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nAture methods  |  VOL.7  NO.12  |  DECEMBER 2010  |  1003

Pil Seok Chae1, Søren G F Rasmussen2, Rohini R Rana3, Kamil Gotfryd4, Richa Chandra5, Michael A Goren6, Andrew C Kruse2, Shailika Nurva5, Claus J Loland4, Yves Pierre7, David Drew3, Jean-Luc Popot7, Daniel Picot7, Brian G Fox6,8, Lan Guan5, Ulrik Gether4, Bernadette Byrne3, Brian Kobilka2 & Samuel H Gellman1

maltose–neopentyl glycol (mnG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

the understanding of integral membrane protein (imP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native imPs. many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. representatives of this maltose–neopentyl glycol (mnG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. mnG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

Integral membrane proteins (IMPs) play crucial roles in many aspects of biology by mediating the transfer of material and sig-nals between cells and their environment. It is estimated that 20–30% of all open reading frames in the human genome encode membrane proteins and that more than 50% of current pharma-ceutical agents target IMPs1. Our understanding of IMP structure and function, however, is hampered by difficulties associated with handling these proteins2. Most IMPs are not soluble in aqueous buffer because they have large hydrophobic surfaces when prop-erly folded; therefore, detergents are required to extract IMPs from the lipid bilayer and to maintain their native states in solu-tion. Mild detergents are widely used for IMP manipulation, but many membrane proteins tend to denature and/or aggregate when

1Department of Chemistry, University of Wisconsin–Madison, Madison, Wisconsin, USA. 2Molecular and Cellular Physiology, Stanford University, Stanford, California, USA. 3Division of Molecular Biosciences, Department of Life Sciences, Imperial College London, London, UK. 4Molecular Neuropharmacology Group, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. 5Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas, USA. 6Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin, USA. 7Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, Centre National de la Recherche Scientifique/Université Paris-7 Unité Mixte de Recherche 7099, Institut de Biologie Physico-Chimique, Paris, France. 8Center for Eukaryotic Structural Genomics, University of Wisconsin–Madison, Madison, Wisconsin, USA. Correspondence should be addressed to S.H.G. (gellman@chem.wisc.edu), B.K. (kobilka@stanford.edu) or B.B. (b.byrne@imperial.ac.uk).Received 25 May; accepted 30 SepteMbeR; publiShed online 31 octobeR 2010; coRRected online 9 noveMbeR 2010 (detailS online); doi:10.1038/nMeth.1526

solubilized with these agents3, making it difficult to conduct func-tional studies, spectroscopic analysis or crystallization trials.

Earlier efforts to develop amphiphiles tailored for IMP appli-cations have involved diverse strategies and achieved vary-ing levels of success. Several peptide-based designs have been explored (peptitergents4, lipopeptide detergents5, short pep-tide surfactants6) but so far have not gained broad acceptance. Amphiphilic polymers (“amphipols”7,8) and discoidal lipid bilay-ers stabilized by an amphiphilic protein scaffold (“nanodiscs”9,10) have proven to be versatile tools for studying IMPs in native-like states in aqueous solution. It is not clear, however, whether either of these approaches can yield high-quality crystals for diffraction analysis, a prominent objective of IMP studies. Furthermore, nei-ther amphipols nor nanodiscs are designed to extract IMPs from biological membranes. Recently reported agents of low molecular weight, such as hemifluorinated surfactants (HFS)8,11 and cholic acid–based amphiphiles12, have shown promising properties, but the scope of their utility remains to be explored. Thus there has been a need for amphiphiles that can extract, stabilize and pro-mote crystallization of IMPs more effectively than do current detergents. Amphiphiles with this combination of capabilities would have to be easily prepared on a large scale, which would be extremely challenging for peptide- or protein-based agents.

Here we report a class of amphiphiles that show favorable behav-ior with a diverse set of membrane proteins. The design of these amphiphiles features a central quaternary carbon, which is intended to place subtle restraints on conformational flexibility13–15. Because the quaternary carbon was derived from neopentyl glycol and because the hydrophilic groups in the examples discussed here are derived from maltose, we designate these compounds maltose–neopentyl glycol (MNG) amphiphiles. The quaternary

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critical micelle concentration (CMC) (Supplementary Fig. 2b). The most successful amphiphile was MNG-3. MNG-3 was superior to DDM as well in terms of maintaining a solubilized form of the muscarinic M3 acetylcholine receptor (M3AchR) in an active state (Fig. 2b). Together, the observations with β2AR-T4L and M3AchR raised the possibility that MNG amphiphiles will be generally use-ful for G protein–coupled receptor (GPCR) stabilization.

