Full Structural Characterization of an Extracellular Polysaccharide Produced by the Freshwater Cyanobacterium Oscillatoria planktothrix FP1

Full Structural Characterization of an Extracellular Polysaccharide Produced by the Freshwater Cyanobacterium Oscillatoria planktothrix FP1 - pdf for free download
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FULL PAPER DOI: 10.1002/ejoc.201000749

Full Structural Characterization of an Extracellular Polysaccharide Produced by the Freshwater Cyanobacterium Oscillatoria planktothrix FP1 Alba Silipo,*[a] Antonio Molinaro,[a] Monica Molteni,[b,c] Carlo Rossetti,[d] Michelangelo Parrilli,[a] and Rosa Lanzetta[a] Keywords: Carbohydrates / Configuration determination / NMR spectroscopy / Structure elucidation / Environmental chemistry Cyanobacteria, also known as blue-green algae or Cyanophyta, represent one of the oldest forms of life on Earth. The organisms exhibit a high degree of biological adaptation and comprise a large group of aquatic and photosynthetic oxygenic prokaryotes that obtain their energy through photosynthesis. Cyanobacterial extracellular polysaccharides are high-molecular-mass hetero-polysaccharides that are present on or released onto the cell surface and are characterized by high variability. In the present work we defined the structure of the extracellular polysaccharide produced by the fresh-

water cyanobacterium Oscillatoria planktothrix FP1. The structural determination, which was achieved by chemical, spectroscopic, and computational analyses, indicates a novel pentasaccharide repeating unit made up of glucose, mannose, and 2-deoxy-D-ribo-hexose, that has never previously been found in Nature. The elucidation of the structural and conformational features of the extracellular polysaccharide from Oscillatoria planktothrix is a first step toward understanding the biology of these important and interesting microorganisms.

Introduction

ute to the maintenance of ecosystem health and are a source of biologically active industrial and pharmaceutical products, however, they also produce a variety of toxins, some of which are implicated in the pathogenesis of severe, chronic, and potentially life-threatening diseases in humans. Cyanobacteria bear cell envelopes that resemble that of Gram-negative bacteria, consisting of an outer membrane located outside the cytoplasmic membrane and the peptidoglycan layer,[4,5] although structural differences can be highlighted. The peptidoglycan layer in cyanobacteria is considerably thicker than that of most Gram-negative bacteria and is complexed with specific polysaccharides. The lipopolysaccharides (LPS) of some cyanobacteria lack the typical components of the enterobacterial LPS;[4–6] it may contain only small amounts or be completely devoid of phosphate, but can also often lack heptose and Kdo (3-deoxy-manno-oct-2-ulosonic acid), the latter being always present in the LPS of Gram-negative bacteria. Frequently, cyanobacterial cells are covered by external surface layers such as S-layers and carbohydrate structures. In respect of carbohydrate structures, several cyanobacteria are able to synthesize and secrete extracellular polysaccharides,[7] which are usually associated with the outer surface of the bacterium and either covalently linked or loosely attached to the cell surface.[7,8] Such bacterial polysaccharides can, in fact, form an amorphous layer of extracellular polysaccharides (EPS) that are released onto the cell surface and surround the cell with no clear means of attachment, often forming a slime. In contrast, capsular polysaccharides are covalently linked to the cell surface of the bacteria through phospholipid mo-

Cyanobacteria, also termed blue-green algae, constitute a large family of phylogenetically coherent photosynthetic bacteria that also include chloroplasts, representing one of the oldest forms of life on earth,[1–3] and estimated to have existed for approximately 3.5 million years. As the first organisms to use oxygenic photosynthesis, cyanobacteria were key players in the early evolution of life. They have a high degree of biological adaptation, inhabit all types of aquatic environment, and are known to occur in marine water, in which they are a significant component of the nitrogen cycle, in fresh and brackish water, lake sand, arid areas, and are an important component of phytoplankton. Moreover, the presence and detrimental action of microorganisms on monuments and the stonework of art has received considerable attention in the last few years. Cyanobacteria contrib[a] Dipartimento di Chimica Organica e Biochimica, Università di Napoli “Federico II”, Complesso Universitario Monte S.Angelo, via Cintia 4, 80126 Napoli, Italy Fax: +39-081-674393 E-mail: [email protected] [b] Bluegreen Biotech s. r. l., Milano, Italy [c] Dipartimento Ambiente e Salute, Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy [d] Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell’Insubria, Varese, Italy Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ejoc.201000749. 5594

View this journal online at wileyonlinelibrary.com

© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Eur. J. Org. Chem. 2010, 5594–5600

