Synthesis, preliminary characterization, and application of novel surfactants from highly branched xyloglucan oligosaccharides

Lionel Greffe2, Laurence Bessueille3, Vincent Bulone3 and Harry Brumer1,2

2 Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, 106 91 Stockholm, Sweden, and 3 Equipe "Organisation et Dynamique des Membranes Biologiques," UMR CNRS 5013, Bâtiment Chevreul, Université Claude Bernard Lyon I, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne cedex, France


1 To whom correspondence should be addressed; e-mail: harry{at}biotech.kth.se

Received on September 19, 2004; revised on November 3, 2004; accepted on November 3, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
A novel class of nonionic, carbohydrate-based surfactants has been synthesized from the plant polysaccharide xyloglucan. Enzymatic hydrolysis of xyloglucan yielded a series of well-defined, highly branched oligosaccharides that, following reductive amination, were readily conjugated with fatty acids bearing C8 to C18 chains under mild conditions. The critical micelle concentration, determined by tensiometry and dye-inclusion measurements, showed a typical dependence on acyl chain length and was sensitive to the degree of galactosylation of the head group. Several compounds from this new group of surfactants, especially those with C14 and C16 chains, were useful for the extraction of membrane-bound enzyme markers from different plant cell compartments in catalytically active form.

Key words: membrane protein extraction / reductive amination / surfactant / xyloglucan / xyloglucan oligosaccharides


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
The use of carbohydrates as polar head groups in amphiphilic surfactants has been widely studied, motivated in large part by industrial requirements for nontoxic, biodegradable products (Biermann et al., 1993Go; Chevalier, 2002Go; Hill and Rhode, 1999Go; Sarney and Vulfson, 1995Go; Steber et al., 1995Go). Furthermore, carbohydrates are attractive starting materials for chemical elaboration due to their status as a renewable resource (Holmberg, 2001Go; Von Rybinski, 1996Go). When conjugated with fatty acids, long-chain alkanols, or other natural hydrophobic species (Johansson and Svensson, 2001Go; Piispanen et al., 2002Go), the resulting compounds are appealing alternatives to surfactants of petrochemical origin. In particular, the alkylglycosides find widespread use in the food, hygiene, and cosmetics industries (Biermann et al., 1993Go; Hill and Rhode, 1999Go; Von Rybinski, 1996Go), as well as in research applications, such as membrane protein extraction (Garavito and Ferguson-Miller, 2001Go; Jones, 1999Go; Le Maire et al., 2000Go), due to their biological compatibility.

One of the most well-studied alkylglycosides, both in terms of general surfactant properties and ability to extract membrane proteins, is n-octyl-ß-D-glucopyranoside, in which a linear C8 hydrocarbon chain is linked via a glycosidic bond to the monosaccharide glucose. The diversity of reactions that can be used to modify the polyhydroxylated sugar ring, as well as the ability to control the regio- and stereoselectivity of those reactions, has resulted in the synthesis of a tremendous range of carbohydrate-based surfactants based on glucose alone (Augé and Lubin-Germain, 2000Go; Stubenrauch, 2001Go). Indeed, the industrial synthesis of octyl-glucoside, which is carried out using specific acid catalysis, gives rise to a mixture of glucose derivatives: glucosides of both anomeric configurations, {alpha} and ß, as well as longer octyl-glucooligosaccharides with mixed sugar linkages (Von Rybinski, 1996Go), each of which possesses unique surfactant properties (Persson et al., 2000Go).

The simple alkylglucosides continue to receive much attention regarding their physical/chemical properties due to their practical importance (Hoffmann and Platz, 2001Go). In particular, recent fundamental studies have sought to elucidate the behavior of the simple alkylglucosides in the context of other well-characterized nonionic surfactants, namely, those based on poly(ethene oxide) head groups (Ericsson et al., 2004Go). Interest in surfactants derived from sugars other than glucose has been extended to include other monosaccharides, as well as linear di-, tri-, and tetrasaccharides (Costes et al., 1995Go; Ferrer et al., 2002Go; Garofalakis et al., 2000Go; Kjellin et al., 2001Go; Plou et al., 2002Go; Söderberg et al., 1995Go). The use of branched oligosaccharide head groups has been little explored, presumably due to the difficulties associated with isolating or synthesizing pure compounds. In the latter case, complex protection/deprotection chemistry is required to control the regioselectivity of glycosylation reactions, which is particularly unattractive for large-scale industrial production.