We turned next to melibiose permease (MelB), which catalyzes the accumulation of α-d-galactopyranosides by a cation-solute symport mechanism17. Treatment of membrane preparations con-taining overexpressed MelB (from Escherichia coli DW2 cells) with solutions containing 1.5 wt % amphiphile or detergent at 0 °C for 10 min quantitatively extracted MelB (Fig. 3). To assess protein thermostability, we incubated the solubilized samples on ice or at elevated temperatures for 90 min before ultracentrifugation. For the conventional detergents (MPA-1, MPA-3 and DDM), we observed MelB aggregation when the protein was incubated at 45 °C for 90 min, and the protein disappeared from solution when subjected to treatment at 55 °C or 65 °C followed by ultracentrifugation (Fig. 3). In contrast, all three MNG amphiphiles provided large amounts of soluble protein even after treatment at 55 °C. In particular, MNG-3 was unique in preventing aggregation at 55 °C.

We assessed the thermostabilities of additional membrane protein systems via a fluorescence assay. N-[4-(7-Diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM)18 can be selectively and covalently attached to side chain thiol groups of solvent-accessible cysteine residues. The maleimide unit of CPM quenches coumarin fluorescence; however, the coumarin unit becomes fluorescent after the maleimide reacts with a thiol. This assay provides insight on unfolding for membrane proteins that have buried cysteine resi-dues in the native conformation, because cysteine side chains that become exposed as a result of unfolding are reactive. We applied the CPM assay to two prokaryotic respiratory complexes, succi-nate:quinone oxidoreductase (SQR)19 and cytochrome bo3 ubiq-uinol oxidase (cytochrome bo3)20 from E. coli, and to the mouse cytidine-5′-monophosphate–sialic acid transporter (CMP-Sia)21. SQR was purified with the conventional detergent C12E9, and cytochrome bo3 and CMP-Sia were purified with DDM. The puri-fied membrane protein–detergent preparations were individually diluted into solutions containing an amphiphile at 10× CMC, and the unfolding in each protein sample was monitored over time at

carbon distinguishes MNG architecture from conventional deter-gent structures and enables the incorporation of two hydrophilic and two lipophilic subunits. We hypothesized that the modula-tion of flexibility and distinctive orientations of hydrophilic and lipophilic surfaces would give MNG amphiphiles properties distinct from those of analogous conventional detergents. These amphiphiles are readily synthesized. We have evaluated their performance with multiple membrane proteins in diverse applica-tions, including maintenance of native IMP folding, association and function, extraction from a native membrane, growth of high-quality crystals and support of cell-free translation.

resultsmnG amphiphile architectureWe performed extensive preliminary studies that identified MNG-1, MNG-2 and MNG-3 (Fig. 1) as showing particularly promising behavior. Each of these amphiphiles features two maltose units in its hydrophilic portion and two n-decyl chains in its lipophilic portion. The lipophilic unit attachment varies, with amide link-ages in MNG-1, ether linkages in MNG-2 and direct connection to the quaternary center in MNG-3. Synthesis of each compound was straightforward and efficient (Supplementary Note). We prepared analogs with conventional detergent architecture, MPA-1 to MPA-4 (for monopodal amphiphile), for comparison with MNG-1 and MNG-2 (Fig. 1). The comparison compounds for MNG-3 are commercially available—n-undecyl-β-d-maltoside (UDM) and n-dodecyl-β-d-maltoside (DDM, currently one of the most widely used detergents in membrane protein research); we also examined the lower homolog decyl-β-d-maltoside (DM) and the higher homolog tridecyl-β-d-maltoside (TDM), both of which are commercially available as well.

mnG amphiphiles stabilize diverse membrane proteinsWe first examined the thermal stability of a human β2 adrenergic receptor–T4 lysozyme fusion protein (β2AR-T4L)16 solubilized with an MNG amphiphile or conventional detergent. Stability was assessed via optical absorption measurements of β2AR-T4L bound to the inverse agonist carazolol (fluorescence emission maximum at 341 nm in the receptor-bound state and at 356 nm after carazolol release from the receptor). The receptor was initially solubilized and purified with DDM, which was then

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MNG-1: X = (CMC ~17 µM ; 0.0019 wt %)

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Figure 1 | Chemical structures of MNG amphiphiles (MNG-1, MNG-2 and MNG-3) and their linear counterparts (MPA-1, MPA-2, MPA-3, MPA-4, DM, UDM, DDM and TDM). The CMC value for each agent, measured via hydrophobic dye solubilization, is indicated in parentheses.

exchanged for the amphiphile or deter-gent to be evaluated. The 341 nm/356 nm peak-intensity ratio was used to monitor the relative amounts of intact and dena-tured β2AR-T4L (Supplementary Fig. 1). We evaluated the effect of amphiphile con-centration on the melting temperature (Tm) of β2AR-T4L (Supplementary Fig. 2 and Supplementary Table 1). All three MNG amphiphiles were superior to DDM and other conventional detergents (including DM and TDM) in their effects on β2AR-T4L thermal stability (Fig. 2a). The concentra-tion ranges that confer optimal stabilization in each case were similar in terms of weight percentage (between 0.05 and 0.1 wt %) but differed somewhat on a scale based on

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40 °C. In addition to DDM, we evaluated MPA-4, DM and sodium dodecyl sulfate (SDS). SDS is widely recognized to be highly dis-ruptive of native protein conformations, and this detergent caused the most rapid and extensive unfolding of each protein among the agents we examined (Fig. 4a and Supplementary Fig. 3). For each protein, the level of fluorescence observed with SDS after 130 min at 40 °C was taken to indicate a limiting denatured state, and a lack of fluorescence was taken to indicate a native state.

