Phase Behaviour of Nature-Like Branched-Chain

UMT 11th International Annual Symposium on Sustainability Science and Management 09th – 11th July 2012, Terengganu, Malaysia

Phase Behaviour of Nature-Like Branched-Chain Glycosides N. I. M. Zahid, R. Hashim, and T. Heidelberg Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia Corresponding author’s e-mail: [email protected] Abstract Glycolipids are remarkable biomolecule which become primary constituents of bio-membranes and may form various biologically relevance phases. Due to their diverse functions in nature, investigation on their self-assembly have obtained great attention from the fundamental point of view as well as for technological aspects. The glycolipid materials are highly in demand but, natural products such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) found in plant membrane are difficult to extract and purify in large quantity. Therefore, synthetic substitutes are preferred such as those of glycolipids, since these are cheap renewable resources, highly biodegradable, environmental friendly materials and have diverse biological activity. In this study, a branched-chain glycolipids based on Guerbet alcohol have been synthesized namely 2-hexyldexyl-α-D-mannopyranoside. The neat compound was measured with optical polarizing microscopy (OPM) and differential scanning calorimetry (DSC) whereas their lyotropic phase behaviour was investigated by contact penetration technique. α-Man-OC10C6 formed inverse hexagonal phase in dry form whereas in water, it gave inverse bicontinuous cubic phase. The presence of non-lamellar phases implies greater potential for the material (α-Man-OC10C6) to be used in several applications such as drug-carrier systems and membrane protein crystallization. Keywords: Glycolipid, liquid crystal, small-angle X-ray scattering, hexagonal phase, bicontinuous cubic phase. Introduction Glycolipids are a remarkable biomolecule which are minor constituents of biological membranes. They have been involved in numerous biological activities with a variety of functions for instance in molecular recognition events [1, 2], membrane fusion [3] and hosting microbial interactions [4]. Due to their diverse functions in nature, investigation on lyotropic mesophases of synthetic glycolipids have obtained great attention from the fundamental point of view as well as for technological aspects [5-9]. The glycolipid materials are highly in demand, but natural products such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) found in plant membrane are difficult to extract and purify in large quantity. Therefore, synthetic substitutes are preferred especially those which can be made from cheap renewable resources, highly biodegradable, environmental friendly materials and have diverse biological activity. In general, the synthetic glycolipids can be divided into two groups: naturally-mimicking membrane lipids analogue (usually with branching alkyl chain) [10, 11] and synthetic sugar-based surfactants APGs (usually with single alkyl chain) [12, 13]. Unlike the single alkyl chain surfactants/water systems (e.g. APGs) which are widely used in industrial products, the self-assembly properties of branched-chain glycolipids are suitable as model systems in understanding biological cell membranes behaviour. These lipids are known to exhibit liquid crystalline self-assembly behavior due to microphase separation of the polar head group from the non-polar hydrocarbon tails [14, 15]. The molecular structures possess a dichotomic nature , hence these are also called amphiphilic. Apart from their chemical structures which determine the liquid crystalline phase behaviour, the formation of these mesophases also depends on temperature only (thermotropic) or both temperature and concentration (lyotropic). By introducing chain branching in the hydrophobic alkyl tail of the synthetic glycolipids usually leads to the formation of inverse and curved (non-lamellar) phases, as reported by many groups with extensively variety of branched-chain design [10, 16-18]. The presence of non-lamellar phases implies greater potential for the material to be used in several applications such as drug-carrier