Xyloglucan is a polysaccharide from the primary cell walls of dicotyledonous plants and is also a primary energy store in certain seeds (Vincken et al., 1997Go). The flour produced from the seed kernel of Tamarindus indica (tamarind) contains ~ 50% (w/w) xyloglucan (Shankaracharya, 1998Go), which is readily extracted and whose structure consists of a ß-(1->4) polyglucose backbone regularly substituted with {alpha}-(1->6) xylopyranose residues (Vincken et al., 1997Go). Enzymatic digestion of tamarind xyloglucan with an endoglucanase that specifically cleaves the glycosidic bond of unbranched glucose in the backbone yields four oligosaccharides based on a Glc4 chain: XXXG, XXLG, XLXG, and XLLG (Fry et al., 1993Go), in the ratio 13:28:9:50 (York et al., 1990Go). (Standard xyloglucan oligosaccharide nomenclature: X represents a Xylp({alpha}1->6)-Glcp unit, L represents a Galp-ß-(1->2)Xylp-{alpha}-(1->6)Glcp unit, and G represents a Glcp residue. When written sequentially, a ß-(1->4) linkage between the Glcp residues is implied, with the reducing end on the right.) Subsequent treatment with a ß-galactosidase can be used to reduce the complexity of the oligosaccharide mixture by selective or complete degalactosylation prior to chromatographic purification (York et al., 1993Go). The relative ease with which these well-defined, highly branched oligosaccharides are produced makes them convenient starting materials for the production of various derivatives (Brumer et al., 2004Go; Gustavsson et al., forthcoming).

Structure–function studies of membrane-bound proteins implicitly require nondenaturing detergents, that is, amphiphiles that can solublize protein structures while simultaneously maintaining properly folded, biologically active protein monomers or complexes. Sugar-based surfactants often satisfy these requirements, and new compounds with improved performance are desired to augment the range of currently available detergents (Chevalier, 2002Go; Gohon and Popot, 2003Go; Le Maire et al., 2000Go). Our interest in the derivatization of xyloglucan oligosaccharides (XGOs) for various applications (Brumer et al., 2004Go; Gustavsson et al., forthcoming), together with ongoing work in our laboratories on protein extraction from plant cell membranes (Colombani et al., forthcomingGo; Lai Kee Him et al., 2001Go, 2002Go; Pelosi et al., 2003Go), has motivated the study of amphiphiles containing highly branched sugar head groups. We present here the chemoenzymatic synthesis and initial characterization of a novel group of N-alkyl glyconamides derived from well-defined, natural oligosaccharides.


    Results and discussion
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 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Synthesis and physical characterisation of XGO-based surfactants
N-alkyl XGO aminoalditols (XGO-Cn) bearing alkyl chains containing 8 to 18 carbon atoms were conveniently prepared in good yield from tamarind seed xyloglucan by a sequence of endoglucanase digestion, reductive amination, and alkylation. The resulting amphiphilic products comprised a mixture of compounds that varied in the degree of galactosylation of the sugar head group according to the ratio of the Glc4Xyl3-based oligosaccharides present in the polysaccharide starting material (Figure 1). To obtain the pure N-steroyl XGO aminoalditols, the mixture of XGOs produced by endoglucanase digestion of tamarind xyloglucan was per-O-acetylated and fractionated on normal phase silica gel. The isomeric per-O-acetylated XLXG and XXLG were not resolved under the chromatography conditions and thus were not processed further. Deacetylation with NaOMe/MeOH, followed by reductive amination and N-alkylation, gave the products XXXG-C18 and XLLG-C18 in good overall yield.



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Fig. 1. XGO derivatives. Aminoalditols (R = H) may be selectively acylated to yield amphiphiles (R = CO(CH2)nCH3, n = 8, 10, 12, 14, 16, 18). XGO nomenclature: XXXG, x = 0, y = 0; XXLG, x = 0, y = 1; XLXG, x = 1, y = 0; XLLG, x = 1, y = 1.

 

To rapidly test the ability of the XGO-based amphiphiles to form micelles and to estimate the critical micelle concentration (CMC), simple dye-inclusion experiments were performed. The extinction coefficient of Coomassie brilliant blue G-250 at 620 nm is dependent on the hydrophobicity of its environment, and this property has been exploited for the determination of the CMC of nonionic surfactants (Rosenthal and Koussaie, 1983Go). As shown in Figure 2 and summarized in Table I, XGO aminoalditols bearing straight-chain N-acyl groups of 12, 14, 16, and 18 carbon atoms had CMC values in the range 3.7 to 0.01 g/L. Due to a limited amount of material, a full CMC determination was not performed for XGO-C10, although inspection of Figure 2 indicates that the CMC value lies close to the final measurement at 14.5 g/L. The CMC of XGO-C8 was too high to be determined practically. The molar CMC value may be approximated by using average molar masses of the XGO-Cn mixtures (Table I); CMC values thus range from 2.5 mM for XGO-C12 to 6.4 µM for XGO-C18. The standard free energy of micellization ({Delta}Gmic) was estimated using Equation 3 (see Materials and methods) and the molar CMC values for each of the XGO-Cn mixtures (Table I).



