Biomacromolecules 2010, 11, 3487–3494
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NMR Study on Hydroxy Protons of K- and K/µ-Hybrid Carrageenan Oligosaccharides: Experimental Evidence of Hydrogen Bonding and Chemical Exchange Interactions in K/µ Oligosaccharides Eric Morssing Vile´n,† Lena C. E. Lundqvist,† Diane Jouanneau,‡ William Helbert,‡ and Corine Sandstro¨m*,† Department of Chemistry, Swedish University of Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden, and Universite´ Pierre et Marie Curie, CNRS, Marine Plants and Biomolecules, UMR 7139, Station Biologique, BP 74, F29682 Roscoff Cedex, France Received August 24, 2010; Revised Manuscript Received October 20, 2010
The hydroxy protons of κ- and κ/µ-hybrid carrageenan oligosaccharides have been studied by NMR spectroscopy in 85% H2O/15% acetone-d6. Hydration and hydrogen bonding interactions in di- (κ), tetra- (κκ), hexa (κκκ), and octa- (κκκκ) κ-oligosaccharides and hexa- (κµκ), octa- (κµµκ), and deca- (κµµµκ) κ/µ-oligosaccharides have been investigated by measuring the chemical shifts, temperature coefficients, and chemical exchange of the hydroxy protons. These NMR parameters indicate that no strong and persistent intramolecular hydrogen bonds involving hydroxy protons stabilize the structure of κ-carrageenan oligosaccharides in aqueous solution. In the κ/µoligosaccharides, the presence of chemical exchange between OH3 of R-D-Gal-6-sulfate (D6S) and OH2 of β-DGal-4-sulfate (G4S) across the β-D-Gal-4-S-(1f4)-R-D-Gal-6-S linkage reveals the existence of a weak hydrogen bond interaction between the two hydroxyl groups. The smaller temperature coefficients of OH2_D6S and OH3_D6S indicate reduced hydration, interpreted as spatial proximity to the 4-sulfate group and O5 ring oxygen of the neighboring G4S residues, respectively. These first experimental results on the conformation of κ/µ-carrageenan oligosaccharides shine light on the potential role of “kinks” in the properties of the three-dimensional carrageenan gel network.
Introduction Carrageenans are found exclusively in Red Algal (Rhodophyta) cell walls. Carrageenans are linear sulfated galactans composed of (1-3) linked β-D-galactopyranose (G) and (1-4) linked R-D-galactopyranose (D).1 They are classified according to the presence of the 3,6-anhydro bridge on the 4-linked galactose residue (DA) and the position and number of the sulfate groups (S). The industrially most important carrageenans are the so-called iota (ι, G4S-DA2S), kappa (κ, G4S-DA), and lambda (λ, G2-D2S,6S).2 λ-Carrageenan is highly sulfated and is industrially used for its viscosity enhancer properties; ι- and κ-carrageenans produce thermoreversible gels and κ-carrageenan can give strong brittle gels.3 Carrageenan polysaccharides from natural sources are often of varying quality with respect to industrial applications and their usability therein. To chemically introduce a 3,6-anhydro bridge in carrageenans via alkali treatment or to enzymatically change the substitution pattern of the polysaccharide with sulfurylases give possibilities to tailor carrageenan properties and qualities. Structural features, such as sulfation patterns or the presence of an anhydro bridge, affect the conformation of the polysaccharides. These different conformations have, in turn, different physicochemical properties that make the poly or oligosaccharides suitable for different types of applications in industry. Therefore, it is important to recognize certain structural
features and the interactions within the oligosaccharide that they give rise to and then relate these to observed physical properties. The κ-carrageenan is composed of (1-3)-β-D-Gal-4-sulfate f (1-4)-R-D-3,6-anhydro-Gal repeating units. The biological precursor, mu-carrageenan (µ-carrageenan), is constituted of the repeating dimeric unit (1-3)-β-D-Gal-4-sulfate f (1-4)-R-DGal-6-sulfate. The dimeric unit in the different carrageenans is called carrabiose or neocarrabiose, depending on which residue is in the reducing end, in this case, κ- or µ-neocarrabiose, respectively (Scheme 1). Consequently, κ/µ-carrageenans consist of both these dimeric carrabiose units in a variable arrangement. Naturally occurring κ-carrageenans are not entirely made up of κ-carrageenan, but also contain traces of its precursor µ-carrageenan. µ-Carrageenans have reduced gel forming ability due to the 4C1-conformation of the R-D-Gal-6-sulfate sugar residue, which interrupts the repeating sequence of carrabiose units that form a helical tertiary structure.2 In a recent study,4 the structures of κ- and κ/µ-oligosaccharides produced by enzymatic digestion of carrageenans were resolved by NMR spectroscopy. Regarding the conformation and hydrogen bond interactions in κ-carrageenans only a few Scheme 1. Idealized Structures of µ-Neocarrabiose (A) and κ-Neocarrabiose (B)
* To whom correspondence should be addressed. E-mail: corine.