All three MNG amphiphiles appeared to be superior to con-ventional detergents at maintaining native protein structure, as indicated by CPM assay results for SQR (Fig. 4a) and comparable results for cytochrome bo3 and CMP-Sia (Supplementary Fig. 3a,b). DDM and MPA-4 were nearly as effective as the MNG amphiphiles, but DM was noticeably inferior. We next used gel filtration analysis to determine whether MNG-3 or DDM could maintain quaternary interactions among the four SQR subunits. After 120 min at 40 °C in the presence of 10× CMC DDM, the native quaternary structure of SQR was almost com-pletely destroyed (Fig. 4b). In contrast, the quaternary structure remained largely intact after 130 min at 40 °C in the presence of 10× CMC MNG-3 (Fig. 4c). We also compared MNG-3 and DDM in an SQR functional assay. SQR must be thermally activated (by incubation at 30 °C for 20 min) to remove bound oxaloacetate from the active site before assay. We incubated activated SQR with either MNG-3 or DDM at 40 °C and then measured the catalytic efficacy of the protein (kcat) immediately after activa-tion (0 min) and at 60-min intervals thereafter. Both MNG-3 and DDM gave fairly high initial kcat values, but SQR activity steadily declined with DDM, whereas activity was maintained or even slightly improved with MNG-3 (Fig. 4d). Overall, these results show that MNG-3 maintains the SQR quaternary assembly in a fully native state and that the MNG amphiphile is superior to DDM in stabilizing catalytically competent SQR.

We turned to the bacterial leucine transporter (LeuT)22 to eval-uate protein stability as a function of time at room temperature (rather than as an ability to resist thermal denaturation). LeuT, a bacterial member of the neurotransmitter:sodium symporter

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family (NSS family) proteins23, was solubilized and purified with DDM and then transferred into individual amphiphile solutions. We assessed LeuT activity in terms of its ability to bind [3H]leucine via scintillation proximity assay24. Preliminary studies, conducted at 10× CMC, indicated that LeuT solubilized with several conven-tional detergents showed a rapid decline in activity, whereas LeuT solubilized with DDM showed a more gradual loss of activity; all three MNG amphiphiles were superior to DDM in terms of maintaining LeuT activity over time (Supplementary Fig. 4a,b). We conducted further studies involving the MNG amphiphiles and DDM with each agent at 0.026 wt % above its CMC (Fig. 5a and Supplementary Fig. 4c). Under these conditions, each MNG amphiphile kept LeuT fully soluble and fully active over the 12-d study period. In contrast, LeuT activity declined to ~65% after 12 d in the presence of DDM (Fig. 5a).

mnG amphiphiles extract imPs from native membranesTo assess the ability of MNG amphiphiles to extract intrinsic proteins from their native membranes, we examined the pho-tosynthetic superassembly of Rhodobacter capsulatus25. These studies used membranes isolated from an R. capsulatus strain that lacks light-harvesting complex II (LHII)26; in this case the

Figure 2 | GPCR stability in MNG amphiphiles or conventional detergents. (a) Tm values of β2AR-T4L plotted in terms of wt % of the MNG amphiphiles (MNG-1, MNG-2 and MNG-3) or conventional detergents (MPA-1, MPA-3, DM, DDM and TDM). β2AR-T4L with bound carazolol (an inverse agonist) was incubated with various agents at the various concentrations at indicated temperatures for 5 min before fluorescence emission measurements. Normalized results are expressed as mean ± s.e.m. (n = 3, 4 or 5). (b) Specific activities (pmol mg−1) of M3AchR in DDM and MNG-3. The activity of the protein was evaluated after the protein was washed and eluted with buffer including DDM or MNG-3, but without CHS, via a binding assay involving the antagonist [3H]N-methylscopolamine, in the absence (t = 0 h, − CHS; first bar) or presence of CHS (t = 0 h, + CHS; second bar). The DDM- and MNG-3–purified M3AchR samples were stored at 4 °C for 15 h, and then activities were measured again in the presence of CHS (t = 15 h, + CHS; third bar). Results are expressed as mean ± s.d. (n = 3).