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Fig. 2. CMC determination of XGO-based surfactants by micellar inclusion of Coomassie brilliant blue G-250. Triangles: XGO-C18, circles: XGO-C16, squares: XGO-C14, stars: XGO-C12, diamonds: XGO-C10.

 

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Table I. Properties of XGO-based surfactants

 

Linear regression analysis (not shown) of {Delta}Gmic versus number of carbon atoms in the acyl chain indicates a contribution of –2.9 kJ/mol per methylene unit when XGO-C12, XGO-C14, and XGO-C16 are considered (r2 = 0.9996). A free energy contribution of ~ –3 kJ/mol per methylene group is typical for single chain surfactant homologs (Eastoe et al., 1996Go), although values closer to –2 kJ/mol per methylene unit have been observed for some sugar-based surfactants (Augé and Lubin-Germain, 2000Go). A comparatively worse fit (r2 = 0.9830) and lower free energy contribution (–2.5 kJ/mol per methylene unit) is obtained when the CMC of XGO-C18 is included in the analysis. The CMC of XGO-C18, which is in the low micromolar range, may thus be overestimated by the dye-inclusion method, if a normal dependence of CMC on chain length is assumed. As shown in Table I, the CMC value obtained by dye-inclusion for the homogenous surfactant XLLG-C18 was identical (0.0099 g/L, 5.9 µM) to that of the XGO-C18 mixture, whereas the CMC value of XXXG-C18 was significantly lower (0.0010 g/L, 0.75 M). "Interestingly, the CMC value for a monosteroyl XGO ester mixture ([Gustavsson, et al., forthcoming], analytical sample kindly provided by M. Gustavsson, Dept. of Biotechnology, KTH) was in the same range, 0.0046 g/l, as determined by dye-inclusion."

More detailed analysis of the surfactant properties of XGO-C14, XGO-C16, XGO-C18, XXXG-C18, and XLLG-C18 was performed by surface tensiometry (Figure 3). CMC values obtained from the break-points of the surface tension isotherms (estimated as the intersection of lines fitted to the descending and flat portions of the curves) were in good agreement with those obtained by the dye-inclusion method (Table I). The minimum surface tension, {gamma}min, was in the range 47–53 mN/m, which is remarkably high compared to many sugar-based surfactants (Augé and Lubin-Germain, 2000Go; Ericsson et al., 2004Go; Ferrer et al., 2002Go; Garofalakis et al., 2000Go; Kjellin et al., 2001Go; Persson et al., 2000Go; Söderberg et al., 1995Go; Waltermo et al., 1996Go). An anomalously high {gamma}min value can result when the Krafft point of the surfactant is passed at high concentration, which effectively results in the precipitation of the surfactant from solution. Clouding of the amphiphiles in this study was never observed, even in stock solutions of at least 10-fold higher concentration than the highest values shown in Figures 2 and 3. Thus it may be assumed that the apparent {gamma}min value reflects the true value. It is likely that the high observed minimum surface tensions result from poor packing of the bulky head groups. Consistent with a decrease in critical packing parameters (Holmberg et al., 2003Go) as a result of head group branching, XLLG-C18 is characterized by a higher CMC, higher minimum surface tension, and a less steep dependence of surface tension on concentration below the CMC than the homologous XXXG-C18. Notably, a slight but constant decrease in the surface tension is observed after the CMC for all surfactants studied, which may be indicative of the slow rearrangement of the bulky head groups at the surface. The lack of minima in the surface tension curves close to the CMC is an indication of the high purity of the surfactants (Persson et al., 2002Go).



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Fig. 3. Surface tension isotherms for XGO-based surfactants. Triangles: XGO-C18, circles: XGO-C16, squares: XGO-C14, +: XXXG-C18, x: XLLG-C18.

