Erminio Murano

Chemical structure and quality of agars from Gracilaria

Agar polymers synthesized by species of the genus Gracilaria constitute a complex mixture of molecules, containing several extremes in structure. Sulphate hemi-esters, methyl ethers and pyruvic ketals can alter in a number of ways the structural regularity of agar based on strictly 3-O-linked β-l-galactopyranose and 4-O-linked α-l-galactopyranose residues. In comparison with agars from Gelidium and Pterocladia, agars from Gracilaria can have higher degrees of sulphation, methoxylation and pyruvylation. The gelling ability of agars from most of Gracilaria species is considerably improved by adopting, before extraction, an alkali pretreatment which converts α-l-galactose 6 sulphate into 3,6-anhydro-α-l-galactose. Native agars obtained from Gracilaria cannot be classified, with few exceptions, as bacteriological grade agar as they have a high content of methoxyls and consequently high gelling temperatures. On the contrary, the genus Gracilaria is considered the most important source of food and sugar-reactive grade agars.Among techniques which can be used to study algal polysaccharides, combined 1H and 13C nuclear magnetic resonance spectroscopy represent the most effective and powerful method for the investigation of the chemical structure of agarocolloids.

Chemical and macromolecular characterisation of agar polymers from Gracilaria dura (C. Agardh) J. Agardh (Gracilariaceae, Rhodophyta)

Abstract Agar polymers were extracted with and without alkali pre-treatment from the red seaweed Gracilaria dura , growing in the northern Adriatic Sea. Chemical analysis combined with 1 H and 13 C NMR spectroscopy showed that agar polysaccharides characterised by low methoxyl and fairly high pyruvate contents were obtained. Alkali pre-treatment markedly reduced the molecular weight and significantly increased the polydispersity of the extracted polymers. The native and alkali-treated agars exhibited comparable gelling and melting temperatures; however, the alkali-treated sample had higher gel strength than that of the native one. Chemical structure, molecular weight and rheological properties of the extracted galactans are discussed with regard to the algal source.

Modified procedures for extraction and analysis of carrageenan applied to the red alga Hypnea musciformis

Dried powder of Hypnea musciformis was extracted with water at pH 7 after an initial short pre-treatment with cold, diluted HCl. Carrageenans were isolated by alcohol precipitation after an amylase treatment and a filtration of the extracts. The yields at 25 and 90 °C were 25 and 75% (w/w) of the dry alga, with molecular weights (Mw) corresponding to 194 000 and 245 000, respectively. The chemical structure was dominated by G4S-DA-(kappa-carrageenan or carrageenose 4′-sulphate). A simple fractionation procedure for kappa-carrageenase hydrolysates, based on stirring in different enthanol/water mixtures, is introduced. NMR analysis showed that oligosaccharides with a repeating DA-G4S structure were the main constituents in the enzymic hydrolysates of the carrageenans from Hypnea musciformis. These oligosaccharides were solubilized in an ethanol concentration from 96 to 48% (v/v). In some enzyme resistant fractions D6S-G4S and DA2S-G4S sequences and D2S,6S unites were detected by 13C-NMR.

Diffusion-Ordered NMR Spectroscopy in the Structural Characterization of Functionalized Carbon Nanotubes

The emerging applications of functionalized carbon nanotubes (CNTs) in various research domains necessitate the use of many different analytical techniques to confirm their structural modifications in a fast and reliable manner. Thus far, NMR spectroscopy has not been among the main tools for characterization of organically modified carbon nanostructures. (1)H analysis is limited because the signals in these derivatives are typically weak and broad, resulting in uncertainties of a few parts per million, and because of the strong interference of residual solvent signals. To overcome these limitations, we investigated the applicability of proton NMR spectroscopy based on gradient-edited diffusion pulse sequences (1D diffusion-ordered spectroscopy, DOSY) in the characterization of CNT derivatives. In general, diffusion NMR experiments allow the separation of NMR signals of different species present in a mixture, according to their own diffusion coefficients, merging spectroscopy information with size analysis. In the present study, a selected set of CNT derivatives was synthesized and analyzed using 1D DOSY experiments by applying strong magnetic field gradients (up to 42.6 G cm(-1)). Colorimetric tests (i.e., Kaiser test) and TGA analysis support the NMR findings, which are related to isolated and/or bundled short SWNTs, on the basis of TEM and AFM characterization. The overall results show that the diffusion-based NMR spectroscopy is a fast and promising approach for the characterization of covalently modified CNT derivatives.

Investigation of the carrageenans extracted from Solieria filiformis and Agardhiella subulata from Mar Piccolo, Taranto

Abstract Dried samples of the red algae Solieria filiformis and Agardhiella subulata from Mar Piccolo, Taranto, were extracted by stirring in water at 25 (Cold), 85 (Hot) and 120 (pressure) °C. For each species, six successive cold, three hot and two pressure extractions were performed and analyzed separately. Water-soluble polysaccharides were isolated after treatment with amylase, alcohol precipitation, dialysis and freeze drying. The total, starch-free yields were 27 and 24% of the dry weight for S. filiformis (SF) and A. subulata (AS), respectively. FTIR and NMR spectroscopic analysis showed that the different cold and hot water extracts had a similar structural backbone, which can be characteristic of gelling type carrageenans with a dominant iota repeating structure (DA2S-G4S). However, some irregularities were detected by NMR, which may be attributed to 6-sulphated 4-linked precursor units. The occurrence of such and other irregularities was more frequent in the SF samples, giving a possible explanation for the enhanced extractability in cold water of carrageenan polymers from this species.