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Figure 3 | SDS-12% PAGE analysis and western blot detection of MelB. MelB samples were subjected to SDS-PAGE analysis, and MelB was detected by western blotting using anti–histidine tag antibody. Each sample contained 10 µg membrane proteins. For extracts generated with each detergent or amphiphile, one sample was subjected to ultracentrifugation (+) and a comparison sample was not (−). As a control, an untreated membrane sample (no ultracentrifugation) was included in each gel.

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875 nm/680 nm absorbance ratio declined over 20 d; however, at every time point, the ratio was higher for the samples solubilized with an MNG amphiphile than for the samples solubilized with a conventional detergent. Overall, the results with R. capsulatus photosynthetic proteins show that MNG amphiphiles can extract a protein quaternary structure intact from its native membrane and then provide superior structural stability over time relative to DDM or other conventional detergents.

We performed additional studies to evaluate the use of MNG-3 for extraction of other proteins from membranes. MNG-3 was comparable to the conventional detergents DDM and TDM, at 1 wt % or 2 wt %, for the extraction of wild-type β2AR from Sf9 insect cell membranes. We evaluated the activity of β2AR recep-tor via a binding assay with antagonist [3H]dihydroalprenolol (Supplementary Fig. 8a). At 1 wt %, MNG-3 was comparable to DDM and slightly inferior to TDM in terms of β2AR activity, but at 2 wt %, MNG-3 was superior to both conventional deter-gents. MNG-3 at 2 wt % yielded a receptor activity comparable to that of 1 wt % TDM. For extraction of LeuT from the bac-terial membrane, MNG-3 proved to be somewhat inferior to DDM, providing only ~60% of the yield obtained with DDM. However, MNG-3–purified protein showed substrate affinity identical to that of DDM-purified protein (Supplementary Fig. 8b). For extraction of a CMP-Sia fusion protein bearing

superassembly comprises the very labile light-harvesting com-plex I (LHI) and the more resilient reaction-center complex (RC). This system is well suited for assessing extraction and stabiliza-tion properties of detergents and amphiphiles because the super-assembly can be detected and its composition can be qualitatively monitored via optical measurements15: intact LHI-RC super-assembly has a strong absorbance at 875 nm and a 875 nm/760 nm absorption ratio >7. (Absorbance at 760 nm arises from bac-teriochlorophyll units that have dissociated from LHI.) We treated intracytoplasmic R. capsulatus membranes enriched in LHI-RC complex with solutions containing 1 wt % detergent or amphiphile for 30 min at 32 °C. MNG-2 and MNG-3 were effective at extract-ing the intact superassembly (strong absorption at 875 nm; 875 nm/760 nm ratio ~9–10; Supplementary Fig. 5). Comparable efficacy was observed with MPA-3 and DDM (875 nm/760 nm ratio ~8.5), but other conventional detergents were less successful. After purification of solubilized samples (Supplementary Fig. 6), we monitored the superassembly stability over time at room tempera-ture based on the 875 nm/680 nm absorbance ratio (absorbance at 680 nm arises from oxidation of bacteriochlorophyll that has been released from LHI). We compared the three MNG amphiphiles to conventional detergents (DDM and MPA-3), with each agent at its CMC (Fig. 5b; comparisons involving other concentrations may be found in Supplementary Fig. 7). For all samples, the

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Figure 4 | Stability of SQR solubilized with MNG amphiphiles or conventional detergents. (a) Results of CPM assays for SQR solubilized with MNG amphiphiles (MNG-1, MNG-2 and MNG-3) or conventional detergents (MPA-4, DDM, DM and SDS) at 10× CMC. The unfolding of the each protein was monitored at 40 °C for 130 min using a microplate spectrofluorometer. (b,c) Gel filtration analysis of SQR in DDM (b) or MNG-3 (c) at 10× CMC. SQR in DDM or MNG-3 was incubated for 120 min at 40 °C (AU, absorbance unit). (d) Time course of SQR activity in MNG-3 or DDM. Each agent was used at 10× CMC (0.01 wt % for MNG-3, 0.087 wt % for DDM) and 50× CMC (0.05 wt % for MNG-3, 0.44 wt % for DDM). Note that 50× CMC MNG-3 is comparable to DDM at 10× CMC in terms of wt %. The catalytic rate constant (kcat) is plotted as a function of incubation time. Data at t = 0 correspond to the activity of SQR following thermal activation performed at 30 °C for 20 min. Protein solubilized with each agent was incubated at 40 °C for a further 120 min, and activity of the protein was measured at the designated times. The kcat values at each time point were calculated by analyzing reaction data according to Michaelis-Menten kinetics.

