Photodynamic effect of meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins on HaCaT keratinocytes.

Sixteen porphyrins, including neutral, anionic and cationic meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins were herein evaluated in terms of their photosensitizing properties against HaCaT keratinocytes. After an initial screening, the cationic porphyrins were studied in more details, by both determining their log POW and performing PDT assays in lower porphyrin concentrations. Porphyrins presenting two or more adjacent positively charged groups, directly linked to the macrocycle meso positions, appeared to be the most effective photosensitizers. The present study also included the dicationic 5,10-diphenyl-15,20-di(1-methylpyridinium-4-yl)porphyrin (14b), which has previously shown promising results on a psoriasis-like in vivo model. Overall results indicated that the beneficial effect related to porphyrins on psoriasis can be related to the decreasing of keratinocyte viability. Furthermore, some of the cationic porphyrins studied appeared as candidates to be utilized as photosensitizers for psoriasis treatment.

Aqueous semisynthesis of C-glycoside glycamines from agarose.

Agarose was herein employed as starting material to produce primary, secondary and tertiary C-glycoside glycamines, including mono- and disaccharide structures. The semisynthetic approach utilized was generally based on polysaccharide-controlled hydrolysis followed by reductive amination. All reactions were conducted in aqueous media and without the need of hydroxyl group protection. We were able to identify optimal conditions for the reductive amination of agar hydrolysis products and to overcome the major difficulties related to this kind of reaction, also extending it to reducing anhydrosugars. The excess of ammonium acetate, methyl- or dimethylamine, and the use of a diluted basic (pH 11) reaction media were identified as important aspects to achieve improved yields, as well as to decrease the amount of byproducts commonly related to reductive amination of carbohydrates. This strategy allowed the transposition of the 3,6-anhydro-α-L-galactopyranose unit (naturally present in the agarose structure) to all glycamines synthesized, constituting an amino-substituted C-threofuranoside moiety, which is closely related to (+)-muscarine.

Semisynthesis and characterization of versatile azide intermediates using sodium alginate and its homopolymeric derivatives as starting material.

Alginate, a polyuronic biopolymer composed of mannuronic and guluronic acid units, contain hydroxyl and carboxyl groups as targeting modification sites to obtain structures with new and/or improved biological properties. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a versatile click reaction for polymer functionalization, but it typically requires a "pre-click" modification to introduce azide or alkyne groups. Here, we described a straightforward chemical path to selectively modify alginate carboxyl groups producing versatile azido derivatives through N-acylation using 3-azydopropylamine. The resulting azide-functionalized polysaccharides underwent click chemistry to yield amino derivatives, confirmed by NMR and FTIR analyses. The 1H NMR spectrum reveals a characteristic triazole group signal at 8.15 ppm. The absence of the azide FTIR band for all amino derivatives, previously observed for the N-acylation products, indicated reaction success. Antibacterial and antioxidant assessments revealed that the initial polysaccharide lacks E. coli inhibition, while the click chemistry-derived amine products exhibit growth inhibition at 5.0 mg/mL. Lower molecular weight derivatives demonstrate superior DPPH scavenging ability, particularly amino-derivatives (24-33 % at 1.2 mg/mL). This innovative chemical pathway offers a promising strategy for developing polysaccharide structures with enhanced properties, demonstrating potential applications in various fields.

Semisynthesis of new sulfated heterorhamnan derivatives obtained from green seaweed Gayralia brasiliensis and evaluation of their anticoagulant activity.