 

The maximum slope of the rapidly decreasing portion of the surface tension isotherm was used to calculate an apparent surface area per molecule for each of the surfactants based on the XGO mixture, as well as the homogenous surfactants XXXG-C18, and XLLG-C18. Although it is unclear how reliable such an approach is for surfactant mixtures, it can be clearly observed that the effective head group size of XLLG-C18, which bears two additional galactose residues on the Glc4Xyl3 core, is larger than that of XXXG-C18 (Figure 3, Table I). Similarly large areas per molecule have been reported previously for long-chain fatty acid esters of di-, tri-, and tetrasaccharides (Ferrer et al., 2002Go; Söderberg et al., 1995Go). Uncertainties in the determination of the CMCs of these two surfactants by the dye-inclusion and surface tensiometric methods make it difficult to judge the effect of the Gal units to the free energy of micellization, {Delta}Gmic, which is in the range 1.4–5.2 kJ/mol (Table I). Previously, the addition of galactose units to linear carbohydrate head groups has been shown to contribute 1.95 kJ/mol to {Delta}Gmic (Söderberg et al., 1995Go).

Application to membrane protein extraction
Carbohydrate-based surfactants bearing branched head groups are rare. To our knowledge, the only example so far described is the natural steroid glycoside, digitonin (Xyl-ß-(1->3)[Glc-ß-(1->3)Gal-ß-(1->2)]Glc-ß-(1->4)Gal-ß-(1->3)-digitogenin; Merck & Co., 1989Go). In addition to its well-known application as a cardiac stimulant, digitonin is routinely used for membrane protein extraction. In particular, digitonin efficiently extracts and preserves the activity of plant ß-glucan synthases, including callose ([1->3]-ß-D-glucan) synthases (Kudlicka and Brown, 1997Go; Okuda et al., 1993Go) and, in some instances, cellulose synthases (Colombani et al., forthcoming; Kudlicka et al., 1995Go, 1996Go; Okuda et al., 1993Go). Other detergents used for this purpose include 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) (Bulone et al., 1995Go; Colombani et al., forthcoming; Dhugga and Ray, 1991Go; Lai Kee Him et al., 2001Go; Li et al., 1997Go; Li et al., 2003Go; Sloan et al., 1987Go; Wu et al., 1991Go) and, less often, n-octyl-ß-D-glucopyranoside (Lai Kee Him et al., 2001Go). To explore the utility of the new surfactants obtained in the present work, the extraction of a number of plant membrane-bound enzymes was performed.

Callose synthase
Among the XGO-Cn compounds tested, XGO-C14 yielded the fraction with the highest activity (Figure 4). The preparations obtained with this detergent had levels of activity at least twice as high as CHAPS extracts but more than 50% lower than digitonin extracts (possibly due to both efficient extraction and activation of the enzyme by digitonin; Li et al., 1997Go). The length of the acyl chain of the XGO is critical to obtain optimal (1->3)-ß-D-glucan synthase activity. Indeed, detergents that bear either a shorter or longer chain than XGO-C14 yielded fractions with significantly lower activity (Figure 4). The importance of the length of the side chains for extracting plant callose synthases in an active form has been previously reported for the enzyme from Arabidopsis thaliana, using detergents from the sulfobetain family (Lai Kee Him et al., 2001Go).



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Fig. 4. Comparison of the levels of enzyme activities recovered in the different detergent extracts. Enzyme markers specific for plasma membrane (callose synthase and ATPase), Golgi (IDPase), endoplasmic reticulum (cytochrome-c reductase), and mitochondrial (cytochrome-c oxidase) membranes were assayed as indicated in Materials and methods. Activities are expressed as percent of activities measured in the control performed in the same conditions but in the absence of detergent. C12, C14, C16, C18 = XGO-C12, XGO-C14,XGO-C16 and XGO-C18, respectively.

 

ATPase
The levels of activity of the vanadate-sensitive ATPase in the various detergent extracts were also measured to verify whether XGO-Cn compounds can be used to extract other enzymes from the plasma membrane (Figure 4). The activities assayed with these compounds were systematically lower than that measured for the control performed in the same conditions but in the absence of detergent, which indicates that either the XGO-Cn compounds do not extract the enzyme from the plasma membrane or that they inactivate the enzyme during extraction. The relatively high levels of vanadate-sensitive ATPase activity (versus control) obtained using CHAPS and digitonin highlight the need to screen for detergents from different families to select those that are able to extract and preserve the activity of interest.

IDPase
The XGO-Cn detergents were also tested for their ability to yield active fractions of membrane-bound markers from intracellular compartments. In the case of the Triton-dependent IDPase, a marker for Golgi membranes (Nagahashi and Nagahashi, 1982Go), the levels of activity recovered in the XGO-C16 extracts were comparable to those obtained with CHAPS and digitonin and significantly higher compared with the activity measured in the control. The other XGO-Cn compounds tested were not able to extract the enzyme in an active form, with the possible exception of XGO-C18, which yielded a fraction with an activity only slightly higher than that of the control.