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a b Figure 5 | Long-term stability of LeuT and R. capsulatus superassembly in MNG amphiphiles or conventional detergents. (a) Time course of activity ([3H]leucine binding) assay for LeuT solubilized with MNG amphiphiles (MNG-1, MNG-2 and MNG-3) and DDM at 0.026 wt % above the critical micelle concentration (CMC) (total concentrations: 0.035 wt % DDM, 0.028 wt % MNG-1, 0.027 wt % MNG-2 and 0.027 wt % MNG-3). LeuT activity was monitored at indicated time points, using a scintillation proximity assay (SPA), for protein stored at the room temperature. Results are expressed as % activity relative to the appropriate day 0 measurement. Normalized results are expressed as mean ± s.e.m. (n = 2). (b) Time course of stability of R. capsulatus superassembly purified with MNG amphiphiles (MNG-1, MNG-2 and MNG-3) or conventional detergents (MPA-3 and DDM) at 1 × CMC. The absorbance ratios (A875/A680) of the detergent or amphiphile samples were followed as a function of time.

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green fluoresc ent protein (GFP) at the C terminus, expressed in Saccharomyces cerevisiae, MNG-3 and DDM provided comparable protein yields (70–80%); in each case, the protein was intact and homogenous (Supplementary Fig. 8c). Overall, based on results from several different systems, MNG-3 appears to be comparable to DDM for extraction of membrane proteins from biological membranes.

We observed that MNG amphiphiles enabled the expression and concomitant solubilization of a membrane protein, bacterio-opsin (BO), from a cell-free wheat germ–based translation system (Supplementary Fig. 9). DDM and other conventional detergents could solubilize only limited amounts of translated BO at 0.1 wt %, and these detergents inhibited cell-free translation at 0.2 wt %. In contrast, 0.2 wt % MNG-2 or MNG-3 was compatible with translation and solubilized most of the BO.

mnG amphiphiles aid in membrane protein crystallizationGrowth of high-quality crystals that allow structure determination is one of the most important and challenging goals of membrane protein research. We examined crystallization of the cytochrome b6 f complex from Chlamydomonas reinhardtii with MNG-3. This protein assembly tends to denature in the presence of most conventional detergents, but DDM can maintain the native structure and has previ-ously enabled crystallization and structure determination of the com-plex via X-ray diffraction27. Cytochrome b6 f crystallization requires 0.2 mM DDM (CMC = 0.17 mM); lower detergent concentrations promote protein aggregation, whereas higher detergent concentra-tion lead to dissociation of subunits. We found that cytochrome b6 f in the presence of 0.5 mM MNG-3 showed stability comparable to that observed in the presence of 0.2 mM DDM. The tolerance for higher concentrations of MNG-3 relative to DDM provided a larger concentration window in which to attempt crystallization of solubi-lized cytochrome b6 f . Protein-containing crystals appeared within 24 h of setting up drops. After a few days, crystals reached a maxi-mum size of 70 × 400 µm (data not shown).

The diffraction data from cytochrome b6 f crystals grown with MNG-3 extended up to ~3.4-Å resolution (Fig. 6 and Supplementary Table 2), a value similar to that commonly obtained for crystals grown with DDM. With DDM, extensive screening yielded crystals diffracting to 2.8 Å27. Fourier differ-ence maps (of crystals with DDM27 versus crystals with MNG-3) showed no notable features in the protein region (data not shown). However, substantial differences (up to 6.8 σ) were observed in regions where detergent molecules have been localized in the crystals grown with DDM. These differences indicate that during the exchange of detergents, MNG-3 was able to displace the most

strongly bound DDM molecules. Further analysis by refinement of the protein structure starting from the DDM structure con-firmed that the electron density of two DDM molecules had van-ished and that a new molecule with a slightly different maltoside headgroup position, presumably MNG-3, occupied this position. This electron density, however, was not sufficiently well defined to allow model building of MNG-3, despite otherwise good refine-ment statistics for the protein (Supplementary Table 2).

Membrane protein crystallization from a lipidic cubic phase (LCP) is an increasingly popular strategy28 that has, notably, led to the recent structure solution of β2AR-T4L16,29,30. We evaluated the ability of MNG-3 to promote LCP-based crystallization of two new forms of this GPCR, a fusion protein with a covalently attached agonist (unpublished data) and an agonist-bound β2AR-T4L stabi-lized by an antibody in an active state (unpublished data). Although efforts to crystallize DDM-solubilized agonist-bound receptor from a monoolein-water LCP yielded crystals (data not shown), it was impossible to grow them large enough to obtain high-resolution diffraction. By contrast, in both cases detergent exchange into MNG-3 facilitated incorporation into the LCP, from which larger crystals suitable for X-ray diffraction analysis were obtained (data not shown). In the latter case, the crystals were ~40 × 5 × 5 µm, and X-ray diffraction data allowed solution of the structure to 3.5-Å resolution. We speculate that the enhanced stability of β2AR-T4L solubilized by MNG-3 relative to the DDM-solubilized form may be crucial for successful transfer of the protein into the LCP.

discussionOur results suggest that MNG amphiphiles will be generally useful for membrane protein biochemistry research. MNG amphiphiles can be readily prepared in multi-gram quantities, and this synthetic accessibility should enable their evaluation with many systems, including efforts directed toward structural analysis (for exam-ple, through nuclear magnetic resonance spectroscopy31 or mass spectrometry32) and the incorporation of MNG amphiphiles into new techniques for membrane protein purification and manipula-tion33. Given the diversity of shapes, sequences and properties of membrane proteins and their assemblies, it seems very unlikely that any single amphiphile will be ideal for all or even a large sub-set of membrane proteins. The ease with which MNG amphiphile structure may be varied should facilitate the development of a suite of agents that, collectively, have broad utility.