Two-Dimensional Diffusion-Ordered NMR Spectroscopy as a Tool for Monitoring Functionalized Carbon Nanotube Purification and Composition

Functionalized carbon nanotube (CNT) derivatives are currently under thorough investigation in different biomedical investigations. In this field of research, the composition of sample either in terms of covalently attached or physisorbed moieties can greatly affect the observed results and hamper the comparison between different studies. Therefore, the availability of a fast and reliable analytical technique to assess both the type of interaction (covalent vs noncovalent) and the composition of CNT conjugates is of great importance. Here we describe that the two-dimensional diffusion-ordered (DOSY) NMR spectroscopy is extremely useful to discriminate between conjugated and unconjugated polyethylene glycol groups in samples obtained by condensation with oxidized single-walled carbon nanotubes (SWNTs). This fast and nondestructive technique allows us to follow the removal of unconjugated polyethylene glycol chains during the purification. In particular, DOSY analysis reveal that about 1/3 (wt %) of the polyethylene glycol used for the condensation remained physisorbed to functionalized SWNTs after dialysis. Complete elimination of physisorbed polyethylene glycol was achieved using diafiltration.

Synthesis of 6-amino-6-deoxyhyaluronan as an intermediate for conjugation with carboxylate-containing compounds: application to hyaluronan–camptothecin conjugates

A novel methodology for making drug conjugates using hyaluronan as a carrier was developed. This strategy involves a completely regioselective two-step synthesis of 6-amino-6-deoxyhyaluronan, which is then easily functionalized with drugs through a suitable linker. The case of hyaluronan-camptothecin conjugates is described, making use of a simple succinate linker. The antitumor activity of new hyaluronan derivatives prepared is at present under evaluation.

Evaluation of steam explosion as pretreatment in agar extraction fromGracilaria dura (C. Agardh) J. Agardh (Gracilariaceae, Rhodophyta)

Steam explosion was investigated as a pretreatment step in the isolation of agar from the macroalgaGracilaria dura. As compared to conventional procedures, the yield of agar obtained using this method on alkali (Na2CO3) conditioned algal material was higher. Extractions performed first at 95 °C and then at 121 °C showed that the major fraction of the agar was extracted at 95 °C, independently of the pretreatment. The efficiency of sulphate hydrolysis during steam explosion ofG. dura previously conditioned in Na2CO3, was similar to that of a NaOH based alkali pretreatment. Except for a lower nitrogen content of the sample obtained after NaOH based alkali pretreatment and a higher 6-O-methyl-β-d-galactose content in the agar after steam explosion, the chemical composition of the agars showed no significant difference. Agars extracted after steam explosion had melting temperature, gel strength and apparent modulus of elasticity lower than those of corresponding ‘native’ and alkali (NaOH) pretreated samples, but comparable to those of a commercial sample.

Hydrogen-bonding and conformation of agarose in methyl sulfoxide and aqueous solutions investigated by 1H and 13C NMR spectroscopy

Abstract The conformation of agarose in dimethyl sulfoxide (Me 2 SO) or in aqueous solutions has been studied by 1 H and 13 C NMR spectroscopy. Partial assignment of the 1 H NMR spectrum of agarose in Me 2 SO- d 6 has been obtained using 1D and 2D techniques. Coupling constant, chemical shift temperature dependence and exchange rate data for the hydroxyl protons have been used to establish the presence of intramolecular hydrogen bonds. The reported data show that the protons of HO-2 in the 3,6- anhydro-α- l -Gal p residue (A) and HO-4 in the β- d -Gal p residue (G) have a less rotational freedom and a higher thermal stability than the other hydroxyl protons. On the basis of the exchange rate data, obtained from 1D NOESY experiments with selective excitation of the residual water signal, an inter-residue hydrogen bond is proposed only for G HO-4 in Me 2 SO, while the involvement of A HO-2 in the agarose-Me 2 SO complex, recently reported, is suggested. The latter hypothesis is supported by the 13 C NMR data obtained in Me 2 SO- d 6 , in a Me 2 SO- d 6 -D 2 O mixture and in D 2 O, which also show that a relatively small conformational rearrangement of the chain segments occurs in aqueous solution. Deuterium isotope effect data indicate the persistence in water of the inter-residue G HO-4 mediated hydrogen bond with the additional involvement of G HO-6. In addition, the negligible isotope effect on the C-2 atom of A would indicate that the corresponding OH group, although free from the agarose-Me 2 SO complex, is not available for further strong interactions. The hydrogen bonding and conformation of agarose in methyl sulfoxide or in aqueous solutions were studied by NMR spectroscopy. Based on 1 H NMR data, an inter-residue H-bond is proposed for HO-4 of β- d -Gal p in Me 2 SO, while the involvement of HO-2 of 3,6- anhydro -α- l -Gal p in an agarose-Me 2 SO complex is suggested. These hypotheses are supported by the 13 C NMR data.

Lipid extracts from different algal species:1H and13C-NMR spectroscopic studies as a new tool to screen differences in the composition of fatty acids, sterols and carotenoids

One- and two-dimensional1H- and13C-NMR spectra of lipid extracts fromUlva rigida, Gracilaria longa, Fucus virsoides andCodium tomentosum collected in the northern Adriatic Sea allowed screening of the content of fatty acid chains, carotenoids, free and acylated cholesterol and chlorophylls. The carotenoid-to-polyunsaturated fatty acid molar ratio was taken as a comparison parameter in samples ofUlva rigida collected in differentloci and seasons; the value was markedly higher in samples from the Lagoon of Venice than from marine coastal waters. The total cholesterol concentration was evaluated by1H-NMR spectroscopy and similar values were found for all species. Two-dimensional heterocorrelated NMR spectroscopy was shown to give characteristic fingerprints of the lipid extracts from algal samples as regards the content in chlorophylls, unsaturated fatty acids and carotenoids.