Marine green algae produce sulfated polysaccharides with diverse structures and a wide range of biological activities. This study aimed to enhance the biotechnological potential of sulfated heterorhamnan (Gb1) from Gayralia brasiliensis by chemically modifying it for improved or new biological functions. Using controlled Smith Degradation (GBS) and O-alkylation with 3-chloropropylamine, we synthesized partially water-soluble amine derivatives. GBS modification increase sulfate groups (29.3 to 37.5 %) and α-l-rhamnose units (69.9 to 81.2 mol%), reducing xylose and glucose, compared to Gb1. The backbone featured predominantly 3- and 2-linked α-l-rhamnosyl and 2,3- linked α-l-rhamnosyl units as branching points. Infrared and NMR analyses confirmed the substitution of hydroxyl groups with aminoalkyl groups. The modified compounds, GBS-AHCs and GBS-AHK, exhibited altered anticoagulant properties. GBS-AHCs showed reduced effectiveness in the APTT assay, while GBS-AHK maintained a similar anticoagulant activity level to Gb1 and GBS. Increased nitrogen content and N-alkylation in GBS-AHCs compared to GBS-AHK may explain their structural differences. The chemical modification proposed did not enhance its anticoagulant activity, possibly due to the introduction of amino groups and a positive charge to the polymer. This characteristic presents new opportunities for investigating the potential of these polysaccharides in various biological applications, such as antimicrobial and antitumoral activities.

A new porphyrin as selective substrate-based inhibitor of breast cancer resistance protein (BCRP/ABCG2).

The ABCG2 transporter plays a pivotal role in multidrug resistance, however, no clinical trial using specific ABCG2 inhibitors have been successful. Although ABC transporters actively extrude a wide variety of substrates, photodynamic therapeutic agents with porphyrinic scaffolds are exclusively transported by ABCG2. In this work, we describe for the first time a porphyrin derivative (4B) inhibitor of ABCG2 and capable to overcome multidrug resistance in vitro. The inhibition was time-dependent and 4B was not itself transported by ABCG2. Independently of the substrate, the porphyrin 4B showed an IC50 value of 1.6 µM and a mixed type of inhibition. This compound inhibited the ATPase activity and increased the binding of the conformational-sensitive antibody 5D3. A thermostability assay confirmed allosteric protein changes triggered by the porphyrin. Long-timescale molecular dynamics simulations revealed a different behavior between the ABCG2 porphyrinic substrate pheophorbide a and the porphyrin 4B. Pheophorbide a was able to bind in three different protein sites but 4B showed one binding conformation with a strong ionic interaction with GLU446. The inhibition was selective toward ABCG2, since no inhibition was observed for P-glycoprotein and MRP1. Finally, this compound successfully chemosensitized cells that overexpress ABCG2. These findings reinforce that substrates may be a privileged source of chemical scaffolds for identification of new inhibitors of multidrug resistance-linked ABC transporters.

Differential inhibition of dengue virus infection in mammalian and mosquito cells by iota-carrageenan.

The antiviral activity against dengue virus-2 (DENV-2) of carrageenans reported here has shown a differential susceptibility of C6/36 HT and Vero cells, taken as models of mosquito and mammalian cells, depending on the structural class of polysaccharides: all polysaccharides blocked DENV-2 infection in monkey Vero cells, but only iota-carrageenans were virus inhibitors in mosquito cells. However, iota-carrageenans were less effective in mosquito cells in comparison with mammalian cells with effective concentration 50 % (EC(50)) values in C6/36 HT cells 4.9-17.5-fold higher than in Vero cells, as determined by virus yield reduction assay. The mode of action of iota-carrageenan in both cell types was strikingly different: in Vero cells the inhibitory activity was exerted only at the initiation of the cycle, affecting virion binding, whereas in mosquito cells DENV-2 adsorption was not affected and comparable levels of inhibition were obtained if the compound was added to cells together with the virus, after 8 h of infection or by cell pre-treatment before infection. Furthermore, iota-carrageenans induced a subtle alteration in mosquito cells, detected by cell proliferation and protein synthesis analyses, suggesting that a probable cellular target may be responsible for the refractory state of mosquito cells to DENV-2 infection produced by this class of polysulfates. The failure of iota-carrageenan to block DENV-2 adsorption to mosquito cells appeared to be related to the low presence of adequate heparan sulfate (HS) in C6/36 HT cell surface and is indicative of a differential participation of HS residues for DENV-2 entry in both types of cells.

Semi-synthesis of N-alkyl-kappa-carrageenan derivatives and evaluation of their antibacterial activity.