Cytochrome-c reductase and oxidase
Cytochrome-c reductase and oxidase were used as endoplasmic reticulum and mitochondria markers, respectively. The endoplasmic reticulum enzyme was extracted in an active form with XGO-C14, the level of activity in the detergent extract being slightly lower than in the case of CHAPS and digitonin but significantly higher than the control. The other detergents tested did not allow the recovery of fractions with a significantly higher activity than in the control. Interestingly, among all detergents tested, XGO-C16 was the most efficient to extract the mitochondrial cytochrome-c oxidase in an active form; even digitonin and CHAPS yielded extracts with lower activities.

Altogether, the data presented in Figure 4 show that some of the XGO-Cndetergents are at least as efficient for the extraction of specific membrane-bound proteins as the most commonly used CHAPS and digitonin. This is exemplified by the results obtained for IDPase (XGO-C16) and cytochrome-c reductase (XGO-C14). In the case of cytochrome-c oxidase, XGO-C16 is even more efficient than CHAPS and digitonin. XGO-C16 may therefore be a useful detergent for the study of mitochondrial enzymes, although such generalizations are tenuous without screening a wider number of activities. For the plant plasma membrane-bound (1->3)-ß-D-glucan synthase, XGO-C14 may find use as a more efficient detergent than CHAPS, which has been commonly used so far.

It is currently difficult to predict on the basis of structure and physical properties which surfactants will be most suitable for the extraction of a specific enzyme from a given cell compartment. It is certainly not only the structure of the surfactant that is important but also the lipid environment, stability, and molecular organization of the enzyme of interest. It is therefore difficult to rationalize the success or failure of a particular surfactant in an extraction experiment. The detergent extraction of active membrane proteins thus remains largely empirical, requiring the screening of a wide range of detergents, followed by selection of the most efficient. The XGO-Cn compounds described provide new tools for such studies.

Conclusion
A novel class of surfactants that bear bulky, highly branched oligosaccharide head groups has been readily synthesized from the plant polysaccharide xyloglucan. Highly specific enzymatic hydrolysis of xyloglucan followed by reductive amination allowed the regioselective acylation of the oligosaccharide products, either individually or in admixture, without the need for complex protecting group chemistry. The resulting amphiphiles exhibited remarkable surfactant properties, and several of them, especially XGO-C14 and XGO-C16, were useful for the extraction of specific membrane-bound enzymes from different cell compartments in catalytically active form. The facility with which well-defined XGOs may be obtained, together with the wide range of chemical and enzymatic methods available for carbohydrate alkylation, thus expands the range of surfactants derived from natural products for a variety of applications.


    Materials and methods
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 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Analytical procedures
Nuclear magnetic resonance (NMR) spectra were recorded at 298 K on a Bruker Avance 500 spectrometer. Proton chemical shifts ({delta}) are reported in ppm downfield from TMS; carbon chemical shifts are reported with reference to internal solvent. High-resolution mass spectra (HRMS, electrospray ionization [ESI+] mode) of polyhydroxylated compounds were recorded from 1:1 methanol/water, containing 0.5 mM NaCl, on a Micromass Q-Tof 2 (Waters, Micromass MS Technologies, Manchester, U.K.), essentially as previously described (Eneyskaya et al., 2003Go). For accurate mass determinations, centroid spectra were generated from time-of-flight (TOF) MS continuum spectra using the [M+Na]+ adduct of cellohexaose (ß-(1->4)Glcp6; Fluka, St. Louis, MO) as an internal standard (m/z 1013.3173). HRMS of per-O-acetylated compounds were recorded from 1:1 CHCl3/MeOH, containing 1% 50 mM NaCl(aq) (i.e., 0.5 mM NaCl final concentration); accurate mass determinations were obtained using the [M+Na]+ adduct of cellobiose octaacetate (Aldrich, Milwaukee, WI) as an internal standard (m/z 701.1905). Optical rotations were measured with a Perkin Elmer 341 polarimeter. Syntheses were monitored by analytical thin-layer chromatography with silica gel 60 F254 precoated plates (Merck, Darmstadt).