[email protected]. † Swedish University of Agricultural Sciences. ‡ Universite´ Pierre et Marie Curie. 10.1021/bm100994x 2010 American Chemical Society Published on Web 11/18/2010
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Scheme 2. Structures of the Mono- and Oligosaccharides 1-10
studies are available, most of them are molecular dynamic simulations (MDS), molecular modeling (MM), or density functional theory (DFT) studies.5-12 Others are done combining MDS or MM with NMR13,14 or combining MM calculations, NMR, and X-ray crystallography.15-17 Concerning µ-carrageenans, there is to our knowledge only one study available on the conformation of trisaccharide using MM38 while studies on the conformation of hybrid κ/µ oligosaccharides have not been reported yet. The presence of water can have a strong influence on the conformation of carbohydrates by hydrogen bonding to the sugar hydroxyl groups and by replacing carbohydrate-carbohydrate hydrogen bonds, and 1H NMR studies of exchangeable hydroxy protons has been proven useful to investigate these hydrogen bond interactions .18-27 To reduce the rate of exchange with water, hydroxy protons are usually studied at low temperature in a solvent mixture of H2O and acetone-d6. These conditions are used in association with a pulse sequence that efficiently suppresses the water signal without affecting the resonances of the exchangeable protons. Thus, in this work we have studied κ and κ/µ-carrageenan oligosaccharides, namely, di, tetra, hexa, and octasaccharide of κ, hexa, octa, and decasaccharide of κ/µ (Scheme 2). The respective constituting monosaccharides have also been studied. The chemical shifts (δ), chemical shift differences (∆δ), temperature coefficients (dδ/dT), rotating frame nuclear overhauser effect (ROE), and chemical exchange of the hydroxy protons have been measured in an attempt to gain insight into hydration, hydrogen bonding, and structural flexibility of these carrageenan oligosaccharides.
Experimental Section Samples. Carrageenan oligosaccharides were available from a previous study.4 Neocarrabiose-4-O-sulfate, neocarrabiose-6-O-sulfate, and methyl-3,6-anhydro-R-D-galactopyranose were purchased from Dextra Laboratories, U.K. The Na+ form of the carrageenans was used. A total of 5 mg of the oligosaccharides was dissolved in 100 µL of H2O/acetone-d6 85/15% and transferred into 2.5 mm NMR sample tubes. The sample tubes were, previous to sample addition, soaked in
Vile´n et al. sodium phosphate buffer, 100 mM and pH 7, and subsequently rinsed with milli-Q water.28 This was done to minimize the addition of glassware impurities to the samples. The pH of the samples was adjusted to optimum values for observation of hydroxy protons, that is, pH 6-7 by addition of diluted sodium hydroxide or hydrochloric acid solutions. NMR. The NMR experiments were performed on a Bruker DRX600 Avance III spectrometer using a 2.5 mm 1H/13C inverse detection probe equipped with a z-gradient. In all experiments, the suppression of the water signal was achieved using the Watergate water suppression scheme.29 The 1H NMR spectra were referenced by setting the residual acetone-d6 signal to δH 2.204 ppm. 1H NMR spectra were recorded at four different temperatures between -10 and +10 °C to obtain the temperature coefficients (dδ/dT) of the hydroxy protons. The chemical shifts and coupling constants were measured at -5 °C. DQF-COSY and TOCSY spectra were acquired using 2k or 4k data points in the F2 dimension and 256 increments in F1. A minimum of eight scans was used and the relaxation delay was set to 1-2 s. The TOCSY spectra were recorded with 15, 30, 40, and 60 ms mixing times. Spectra obtained with a mixing time of 60 ms were used for determination of δ and dδ/dT. ROESY and NOESY spectra were recorded with mixing times of 100 and 500 ms. The 2D spectral data was zero-filled one time in F2 and two times in F1 before applying a π/4 shifted sinesquared bell function in both dimensions, unless otherwise stated. Nomenclature. The abbreviation of the hydroxy protons is based upon a nomenclature used by Knutsen et al. in 1994.30 R-D-3,6Anhydro-Gal, β-D-Gal-4-sulfate, and R-D-Gal-6-sulfate are abbreviated as DA, G4S, and D6S, respectively. G4S is the reducing end sugar in all κ- and κ/µ-carrageenan samples. Hydroxy protons are numbered with the number of their respective ring carbon. Sugar residues are numbered according to their order in the chain, counting from the reducing end (Scheme 2).
Results and Discussion Six NMR parameters can normally be obtained from the observation of hydroxy protons: the chemical shifts, vicinal coupling constants, temperature coefficients, rate of exchange with water, dipolar interaction, and chemical exchange. These parameters give information on conformation, hydration, and hydrogen bonding. In this work, the coupling constants 3JCH,OH could not be measured except for disaccharide 1 (κ), tetrasaccharide 2 (κκ), and monosaccharide 8 (DA; Supporting Information, S1). In the larger oligosaccharides, spectral overlap, and broad hydroxy proton resonances precluded accurate measurement of 3JCH,OH. Also, the rate of exchange of hydroxy protons could not be obtained due to spectral overlap occurring in the 2D NOESY spectra of the larger oligosaccharides. Chemical shifts and temperature coefficients are however sufficient parameters to gain information on hydration and hydrogen bonding because they depend on both interactions with water molecules and intramolecular interactions. In strongly hydrated systems, such as carbohydrates, the chemical shift of a hydroxy proton signal is a balance between two opposite contributions: a downfield shift due to hydrogen bonding and an upfield shift due to reduced hydration.31 The temperature dependence of exchangeable protons is also commonly used as a tool for studies of intra- and intermolecular hydrogen bond interactions. Thus, the chemical shift of a hydroxy proton involved in a hydrogen bond or with reduced hydration is less affected by temperature changes due to decreased interaction with the solvent. Hydroxy protons with large |dδ/dT| (>11 ppb/°C) are fully hydrated, whereas hydroxy protons with |dδ/dT| (