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Figure 6 | Image and X-ray diffraction pattern from crystals of cytochrome b6 f –MNG-3 complexes. X-ray diffraction by a cytochrome b6 f crystal obtained in the presence of MNG-3. Left panel represents a portion of the pattern (0.5° oscillation range). Resolution limits are marked with arrows (the white cross is due to the tiling of the detector). Top right, enlargement of the yellow square with two strong spots near the resolution limit. A section through the two strong spots is shown in the lower right corner (a.u., arbitrary unit).

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6. Zhao, X. et al. Designer short peptide surfactants stabilize G protein-coupled receptor bovine rhodopsin. Proc. Natl. Acad. Sci. USA 103, 17707–17712 (2006).

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Many important questions remain to be addressed in subse-quent studies. For example, it will be valuable to assess the sizes of micelles formed by MNG amphiphiles and how micelle size is affected by changes in amphiphile structure. In addition, it will be useful to determine average numbers of amphiphile molecules per micelle. But even before these properties are elucidated, we anticipate, based on this first characterization study, that the MNG amphiphiles will become useful tools for membrane pro-tein manipulation.

methodsMethods and any associated references are available in the online version of the paper at http://www.nature.com/naturemethods/.

Note: Supplementary information is available on the Nature Methods website.

AcknoWledGmentsThis work was supported by US National Institutes of Health (NIH) grant P01 GM75913 (S.H.G.), NS28471 (B.K.), by the Lundbeck Foundation (S.G.F.R., C.J.L. and U.G.), by the Danish National Research Council (C.J.L., U.G.), by the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. HEALTH-F4-2007-201924, EDICT Consortium (K.G., U.G. and B.B.) and by NIH grant GM083118 and NIH Protein Structure Initiative grants U54 GM-074901 (J.L. Markley, PI; B.G.F.) and U54 GM094584 (B.G.F.). This work was also supported by grant no. R21HL087895 from the US National Heart, Lung, and Blood Institute, by the Texas Norman Hackerman Advanced Research Program under grant no. 010674-0034-2009 (to L.G.) and by the Center for Membrane Protein Research, Texas Tech University Health Sciences Center. R.R.R. was funded by the Defence Science and Technology Laboratory. We thank P. Laible (Argonne National Laboratory, Chicago) for supplying membrane preparations from R. capsulatus. We acknowledge SOLEIL (Saint-Aubin, France) for provision of synchrotron radiation facilities, and we would like to thank B. Guimaraes for assistance in using the beamline Proxima 1. We also thank R. Kaback (University of California, Los Angeles) and G. Leblanc (Institut de Biologie et Technologies–Saclay) for the MelB expression system. M.A.G. acknowledges support from the US National Science Foundation East Asia and Pacific Summer Institutes Fellowship program. We thank G. Cecchini (University of California, San Francisco) and J. Ruprecht (Medical Research Council Mitochondrial Biology Unit, Cambridge) for the purified SQR and the details of the SQR functional assay, and we acknowledge the assistance of P. Nixon in the analysis of the SQR functional data.

Author contriButionsP.S.C. designed the MNG amphiphiles, with contributions from S.G.F.R., B.K. and S.H.G. P.S.C. synthesized the amphiphiles. P.S.C., S.G.F.R., R.R.R., K.G., R.C., M.A.G., A.C.K., S.N., Y.P. and D.P. designed and performed the research and interpreted the data. C.J.L., D.D., B.G.F., L.G., U.G., J.-L.P., B.B., B.K. and S.H.G. contributed to experimental design and data interpretation. P.S.C. and S.H.G. wrote the manuscript, with oversight from S.G.F.R., R.R.R., K.G., R.C., M.A.G., A.C.K., S.N., C.J.L., Y.P., D.D., J.-L.P., D.P., B.G.F., L.G., U.G., B.B. and B.K.

comPetinG FinAnciAl interestsThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemethods/.

Published online at http://www.nature.com/naturemethods/. reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.

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2. Lacapère, J.J., Pebay-Peyroula, E., Neumann, J.M. & Etchebest, C. Determining membrane protein structures: still a challenge! Trends Biochem. Sci. 32, 259–270 (2007).