CMC determination by dye-inclusion using Coomassie brilliant blue G-250 was performed at 298 K, essentially as previously described (Rosenthal and Koussaie, 1983Go). Surface tension measurements were performed at 298 K with a Krüss K12 tensiometer, employing the Wilhelmy plate method. The measurements were carried out by using a sand-blasted platinum plate to ensure zero contact angle. The plate was cleaned using bichromosulfuric acid prior to each experiment and was rinsed with ethanol and dried with nitrogen gas between each measurement. The surface excess, {Gamma} (mol/m2), was calculated using the Gibbs adsorption isotherm,

(1)
where R is the ideal gas constant (8.314 J/[mol.K]), T is the absolute temperature, and d{gamma}/dln C was estimated from the slope of plots of surface tension, {gamma} (N/m), versus ln(surfactant concentration) as Capproaches the CMC. The cross-sectional area per molecule, A (m2), was subsequently calculated from

(2)
where NA is Avogadro’s number (Ferrer et al., 2002Go; Garofalakis et al., 2000Go; Kjellin et al., 2001Go; Söderberg et al., 1995Go). The standard free energy of micellization was calculated from CMCs determined by the dye-inclusion method using the relationship (Augé and Lubin-Germain, 2000Go; Eastoe et al., 1996Go)

(3)

The water used for the surfacant characterization experments was purified on a Millipore Milli-Q system and had a surface tension of 72 mN/m.

Synthesis of XGO-Cn, n = 8, 10, 12, 14, 16, 18
XGOs
Tamarind powder (D.N. Palani, Mumbai, India) (10 g) was vigorously stirred in water (500 ml) at 95°C until a homogenous mixture was obtained. The suspension was then cooled to 35°C, and crude cellulase from Trichoderma reesi (Fluka) was added (60 mg, 300 U). The resulting mixture was incubated at 35°C for 24 h with gentle stirring. The cellulase was removed by adsorption on activated charcoal (6 g) followed by centrifugation (40 min, 10,000 x g). The supernatant was filtered through celite, concentrated, and freeze-dried to give a mixture of XGOs (5.1 g) with the composition XXXG/XXLG/XLXG/XLLG 15:32:7:46 (mol/mol) (Henriksson et al., 2003Go). Average molar masses for compounds derived from this mixture were based upon the molar ratios of the individual components, which were similar to those reported previously (13:28:9:50; York et al., 1990Go).

XGO aminoalditol derivatives (XGO-NH2)
The XGO mixture (4.8 g, 3.8 mmol) was dissolved in saturated NH4HCO3 (aq) (100 ml). Following the addition of sodium cyanoborohydride (4.8 g, 76 mmol), the reaction was stirred at room temperature in the dark for 7 days (Bourquin et al., 2002Go; Fry, 1997Go). The reaction was then filtered to remove solid NH4HCO3, and acetic acid was added until the solution reached pH 3.5. After evaporation and coevaporation with methanol (3 x 50 ml), the crude product was redissolved in 1:1 HOAc/MeOH (50 ml) and precipitated with 1:1 Et2O/EtOAc (150 ml). The solid was collected by filtration, redissolved in deionized water, and purified on a cationic exchange column (Resin Dowex 50Wx4–400, gradient 0–5% NH3(aq)), yield 3.36 g, 70%. 1H, 13C NMR and HRMS data are available as supporting information.

General procedure for the acylation of XGO-NH2
Aliphatic acids were purchased from Aldrich (all were 99.5+% pure according to the supplier) and were used without further purification. XGO-NH2 (50 mg, average molecular mass 1174 g/mol, 42.5 µmol) was dissolved in dry DMF (625 ml). Solutions of aliphatic acid (0.168 mmol/ml, 250 ml, 1 eq.), DMAP (0.168 mmol/ml, 125 ml, 0.5 eq.), and EDC (0.168 mmol/ml, 500 ml, 2 eq.) in dry DMF were then added in sequence. The reaction mixture was stirred overnight and evaporated. The crude product was purified on a C18 reversed phase column (Supelclean ENVI18, Supelco Bellefonte, PA, USA) by stepwise elution with increasing concentrations of CH3CN in water. Product yields after purification are summarized in Table II; 1H, 13C NMR and HRMS data are available as supporting information.


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Table II. Yields of products from the acylation of XGO-NH2 with fatty acids

 

Production of monodisperse N-steroyl XGO aminoalditols XXXG-C18 and XLLG-C18
Characterization data (1H, 13C NMR, HRMS, optical rotation) for the purified compounds described next are available as supporting information.