In this article, we describe the semi-synthesis of N-alkyl-kappa-carrageenan derivatives and their antibacterial activity against Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), and Pseudomonas aeruginosa (ATCC 9027). Kappa-carrageenan was submitted to partial acid hydrolysis promoting the selective cleavage of α-glycosidic bonds involving 3,6-anhydro-α-D-Galp units, giving rise to reducing low-molecular weight polysaccharide fragments, which were reacted with alkylamines of varying chain lengths by reductive amination. The carrageenan derivatives were characterized by HPSEC-MALLS-RID and 1D and 2D 1H and 13C NMR spectroscopy. The antibacterial activity of N-alkyl-kappa-carrageenan derivatives was compared with N-alkyl-(1-deoxylactitol-1-yl)-amines using a microdilution test, which indicated that inhibitory activity was dependent on the degree of substitution by hydrophobic groups at the polysaccharide structure. Comparing the effect of different N-alkyl chains, those with longer chains showed higher activity.

Semi-synthesis of hybrid ulvan-kappa-carrabiose polysaccharides and evaluation of their cytotoxic and anticoagulant effects.

In this study we described the synthesis of a hybrid polysaccharide harboring moieties of ulvan and kappa-carrabiose. Alkylamines (1,3-diaminopropane and 1,6-diaminohexane) were selectively inserted into ß-D-GlcAp and α-L-IdoAp units in the ulvan structure via an amide bond formation producing ulvan-amide derivatives F-DAP (N% = 1.77; Mw = 208 kg mol-1) and F-DAH (N% = 1.77; Mw = 202 kg mol-1), which were reacted with kappa-carrabiose via reductive amination to produce hybrid ulvan-kappa-carrabiose polysaccharides F-DAP-Kb (N% = 1.56; Mw = 206 kg mol-1) and F-DAH-Kb (N% = 1.16; Mw = 200 kg mol-1). All the ulvan derivatives were characterized by 1H and 13C NMR spectroscopy and did not show cytotoxicity against human dermal fibroblasts (HDFa) at the concentrations of 25, 100, and 500 µg mL-1, neither anticoagulant properties at the range of 10-150 µg mL-1. Therefore, the ulvan-amide derivatives and the hybrid ulvan-kappa-carrabiose polysaccharides showed good biocompatibility in vitro, presenting as worthy candidates for tailoring scaffolds for biomedical applications.

Synthesis of C6-amino agarose and evaluation of its antibacterial activity.

In this paper, the biologically inert agarose was selectively modified at C6 of ß-d-Galp units to produce an amino derivative with antibacterial property. The synthetic route involved the preparation of tosyl and azido agarose intermediates. All the polysaccharide derivatives were characterized by mono- and bidimensional 1H and 13C NMR and FT-IR analysis. A water-soluble amino polymer (Mw = 39,000 g mol-1, DSamino = 0.50) was produced by partial acid hydrolysis showing bactericidal and bacteriostatic activity against P. aeruginosa (ATCC 9027), S. aureus (ATCC 6538), and E. coli (ATCC 25922), with MIC values lower than 2.5 mg mL-1 and MBC values ranging from 2.5 to 5.0 mg mL-1.

Chemical structure of native and modified sulfated heterorhamnans from the green seaweed Gayralia brasiliensis and their cytotoxic effect on U87MG human glioma cells.

A water-soluble sulfated heterorhamnan (Gb1) was isolated from the green seaweed Gayralia brasiliensis and purified by ultrafiltration, yielding a homogeneous polysaccharide (Gb1r). Both fractions contained rhamnose, xylose, galacturonic and glucuronic acids, galactose, and glucose. Chemical and spectroscopic methods allowed the determination of Gb1 and Gb1r chemical structure. Their backbones were constituted by 3-, 2-, and 2,3-linked rhamnosyl units (1:0.49:0.13 and 1:0.58:0.17, respectively), which are unsulfated (13.5 and 14.6%), disulfated (16.6 and 17.8%) or monosulfated at C-2 (8 and 8.6%) and C-4 (24.5 and 23.4%). Gb1 was oversulfated giving rise to Gb1-OS, which presented ~2.5-fold higher content of disulfated rhamnosyl units than Gb1, as determined by methylation analyses and NMR spectroscopy. Gb1 and Gb1-OS potently reduced the viability of U87MG human glioblastoma cells. Gb1 caused cell cycle arrest in the G1 phase, increased annexin V-stained cells, and no DNA fragmentation, while Gb1-OS increased the percentage of cells in the S and G2 phases and the levels of fragmented DNA and cells double-stained with annexin V/propidium iodide, suggesting an apoptosis mechanism. The results suggest that the different effects of Gb1 and Gb1-OS were related to differences in the sulfate content and position of these groups along the polysaccharide chains.

