Partial and total C-6 oxidation of gelling carrageenans. Modulation of the antiviral activity with the anionic character

Highlights

• TEMPO (a nitroxyl radical) oxidizes ι-carrageenans exclusively on C-6.

• For κ-carrageenan oxidation of C-2 of 3,6-AnGal also occurs.

• The side reaction can be averted by further borohydride reduction.

• The optimal reaction conditions for TEMPO oxidation were found.

• Partial C-6 oxidation enhances the antiviral activity of κ-carrageenan.

Abstract

The optimal conditions for the full C-6 oxidation of κ- and ι-carrageenans using (2,2,6,6-tetramethylpiperidinyl)oxy (TEMPO) in the presence of sodium hypochlorite and sodium bromide were assessed. The fully oxidized products were characterized by NMR spectroscopy. Partially oxidized products were also obtained and analyzed by chemical and spectroscopical methods. The antiviral activity of carrageenans against herpes simplex virus HSV-1 and HSV-2 determined by plaque reduction assay, was not largely affected by full oxidation of the polysaccharides, but an increase in activity was detected by partial oxidation. A specific overoxidation on C-2 of the 3,6-anhydrogalactose moiety of κ-carrageenan was identified, solved experimentally and rationalized through the application of molecular modeling.

Introduction

Many natural polysaccharides have been used in the food industry, as well as in other applications (Stephen & Churms, 2006). Polysaccharides from marine sources (alginates, agarans and carrageenans) are especially chosen for industrial applications due to their availability, and their (usually) regular structure (Stortz & Cerezo, 2000). Carrageenans have structures based on linear chains of alternating 3-linked β-d-galactopyranosyl residues and 4-linked α-d-galactopyranosyl or 3,6-anhydrogalactopyranosyl residues, substituted with sulfate esters in different positions. The polyanionic characteristics of carrageenans allow them to carry many proven biological activities, such as antitumor (Bondu et al., 2010, Haijin et al., 2003;), anticoagulant (Carlucci et al., 1997, Wijesekara et al., 2011), and especially antiviral (Carlucci et al., 1997, Damonte et al., 2004, Talarico et al., 2004, Tischer et al., 2006).

Carrageenans are classified according to their idealized structure, and named by specific Greek letters. The most important gelling carrageenans (κ- and ι-) are 4-sulfated on the galactose moiety, have a 4-linked 3,6-anhydrogalactose moiety and differ only by the sulfation pattern of O-2 of this anhydro residue (sulfated in ι-, not sulfated in κ-).

A number of different chemical modifications of carrageenans have been carried out. The simplest and best known, even at an industrial level, is the alkaline treatment which converts by an intramolecular nucleophilic attack 6-sulfated α-d-galactose units into 3,6-anhydro-α-d-galactose moieties (Ciancia et al., 1993, Navarro and Stortz, 2005). Other modifications included oversulfation (Yuan et al., 2005), phosphorylation (Yuan et al., 2005), replacement of sulfate groups by seleniate groups (Campos, Kawano, da Silva Jr., & Carvalho, 2009), O-maleolylation (Jiang & Guo, 2005), and O-succinylation (Jiang, Guo, & Chen, 2007), used to increase and/or modify the anionic properties, and thus their interactions with biological receptors.

Oversulfation, phosphorylation, and introduction of spacers terminating in carboxyl groups were thus the most common ways to increase the anionic charge of carrageenans, and then improve their biological activities. A simpler way might be the oxidation of the primary hydroxyl group of the galactose units to generate a C-6 carboxyl group (galacturonic acid). The most successful reagent for this reaction has been (2,2,6,6-tetramethylpiperidinyl)oxy or TEMPO, a stable water-soluble nitroxyl radical which can be used in catalytic amounts by the addition of another oxidant (usually NaOCl or commercial bleach) which regenerates the nitroxyl oxidant (Bragd, van Bekkum, & Besemer, 2004). With an adequate pH regulation to avoid oxidation by the hypochlorite, selective C-6 oxidation was achieved for different polysaccharides like cellulose (Follain et al., 2008, Saito and Isogai, 2005, Xu et al., 2012) and starch (Bragd et al., 2001, de Nooy et al., 1995, Kato et al., 2003, ter Haar et al., 2010, Thaburet et al., 2001), among many others. Recently, the C-6 oxidation of agarose (Su et al., 2013), a polysaccharide related to carrageenan, was reported. The modified polysaccharides showed improved solubility properties (Chang & Robyt, 1996), metal chelating abilities (Muzzarelli et al., 1999, Saito and Isogai, 2005) and the possibility of introducing new functional groups through amidation (Follain et al., 2008, Su et al., 2013) or esterification (Muzzarelli et al., 1999). Besides, the biological activity was modified by the introduction of new anionic groups (Bae et al., 2011, Delattre et al., 2015, Elboutachfaiti et al., 2011, Wang et al., 2011).

Herpes simplex virus (HSV) types 1 and 2 may cause a broad range of human diseases, including oral and genital infections, keratoconjunctivitis and encephalitis, with different degrees of severity (Whitley & Roizman, 2009). As prolonged therapies with acyclovir, the most successful antiherpetic drug, have resulted in the emergence of drug-resistant variants (Piret & Boivin, 2011), the development of new compounds with different targets is required. In particular, sulfated polysaccharides like carrageenans have shown a potent anti-HSV activity (Damonte et al., 2004). Thus, the improvement of their antiviral effectiveness becomes of considerable interest.

With the aim of introducing modified materials with enhanced biological or functional properties, we have carried out a detailed study of the optimal conditions for achieving different degrees of C-6 oxidation of the κ-carrageenan from Hypnea musciformis (Cosenza, Navarro, Fissore, Rojas, & Stortz, 2014). We also report the study of a side reaction and its rationalization through molecular modeling, the extension of the reaction to ι-carrageenan, the chemical and spectroscopical characterization of all the products, and the assessment of the anti-herpes simplex virus (HSV) activity of fully and partly oxidized carrageenans.