Per(OAc)-XGOs
XGOs (4 g) were acetylated in 1:1.5 v/v acetic anhydride/pyridine (125 ml). After 14 h at 60°C, the reaction mixture was cooled to 0°C, quenched by the addition of MeOH (50 ml), and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (stepwise elution with toluene-acetone, 75:25 v/v, 65:35 v/v, and 50:50 v/v) to produce pure acetylated XGO per(OAc)-XXXG (416 mg) and per(OAc)-XLLG (1.9 g). Per(OAc)-XLXG and per(OAc)-XXLG (1.2 g) were not resolved under these conditions. The total mass of per(OAc)-XGOs obtained (3.5 g) corresponds to an overall yield of 50%.

XXXG
Sodium methoxide in methanol (1 M, 833 µl) was added to a solution of per(OAc)-XXXG (435 mg, 0.23 mmol) in methanol (125 ml). The mixture was stirred at room temperature for 3 h, neutralized with Amberlite IRN 120(H+) resin, concentrated, coevaporated with water, purified on C18 (Supelclean ENVI18, Supelco, Bellefonte, PA, USA) column, and freeze-dried to give XXXG (200 mg, 82%).

XXXG-NH2
XXXG (150 mg, 0.141 mmol) was dissolved in saturated ammonium bicarbonate solution (10 ml). Following the addition of sodium cyanoborohydride (150 mg, 3 mmol), the reaction was stirred at room temperature in the dark for 7 days. The reaction was then filtered to remove solid NH4HCO3, and HOAc was added until the solution reached pH 3.5. After evaporation and coevaporation with MeOH (3 x 50 ml), the crude product was purified on a cation exchange column (Resin Dowex 50Wx4–400, gradient 0–5% NH3(aq)), yield 75 mg (50%).

XXXG-C18
XXXG-NH2 (50 mg, 47 µmol) was dissolved in dry DMF (575 µl). Solutions of octadecanoic acid (0.168 mmol/ml, 287.5 µl, 1.05 eq.), DMAP (0.168 mmol/ml, 144 ml, 0.5 eq.), and EDC (0.168 mmol/ml, 575 ml, 2 eq.) in dry DMF were then added in sequence. The reaction mixture was stirred overnight and evaporated. The crude product was purified on a C8 reversed phase column (Supelclean ENVI8, Supelco, Bellefonte, PA, USA) by stepwise elution with increasing concentrations of CH3CN in water (52 mg; 83%).

XLLG
Sodium methoxide in methanol (1 M, 833 µl) was added to a solution of per(OAc)-XLLG (410 mg, 0.23 mmol) in methanol (125 ml). The mixture was stirred at room temperature for 3 h, then water was added to dissolve the precipitate formed during the course of the reaction. The reaction was worked up as described for XXXG, yield 206 mg (90%).

XLLG-NH2
XLLG-NH2 was produced from XLLG (145 mg, 0.104 mmol) exactly as described for XXXG-NH2, yield 81 mg (56%).

XLLG-C18
XLLG-NH2 (50 mg, 36 µmol) was dissolved in dry DMF (440 ml). Solutions of octadecanoic acid (0.168 mmol/ml, 221 ml, 1.05 eq.), DMAP (0.168 mmol/ml, 110 µl, 0.5 eq.), and EDC (0.168 mmol/ml, 441 ml, 2 eq.) in dry DMF were then added in series. The reaction mixture was stirred overnight and evaporated. The crude product was purified on a C8 reversed phase column (Supelclean ENVI8, Supelco, Bellefonte, PA, USA) by stepwise elution with increasing concentrations of CH3CN in water (50.3 mg; 84%).

Application of the XGO-Cn surfactants to the extraction of membrane-bound enzyme markers from various cell compartments
Source of enzymes
The XGO-Cn surfactants were tested for their ability to extract membrane-bound enzymes in an active form, using microsomal fractions from suspension-cultured cells of the hybrid aspen Populus tremula x P. tremuloides. Hybrid aspen cells (Colombani et al., forthcoming) were maintained as suspension cultures in a modified MS medium supplemented with sucrose (30 g/l), kinetin (20 mg/l) and 2,4-dichlorophenoxyacetic acid (1 g/l). They were grown at 24°C under agitation on a rotary shaker (130 oscillations per min) and successive 12-h night and day periods. The cells were harvested in exponential phase 14 days after inoculation of fresh medium and extensively washed with distilled water by vacuum filtration.