Production, characterization, and biological activity of a chitin-like EPS produced by Mortierella alpina under submerged fermentation.

The production of a chitin-like exopolysaccharide (EPS) was optimized through experimental design methods, evaluating the influence of urea, phosphate, and glucose. Under optimized conditions, up to 1.51 g/L was produced and its physicochemical characteristics were evaluated by chromatography, NMR, and FTIR spectroscopy, and rheological techniques. The results showed a homogeneous EPS (Mw 4.9 × 105 g mol-1) composed of chitin, linear polymer of ß-(1→4)-linked N-acetyl-d-glucosamine residues. The acetylation degree as determined by 13C CP-MAS NMR spectroscopy was over 90 %. The EPS biological activities, such as antioxidant effect and antitumor properties, were evaluated. To the best of our knowledge, this is the first study on the production of a new alternative of extracellular chitin-like polysaccharide with promising bioactive properties from the filamentous fungus M. alpina.

Conformational analysis of ulvans from Ulva fasciata and their anticoagulant polycarboxylic derivatives.

This study investigates conformational aspects of ulvans (F2) and their polycarboxyl derivatives obtained through periodate-chlorite oxidation (C3) followed by DEAE-Sephacel fractioning (C3b and C3c). Size exclusion chromatography coupled with laser light scattering and viscometric detection, in addition to circular dichroism (CD) and molecular modeling analyses, suggested that F2 had a compact sphere conformation with a helical motif as secondary structure. In contrast, all the polycarboxyl ulvans showed a random coil conformation, although C3c (NaSO3- 21.0%; COO- 1.81 mmol·g-1; Mw 18 kg·mol-1) had a more rigid and constrained backbone than C3 (NaSO3- 21.0%; COO- 1.81 mmol·g-1; Mw 49 kg·mol-1), largely due to its higher sulfate and carboxyl content. Despite the higher ionic character of C3c, its anticoagulant activity (ACA), determined by activated partial thromboplastin time (APTT) assay, was not improved compared to that of C3. Moreover, C3b (NaSO3- 14.1%; COO- 1.23 mmol·g-1; Mw 8.1 kg·mol-1) had higher activity than F2 (NaSO3- 20.6.%; COO- 0.36 mmol·g-1; Mw 123 kg·mol-1), even with its lower sulfate content and molar mass. These results suggest that the ACA of polycarboxyl ulvans relies on carboxyl and sulfate content and may depend, in addition, on a proper flexible conformation.

Production of carbohydrate building blocks from red seaweed polysaccharides. Efficient conversion of galactans into C-glycosyl aldehydes.

Agarans and carrageenans are abundant natural polysaccharides which are obtained on a large scale by water extraction from a variety of red seaweeds. These galactans, in addition to being valuable products for the pharmaceutical and food industries, are low cost starting materials for the preparation of useful and rare carbohydrate-based building blocks whose access by total synthesis is difficult and expensive. The semisynthesis of two sets of C-glycosyl aldehydes with l- and d-configuration from agarose and kappa-carrageenan respectively is described. Succinctly, the partial acid-catalyzed mercaptolysis of the two galactans under mild conditions afforded agarobiose and carrabiose (beta-d-Galp-(1-->4)-3,6-anhydro-aldehydo-l- and d-galactose, respectively) derivatives. Complete depolymerization of agarose and kappa-carrageenan under harsher conditions produced 3,6-anhydro l- and d-galactose aldehyde derivatives. Chain shortening of these products via alditol formation and oxidative carbon-carbon bond cleavage furnished C-formyl alpha-l- and alpha-d-threofuranosides. The above C-glycosyl aldehydes were all prepared on a meaningful preparative scale starting from gram quantities of galactans. Finally, a new procedure for the preparation of the 2,3-O-benzyl l-threofuranose was established by Baeyer-Villiger oxidation of the benzylated C-formyl alpha-l-threofuranoside here prepared from agarose.