Polysaccharides from Oscillatoria planktothrix FP1

lecules. Cyanobacterial polysaccharides can be homo- or hetero-polymeric and can also carry non-glycidic appendages such as phosphate, sulfate, lactate, acetate, and glycerol.[7] Often, they possess a significant level of hydrophobicity, due to the presence of acetyl groups, peptide moieties, and deoxysugars, which confer to the polysaccharide emulsifying properties and lead to some interesting rheological characteristics. Bacterial polysaccharides mediate direct interactions between the microorganism and its immediate environment and are involved in a number of biological processes; they also play a role in the virulence of many animal and plant pathogens. Furthermore, cyanobacterial polysaccharides act as protecting agents against dehydration due to their ability to entrap and accumulate water, creating a gelatinous layer around the cells that regulates water uptake and loss, thus stabilizing the cell during periods of desiccation, and protecting cells against phagocytosis, antibody recognition and lysis. They have also been implicated in several processes, such as metal chelation, due to the frequent presence of negatively charged sugars and substituents, or can even prevent direct contact with toxic heavy metals. Moreover, these polysaccharides can modify the rheological properties of water, act as thickening agents, and have the capacity to protect cyanobacterial cells from the harmful effects of UV irradiation; they also have a role in adhesion and locomotion and can protect cells from biomineralization processes. These polymers are of interest for potential biotechnological exploitation as, for example, viscosying or suspension agents in the food, cosmetic, and textile industries, for their capacity to modify the flow properties of water, and as metal ion sequestrating agents in the pharmaceutical industry.[7] In the present work, we have defined for the first time the structure and the solution conformation of the exopolysaccharide produced by the freshwater cyanobacterium Oscillatoria planktothrix FP1. This strain produces a polymer with a branched pentasaccharide repeating unit that is characterized by the presence of glucose, mannose, and 2-deoxy-ribo-hexose. The latter, to the best of our knowledge, is the first such naturally occurring example of its kind found in Nature. Due to the lack of reference compounds, its absolute configuration was determined by 2D NMR spectroscopy; the findings were supported by molecular mechanical and dynamic calculations[9,10,11] and by the application of Klyne’s rule.[12]

Structural Elucidation of Acid Hydrolyzed Product (OS) Due to the insolubility of the exopolysaccharide in aqueous solvent and considering the presence of an acid-labile deoxysugar, it was depolymerized under mild acid hydrolysis and purified by gel-permeation chromatography to give the oligosaccharide (OS) product. The monosaccharide analysis of OS gave the same results as for intact polymer, meaning that the compositional integrity of the sugar remained unchanged during chemical treatment. The oligosaccharide product was characterized by full 2D NMR analysis; the 1H NMR spectrum of the OS product is shown in Figure 1 (a). A combination of homo- and hetero-nuclear 2D NMR experiments (DQF-COSY, TOCSY, T-ROESY, 1 H,13C-HSQC, 1H,13C-HMBC) were conducted to assign all the spin systems and to define the saccharidic sequence. Complete assignment of the sugar residues was accomplished by attributing the proton resonances from the DQF-COSY and TOCSY spectra, and by subsequently

Results and Discussion Chemical Analysis The compositional analysis of the isolated exopolysaccharide from Oscillatoria planktothrix FP1 (Exo-Cy) showed the presence of mannose, glucose, and an unidentifiable 2deoxy-hexose. The absolute configurations of both glucose and mannose were determined to be  by GLC of the acetylated (S)-2-octyl glycosides and comparison with authentic standards. Methylation analysis showed the presence of 2,4-substituted-Manp, 4-substituted-Glcp, terminal glucose, and 4,5-substituted-2-deoxy-hexose. Eur. J. Org. Chem. 2010, 5594–5600

Figure 1. (a) 1H NMR spectrum (600 MHz) of the oligosaccharide (OS) derived from acid treatment of the exopolysaccharide of Oscillatoria planktothrix FP1. Key NMR signals are as indicated in Table 1; (b) Expansion of the anomeric region of the 1H NMR spectrum and of the F2-coupled HSQC spectra in which the diagnostic heteronuclear anomeric constants are shown.

© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjoc.org

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FULL PAPER

A. Silipo et al.

correlating each proton to its related carbon atom by reference to the HSQC spectrum. The anomeric configuration of each monosaccharide unit was assigned on the basis of the 3JH-1,H-2 coupling constant values obtained from the DQF-COSY experiment, the 1JC1,H1 constants (Figure 1, b)[13] derived from F2-coupled HSQC measurements, and were confirmed by the intra-residual NOE contacts observed in the T-ROESY spectrum (Figure...

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