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4. Schafmeister, C.E., Meircke, L.J.W. & Stroud, R.M. Structure at 2.5 Å of a designed peptide that maintains solubility of membrane proteins. Science 262, 734–738 (1993).

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radioligand using a Sephadex G-50 column (GE Healthcare). All binding reactions were performed in buffer containing 0.1% of the same detergent used in solubilization, and G-50 columns were likewise equilibrated and eluted in this buffer (100 mM NaCl, 20 mM HEPES, pH 7.5, 0.1% detergent). Nonspecific binding was determined in the same manner but with the addition of 10 µM alprenolol to the binding reaction. All binding reactions were performed in triplicate, and protein was diluted before binding such that bound radioligand never exceeded 10% of the total.

Muscarinic M3 acetylcholine receptor activity assay. M3 mus-carinic acetylcholine receptor with the third intracellular loop replaced by T4 lysozyme and an N-terminal Flag epitope tag added was expressed in the same manner as described above, but with the addition of 1 µM atropine to culture medium upon infection. Cells were lysed as described. Following centrifugation, the lysed cell pellet from 1 liter of culture was mixed with 200 ml M3 solubilization buffer (400 mM NaCl, 0.03% cholesterol hemi-succinate, 20 mM HEPES pH 7.5, 0.2% sodium cholate, 1% DDM, 5 µg ml−1 leupeptin, 320 µg ml−1 benzamidine). After being dounce homogenized 20 times with a tight pestle, the material was stirred at 4 °C for 1 h. 400 ml of dilution buffer containing 0.1% DDM, 20 mM HEPES pH 7.5, 30 mM NaCl, 3 mM CaCl2, 5 µg ml−1 leupeptin and 320 µg ml−1 benzamidine was added with stirring, and the mix was then centrifuged to remove insoluble material. The resulting supernatant was split in half and flowed over Flag affinity columns to bind the protein. Protein was then washed with either 50 ml of high-salt DDM buffer (0.1% DDM, 20 mM HEPES pH 7.5, 500 mM NaCl, 0.01% CHS 5 µg ml−1 leupeptin, 320 µg ml−1 benzamidine) or exchanged into MNG-3 buffer (0.1% MNG-3, 20 mM HEPES, 500 mM NaCl, <0.01% CHS, 5 µg ml−1 leupeptin, 320 µg ml−1 benzamidine). The exchange was performed over the course of 1.5 h by increasing MNG-3 and decreasing DDM concentrations in 0.005% increments until the final 0.1% concentration of MNG-3 was reached. Columns were then washed with 50 ml of DDM buffer without CHS or with MNG-3 buffer without CHS (with MNG-3 decreased to 0.01%), over the course of 50 min. These and all other wash buffers con-tained 2 mM CaCl2. Columns were eluted in washing buffer with 5 mM EDTA and no CaCl2 with 0.2 mg ml−1 Flag peptide added. Elution volumes for MNG-3 and DDM were identical. After elu-tion, binding reactions were performed as described above but using a saturating concentration of [3H]N-methylscopolamine (10 nM). Nonspecific binding was measured in the presence of 10 µM atropine. Protein assay was done as described above. All M3 binding reactions and G-50 elution with MNG-3 were performed at 0.01% concentration. Binding was measured again after overnight incubation at 4 °C.

MelB thermostability assay. Vector pK95 ΔAHB/WT MelB/CH6, encoding the wild-type MelB with a hexahistidine (His6) tag at the C terminus, and E. coli DW2-R cells (ΔmelB and ΔlacZY) were gifts from G. Leblanc (Institut de Biologie et Technologies–Saclay). Overexpression of MelB was performed as described34. Cells were harvested, resuspended in a buffer containing 20 mM Tris, pH 7.5, 200 mM NaCl and 10% glycerol, broken by French press and centri-fuged at 20,000g for 15 min to remove unbroken cells. Membranes were then harvested from the supernatant by ultracentrifugation at 43,000 r.p.m. for 3 h in the Beckman Ti 45 rotor. The pellets

online methodsDetergents and amphiphiles. We purchased conventional detergents (DM, UDM, DDM, TDM, LDAO, SDS and OG) from Anatrace. We obtained the starting materials and reagents used for preparation of MNG amphiphiles from Sigma-Aldrich. We used all of these agents without purification. Details of the syntheses of MNG amphiphiles can be found in the Supplementary Note.