Dihydropyridine C-glycoconjugates by organocatalytic Hantzsch cyclocondensation. Stereoselective synthesis of alpha-threofuranose C-nucleoside enantiomers.

The Hantzsch reaction of C-glycosyl aldehyde/enamino ester/beta-ketoester systems under l-proline catalysis to give dihydropyridine C-glycoconjugates is reported. Asymmetric cyclocondensations of differentially substituted enamine and beta-dicarbonyl components with formyl alpha-L-C-threofuranoside and with the alpha-D-isomer were also carried out. Each reaction occurred with high yet opposite stereoselectivity (de >95%) so that the pair of alpha-threofuranose C-nucleoside enantiomers was prepared.

Effects of carboxyl group on the anticoagulant activity of oxidized carrageenans.

In this paper, carrageenans having distinct sulfation patterns (κ-, ι-, ι/ν-, θ- and λ-carrageenans), were fully or partially oxidized at C-6 of the ß-d-Galp units using 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and trichloroisocyanuric acid (TCCA) in bicarbonate buffer. The modified carrageenans were characterized by mono- and bidimensional 1H and 13C NMR spectroscopy. The influence of the sulfate and carboxyl groups onto anticoagulant activity was evaluated using Activated Partial Thromboplastin Time (aPTT) in vitro assay. The results showed a synergic effect of the carboxyl groups on the anticoagulant activity, which was dependent on the regiochemistry of the sulfate groups in the polysaccharide backbone. Sulfate groups at C2 of the ß-d-GalAp units appeared to positively influence the anticoagulant effect in comparison to C4-sulfate samples. Also, the partially oxidized κ-carrageenan derivative (κLO) showed better anticoagulant effect than the fully oxidized carrageenan (κHO).

Monitoring of κ-carrageenan depolymerization by capillary electrophoresis and semisynthesis of oligosaccharide alditols.

Different hydrolysis conditions to produce κ-carrageenan oligosaccharide alditols were studied and the depolymerization process monitored by capillary electrophoresis (CE). Semisynthesis, ion-exchange and exclusion chromatography were used to obtain and isolate sulfated di-, tetra- and hexasaccharide alditols, the last being fully characterized for the first time. Those derivatives were used as standards to validate a new quantitative CE analytical method which was used to compare two different partial hydrolysis methodologies: an acid hydrolysis followed by reduction and a one-pot reductive hydrolysis using 4-methylmorpholine borane. The resulting depolymerization profiles were quite different from each other. Optimal hydrolysis conditions to produce high yields of specific sulfated oligosaccharides as well as particular mixtures of oligosaccharide alditols were determined. Moreover, using the novel CE method, we were able to distinguish up to eight different oligosaccharides in the hydrolysate mixtures.

Chemical structure and snake antivenom properties of sulfated agarans obtained from Laurencia dendroidea (Ceramiales, Rhodophyta).

Aqueous and KCl-soluble polysaccharides were extracted from Laurencia dendroidea (Rhodomelaceae, Ceramiales) and their chemical profile was accessed by anion-exchange chromatography, chemical and spectroscopic analyses. The homogeneous agaran DHS-4 (181.3 × 103 g. mol-1, 21.3% of NaSO3) presents A units mostly 2-sulfated (18.9 mol%), nonsubstituted (15.3 mol%) and 6-O-methylated (10.1 mol%), while B units are l-sugars composed predominantly by galactose 6-sulfate precursor units (19.2 mol%) and 3,6-anhydrogalactose (13.8 mol%), besides non-precursor galactose 6-sulfate units bearing d-xylose substituents on C-3 (8.1 mol%). The crude KCl-soluble DHS agaran (20.5% of NaSO3) inhibited proteolysis and hemolysis induced by Lachesis muta and Bothrops jararaca venoms. DHS was able to inhibit up to 75% the L. muta venom hemorrhagic effect and to reduce the lethality displayed by B. jararaca venom, increasing the mice survival time up to 3 times. Therefore, this agaran has high potential to be used as an additional tool to treat snakebite envenomation.