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systems and membrane protein crystallization. Previously, a class of branched chain glycolipids with asymmetric chain branching design was prepared and studied extensively [15, 19] for their properties [20, 21] and potential applications [22, 23]. In these studies, detailed molecular structure of especially the sugar headgroup was found to be very important in controlling the phase and application behaviour. Subtle changes in anomeric and epimeric isomers lead to pronounced changes in the observed phases [21]. Here, we report the preparation and phase characterization studies on an anomerically pure branchedchain glycolipids (2-hexyl-dexyl-α-D-mannopyranoside, α-Man-OC10C6, Fig. 1) synthesized using a glycosidation procedure from Guerbet alcohol reacting with mannose which is the C2 epimer of glucose. The aim of this study was to investigate the effect of C2 epimer on the liquid crystal phase behaviour. We had applied a number of physical techniques to obtain information on the phase behaviour, including the optical polarizing microscopy (OPM) for texture information, in both thermotropic and lyotropic conditions. The thermal properties of the gel to liquid-crystalline phases was measured using differential scanning calorimetry (DSC). OHOH O HO HO O

Fig. 1 Chemical structures of the 2-hexyl-dexyl-α-D-mannopyranoside (α-Man-OC10C6). Materials and Methods Preparation of the compound. The synthesis of the compound which involved three steps reaction were described elsewhere [15, 24]. The anomeric purity of the materials was confirmed by three different techniques namely nuclear magnetic resonance (NMR), thin layer chromatography (TLC) and optical rotation and is at least 98%. Optical Polarising Microscopy (OPM). The thermotropic liquid crystalline behavior was characterized by using a Mettler Toledo FP82HT hot stage and viewed with an Olympus BX51 microscope fitted with crossed polarizing filters. The microscope was connected to Olympus camera for image capture. A magnification factor of 20 was used. The material was heated until it reached isotropic point and the mesophase image formed was taken upon slow cooling at rate of 1°C min -1 for better texture. Lyotropic mesophase behavior was investigated via water penetration scan technique [25]. A small amount of the sample was placed on a glass slide and covered with a cover slip. The sample was heated to the isotropic point and cooled down to the room temperature. Then, a drop of water was placed on the slide at the edge of the cover slip and the water slowly moved into the sample to form a concentration gradient that ranged from pure water to neat surfactant by capillary action. Differential Scanning Calorimetry (DSC). The transition temperatures and enthalpies were determined by a Mettler Toledo DSC822e equipped with Haake EK90/MT intercooler. A rate of 5°C min-1 was used for both heating and cooling and the measurement was conducted with three cycles to ensure reproducibility results. The sample was weighed between 4-8 mg in an aluminum pans. The weighed sample was dried over phosphorus pentoxide placed under vacuum at 50°C for 48 hours before being covered to remove any moisture absorbed during preparation of the pan. The sample was then reweighed immediately before the measurement was started. Results and Discussion Thermotropic Phase Behaviour. In this section, the mesophase and self-assembly properties formed by α-Man-OC10C6 are described. Physically, α-Man-OC10C6 existed as yellowish gel-like syrup at the room temperature. The dry sample of α-Man-OC10C6 gave strong birefringence under OPM at the room temperature especially at the sample edge. Upon heating, the sample started to melt at 67 °C and become completely isotropic at 77 °C. On cooling the sample, the birefringent texture reappeared e-ISBN 978-967-5366-93-2

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at 73 °C and no further changes were obtained upon cooling the material down to the room temperature. The mesomorphic texture exhibited by α-Man-OC10C6 is shown in Fig. 2. The highly birefringent fan-shaped texture with larger homeotropic area is the characteristic of a hexagonal or columnar phase [26].

Fig. 2 The liquid crystalline texture at 30°C upon cooling for α-Man-OC10C6 (x 20). DSC confirmed the transition temperatures obtained from OPM for dry α-Man-OC10C6 (Fig. 3). Slight differences in temperatures between the DSC and OPM were due to different rates of decomposition in aluminum pans and glass slides [27]. α-Man-OC10C6 showed melting at around 75 °C with ΔH = 0.42 kJ mol-1. No other phase transitions were observed upon subsequent heating and cooling. We can conclude α-Man-OC10C6 only exhibits one mesophase. On cooling, one exothermic peak appeared at around 73°C. The enthalpy of the phase transition from the liquid crystal to the ^exo ID - alphamanno#3- 1 19012011b 19.01.2011 10:53:54 isotropic liquid is relatively small, indicating that the mesophase is disordered and liquidlike [27]. mW