Preparation of microsomal membranes and detergent extraction of membrane-bound proteins
The following steps were performed at 4°C. Cells harvested as described were resuspended in 100 mM 4-morpholine propane sulfonic acid/NaOH buffer, pH 7.0, containing 2 mM ethylenediamine tetra-acetic acid and 2 mM ethylene glycol bis(2-aminoethyl ether)-tetra acetic acid (1 ml of extraction buffer per g of fresh cells). They were disrupted at 120 bars for 15 min in a cell disruption bomb (Parr Instrument, Moline, IL), and the microsomal fraction was isolated by differential centrifugation as previously described (Lai Kee Him et al., 2001Go). The pellet of membranes was resuspended in extraction buffer containing 10% glycerol. The protein content in the suspension was measured using the Bradford dye-binding assay (Bradford, 1976Go). The membrane suspension was then diluted to obtain a final protein concentration of 4 g/l. CHAPS, digitonin, and the XGO-C12, XGO-C14,XGO-C16, and XGO-C18detergents were used at 5 mg/ml, 10 mg/ml, 3.7 mg/ml, 0.45 mg/ml, 50 µg/ml, and 10 µg/ml, respectively, to extract the membrane-bound proteins. Extractions were performed under continuous stirring for 30 min in the presence of either of the six detergents tested. The preparations were then centrifuged at 150,000 x g for 1 h, and the supernatant was used as the source of solubilized enzymes, after protein determination as indicated.

Assay of enzymatic markers
Plant (1->3)-ß-D-glucan (callose) synthases are located in plasma membranes (Briskin et al., 1987Go; Quail, 1979Go). They catalyze the incorporation of glucose from UDP-glucose into a strictly linear (1->3)-ß-D-glucan, which is insoluble in 66% ethanol. They can be assayed by measuring the incorporation of radioactive glucose in the insoluble polymer using UDP-D-[U-14C]glucose as a substrate. The assay mixture used had a final volume of 200 ml and contained 50 ml of detergent extract, 100 mM 4-morpholine propane sulfonic acid/NaOH buffer (pH 7.0), 20 mM cellobiose, 8 mM CaCl2, 0.16 µM UDP-D-[U-14C]glucose (11,318 MBq/mmol), and 1 mM UDP-glucose (final concentrations). The reactions were stopped after a 45-min incubation by the addition of 400 ml absolute ethanol. After an overnight precipitation at –20°C, the insoluble radioactive polysaccharides were recovered by filtration on glass-fiber filters (GF/C, Whatman), which were then successively washed with 4 ml water and 66% ethanol. The radioactivity retained on the filters was measured in 4 ml scintillation cocktail, using a Wallac liquid scintillation counter.

The activity of Triton-dependent inosine diphosphatase (IDPase) was determined as described by Gibeaut and Carpita (1990)Go in the absence and in the presence of 0.01% (v/v) Triton X-100. The activity corresponding to the Golgi IDPase (Nagahashi and Nagahashi, 1982Go) is calculated by subtracting the activity measured in the absence of Triton to the one measured in the presence of the detergent. The release of inorganic phosphate was measured according to the method of Ames (1966)Go in the presence of 0.075% of sodium dodecyl sulfate.

The activity of vanadate-sensitive ATPase, a marker for plasma membrane (Gallagher and Leonard, 1982Go), was measured as described by Widell and Larsson (1990)Go, except that vanadate was used at 200 µM. All assays contained 1 mM sodium azide, 1 mM sodium molybdate, and 50 mM potassium nitrate as inhibitors of mitochondrial ATPase, acid phosphatase, and tonoplast ATPase, respectively. The release of inorganic phosphate was determined as indicated for the Golgi IDPase assay.

Cytochrome-c oxydase and the antimycin A-insensitive NADH-cytochrome-c reductase were used as mitochondrial and endoplasmic reticulum markers, respectively, and assayed as described by Briskin et al. (1987)Go.


    Acknowledgements
 
The authors express their gratitude to Prof. Tuula Teeri (Dept. of Biotechnology, KTH) for facilitating the collaboration between the Stockholm and Lyon groups. We thank Dr. Mark Rutland (Dept. of Surface Chemistry, KTH) for the use of surface tensiometry equipment, assistance with data analysis, and critical comments on the manuscript. We also thank Luis Bastardo and Marcus Persson (Dept. of Surface Chemistry, KTH) for experimental assistance. The P. tremulax P. tremuloides cell cultures used in this study were established by Drs. Torkel Berglund and Anna Ohlsson (Dept. of Biotechnology, KTH).


    Abbreviations
 
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate; CMC, critical micelle concentration; DMF, N, N-dimethyl formamide; DMAP, 4-(dimethylamino) pyridine; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI, electrospray ionization; HRMS, high-resolution mass spectrometry; NMR, nuclear magnetic resonance; TOF, time-of-flight; XGO, xyloglucan oligosaccharide


    References
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 Abstract
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 Results and discussion
 Materials and methods
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