming numerous hydrogen bonds and a sugar ring that makes ring-stacking interactions with Trpll7 of CysMR. Using a fluorescence-based assay, we characterized the binding affinities between CysMR and its ligands, and rationalized the derived affinities based upon the crystal structures. These studies reveal the mechanism of sulfated carbohydrate recognition by Cys-MR and facilitate our understanding of the role of Cys-MR in MR recognition of its ligands.

Short-term preservation of Brachionus calyciflorus: effects of storage on protein, lipid and carbohydrate contents

The short-term preservation of Brachionus calyciflorus for 45 days at three different temperatures (4, -4 and -20°C) led to decrease in protein, lipid and carbohydrate contents in all the three cases. However, the rate of deterioration was much higher at 4C than at -4 and -20°C. At 4C, protein, lipid and carbohydrate contents reduced by 76.78, 81.11 and 62.83%, respectively, and at -4°C, these were 27.94, 37.46 and 18.42%, respectively, whereas at -20°C, the deterioration was limited to 9.28, 16.44 and 11.35%, respectively, when compared with the control values. Thus, preservation at -20°C is comparatively better as it exerts limited effect on the protein, lipid and carbohydrate contents of B. calycijlorus.

Effects of control of C/N ratio by low-cost carbohydrate addition on water quality and pond ecology in freshwater prawn Macrobrachium rosenbergii post-larvae nursing system

An experiment was conducted to evaluate the effects of control of carbon/nitrogen ratio (C/N ratio) by addition of low cost carbohydrate to the water column on water quality and pond ecology in freshwater prawn Macrobrachium rosenbergii post-larvae nursing system. In this experiment, two level of dietary protein 20% and 35% without carbohydrate addition (‘P20' and ‘P35') and with carbohydrate addition (‘P20+CH' and ‘P35+CH') were compared in small ponds of 40 m² area stocked with 20 post-larvae (0.021 ± 0.001g) per m² . Maize flour was used as low cost carbohydrate and applied to the water column followed by the first feeding during the day. The addition of carbohydrate significantly reduced (p< 0.05) ammonia-nitrogen (NH sub(3)-N) and nitrite-nitrogen (NO sub(2) - N) of water in P20 + CH and P35 + CH treatments. It significantly increased (p< 0.05) the total heterotrophic bacteria (THB) population both in water and sediment. Fifty nine genera of plankton were identified belonging to the Bacillariophyceae (11), Chlorophyceae (21), Cyanophyceae (7), Dinophyceae (1), Rotifera (7) and Crustacea (9) without any significant difference (p>0.05) of total phytoplankton and zooplankton among the treatments. Survival rate of prawn was significantly lowest (p<0.05) in P20 and no significant difference (p>0.05) was observed between P20+CH and P35 treatments. Control of C/N ratio by the addition of low-cost carbohydrate to the pond water column benefited the freshwater prawn nursing practices in three ways (1) increased heterotrophic bacterial growth supplying bacterial protein augment the prawn post-larvae growth performances, (2) reduced demand for supplemental feed protein and subsequent reduction in feed cost and (3) reduced toxic NH sub(3)-N and NO sub(2)-N levels in pond nursing system.