First isolation and structural determination of cyclic beta-(1-->2)-glucans from an alga, Chlorella pyrenoidosa.

The aqueous extract of the edible green microalgae Chlorella pyrenoidosa is of interest because of its immunostimulatory activity. Some components in the extract have been identified previously, namely a unique type of arabinogalactan and a galactofuran. Further fractionation of this extract was accomplished by treating the aqueous solution of the fraction precipitated by addition of 1.5vol of 95% ethanol with cetyltrimethylammonium bromide. The residue obtained by concentration of the supernatant was fractionated further by anion-exchange chromatography and size-exclusion chromatography on Sephadex G-100. Two fractions from the latter column were retained, of which one was a starch-like alpha-(1-->4)-linked d-glucan with some alpha-(1-->6) branches, and the other contained a starch plus a mixture of beta-(1-->2)-d-glucans. ESI mass spectrometry was used to show that the mixture contained both cyclic and linear beta-(1-->2)-d-glucans in a cyclic:linear ratio of 64:36, based on intensities of mass spectral peaks. For the cyclic beta-(1-->2)-d-glucans, ring sizes ranged from 18 to 35 monosaccharides with the ring containing 21 glucose units (54% of the cyclic glucans) being greater than three times more abundant than the next most abundant component, the ring containing 22 glucose units (15%). No rings containing 20 glucose units were present. This is the first observation of cyclic beta-(1-->2)-d-glucans in algae, as far as we are aware. For the linear beta-(1-->2)-d-glucans, the component containing 20 glucoses was most abundant (35% of the linear glucans), while the component containing 21 glucose units was the next most abundant (17%). These relatively low-molecular-weight glucans had low immunostimulatory activity.

Modification of ulvans via periodate-chlorite oxidation: Chemical characterization and anticoagulant activity.

Native (F2) and carboxyl-reduced (R) ulvans from Ulva fasciata were sequentially oxidized with periodate-chlorite affording the polycarboxyl ulvans C1, C2 and C3 (1.20, 1.41 and 1.81 mmol g-1 of COOH, respectively; 19.7, 21.3 and 21.0% of NaSO3, respectively) and R-C3 (1.86 mmol g-1 of COOH; NaSO3 = 22.7%), respectively. APTT assay (polysaccharide fractions at 150 µg mL-1) showed clotting time of 45.6 s for F2 fraction. For polycarboxyl ulvans C1, C2, C3 and R-C3 the clotting times were 101.0, 122.2, 222.0 and 227.0 s, respectively. Comparison of the APTT assay results using ulvans chemically modified by carboxyl-reduction, desulfation, periodate oxidation and/or chlorite oxidation showed the anticoagulant activity of polycarboxyl ulvans is dependent of the sulfate groups present in the native polymer. In addition, the increase of the anticoagulant activity was accompanied by the increasing of the carboxyl groups and the content of this acidic substituent seems to be more important than its positioning.

Sulfated xylomannans isolated from red seaweeds Chondrophycus papillosus and C. flagelliferus (Ceramiales) from Brazil.

Sulfated xylomannans were isolated from two species of genus Chondrophycus by aqueous extraction followed by KCl fractionation. Structural determination of the native, desulfated and Smith-degraded KCl-precipitated polysaccharides carried out by composition and methylation analysis and NMR spectroscopy (1D and 2D experiments) showed the following general structure: [see text] These xylomannans present different degrees of branching (15-25%) by beta-D-Xylp (70-80%) and beta-D-Manp-2-S (20-30%) and molecular weights (33-222kDa). This is the first report of the presence of a sulfated xylomannan in species of order Ceramiales.