Sample: A-manno 3-1 18012011b, 6.9900 mg Curve: A-manno 3-1 18012011b, 19.01.2011 10:48:22

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Integral normalized Onset Peak Left Limit Right Limit Heating Rate

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7.21 mJ 1.03 Jg^-1 74.79 °C 73.30 °C 76.10 °C 69.69 °C -5.00 °Cmin^-1

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STAR e SW 8.10 rate, 5 °C min-1) .

Lyotropic Phase Behaviour. The lyotropic phase sequences for α-Man-OC10C6 was determined via water contact penetration scans under OPM. This method provides a qualitative phase sequence with increasing water content but provides no quantitative information on concentration boundaries. The observed phase sequence for α-Man-OC10C6 with increasing water content is provided in Fig. 4. Upon addition of water at room temperature, a viscous isotropic cubic phase (QII) grew slowly to replace the inverse hexagonal phase (HII).

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H2 O QII HII

Fig. 4 Water penetration scans of α-Man-OC10C6 (x 20). Table 1: Thermotropic phase transition temperatures above 25 °C and lyotropic phases for α-ManOC10C6 , α-Man-OC10 and α-Glc-OC10C6. Compound α-Man-OC10C6 α-Man-OC10 [28] α-Glc-OC10C6 [21]

Thermotropic phases

Lyotropic phases

HII - L2 : 77 °C Lα - L1 : 149 °C Lα - L2: 47.5 °C

HII, QII Lα, QIG Lα, QII

In order to understand the molecular structure-property behaviour, we compared the phase behaviour of branched chain glycolipid (α-Man-OC10C6) to the single chain glycolipid (α-Man-OC10) of same number of carbon atom within the chain (10 carbon atoms). As shown in Table 1, the dry sample of straight chain α-Man-OC10 formed lamellar phase. It also have a higher clearing point compared to that of branched chain glycosides (i.e. α-Man-OC10C6 and α-Glc-OC10C6). It shows that the chain branching effectively reduce the clearing temperature, as expected compared to the alcohol analogue [29]. In hydrated sample, a normal bicontinuous cubic phase with a crystallographic space group Ia3d (QIG ) was formed for the α-Man-OC10 [28]. This implies that the straight chain glycolipid favours lamellar phase (in dry) and the normal liquid crystalline phases in aqueous. On the other hand the Guerbet glycosides with extra chain branching, hence larger chain hydrophobicity and critical packing parameter, CPP > 1 lead to the formation of reverse non-lamellar phases which are useful in many fundamental and technical fields [23, 30, 31]. The higher clearing temperature of α-Man-OC10C6 compare to α-Glc-OC10C6, suggests a greater intralayer hydrogen bond within the sugar headgroup region of the former compare to the latter [32]. Since α-Man-OC10C6 is the C2 epimer of α-Glc-OC10C6, being only slightly different at the C2 position where the hydroxyl group orients axially for the former and equitorially for the latter, implying that a small difference in the hydroxyl group orientation leads to a 30 °C difference in the transition temperatures. Morever, in the dry state α-Man-OC10C6 gives HII while α-Glc-OC10C6 only Lα. Thus, the effect of C2 epimer in α-Man-OC10C6 is to increase the propensity to form curve phases. For the lyotropic phase behaviour, the subtle change at the C2 position of mannoside and glucoside does not affect the structure of inverse bicontinuous cubic phase formed since both compounds gave the same QII phase in water. Conclusion In conclusion, we have shown that subtle change in molecular structure of C2 epimer (i.e mannoside and glucoside) is found to be very important in controlling the liquid crystal phase behaviour. Acknowledgement We are thankful to UM.C/HIR/MOHE/SC/11 and UM.C/625/1/HIR/MOHE/05 for the financial support.

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