b2AR stabilization and solubilization studies. The β2AR-T4L was expressed in Sf9 insect cells, solubilized and purified in DDM as previously described16. Briefly, the receptor was puri-fied by M1 Flag antibody affinity chromatography before and after an alprenolol-Sepharose chromatography step. Carazolol was bound to the receptor on the second M1 resin after exten-sive washing in HEPES low-salt buffer (0.1% DDM, 100 mM NaCl, 20 mM HEPES, pH 7.5) containing 30 µM carazolol. The eluted and carazolol-bound receptor was dialyzed against the same buffer containing 1 µM carazolol to reduce free cara-zolol concentration. The β2AR-T4L was spin concentrated to 7 mg ml−1 (~140 µM) using a 100-kDa molecular-weight-cut-off (MWCO) Vivaspin concentrator (Vivascience). For stability measurements, the carazolol-bound receptor was diluted below the CMC for DDM by adding 3 µl of the concentrated recep-tor in a quartz cuvette containing 600 µl buffer (100 mM NaCl, 20 mM HEPES, pH 7.5) with detergents and amphiphiles at various concentrations ranging from 1.5× to 250× CMC. The cuvette was placed in a Spex FluoroMax-3 spectrofluorometer (Jobin Yvon Inc.) under Peltier temperature control. Fluorescence emission from carazolol was obtained after 5-min incubations from 25 °C to 85 °C in 12 successive 5 °C increments. Excitation was set at 325 nm, and emission was measured from 335 nm to 400 nm with an integration time of 0.3 s nm−1 using a bandpass of 1 nm for both excitation and emission. The 341 nm/356 nm peak ratio was calculated and graphed using Microsoft Excel and GraphPad Prism software. For solubilization study from cell membranes, Sf9 cells were grown to a density of approximately 4 × 106 per ml and then infected with baculovirus expressing the wild-type β2AR (truncated after residue 365). After 48 h cells were harvested by centrifugation. No exogenous ligand was present during protein expression. Cells were resuspended, incubated in ice-cold lysis buffer (1 mM EDTA, 10 mM Tris, pH 8) contain-ing protease inhibitors (5 µg ml−1 leupeptin and 320 µg ml−1 benzamidine) for 5 min and then pelleted by centrifugation (10 min, 4 °C, 16,000g). The pellet was resuspended in PBS saline and aliquoted into 1.5-ml tubes so that each tube contained 35 mg of wet pellet after centrifugation and removal of supernatant. Solubilization buffers contained 100 mM NaCl, 20 mM HEPES, pH 7.5, 5 µg ml−1 leupeptin, 320 µg ml−1 benzamidine, and either 1 wt % or 2 wt % of the indicated detergent. 300 µl of solubiliza-tion buffer was added to each pellet, which was resuspended and homogenized by forcing the mixture through a 27-gauge syringe 30 times. Tubes were rocked at 4 °C for 1.5 h and then centri-fuged at 16,000g to pellet insoluble material. The supernatant was then checked for total protein content by using a Bio-Rad DC protein assay calibrated against a BSA standard. Measurements were performed in triplicate and then averaged. Active protein content was determined by incubating soluble material with the antagonist [3H]dihydroalprenolol at a single saturating concen-tration (10 nM) for 40 min and then separating bound from free

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were resuspended in the same buffer, frozen in liquid N2 and then stored at −80 °C until use. A protein assay was performed with a BCA kit (Thermo Scientific). For solubilization and thermostability assay, membrane samples containing MelB at a final protein con-centration of 10 mg ml−1 in a solubilization buffer (20 mM Tris, 200 mM NaCl, 10% glycerol, 20 mM melibiose, pH 7.5) were incu-bated with 1.5% (w/v) of a given amphiphile or detergent at 0 °C for 10 min and subsequently placed at a given temperature (0, 45, 55 or 65 °C) for 90 min. Samples were collected and then ultracentrifuged at 355,590g in a Beckman OptimaTM MAX ultracentrifuge using a TLA-100 rotor for 45 min at 4 °C. 10 µg protein, from untreated membrane or detergent extracts, and equal volumes of the solu-tions after ultracentrifugation were analyzed by 12% SDS-PAGE and immunoblotted with pentahistidine antibody conjugated to horseradish peroxidase (His5-HRP; Qiagen).

SQR, Cyt bo3 and CMP-Sia thermostability assay and SQR size exclusion chromatography (SEC) analysis and functional assay. The thermostability assay method was performed as described18 with the following minor modifications. CPM dye (Invitrogen), stored in DMSO (Sigma), was diluted in dye buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 0.03% DDM, 5 mM EDTA). All the detergents were used at 10× CMC values in test buffer (20 mM Tris (pH 7.5), 150 mM NaCl). The test proteins—SQR, Cyt bo3 and CMP-Sia (10 mg ml−1)—were diluted (1:150) in test buffer in Greiner 96-well plates, and 3 µl of diluted CPM dye was added to each test condition. The reaction was monitored for 130 min at 40 °C using a microplate spectrofluorometer set at excitation and emission wavelengths of 387 nm and 463 nm, respectively. Relative maximum fluorescence was used to calculate per-centage of relative folded protein remaining after 130 min at 40 °C. Relative unfolding profiles of proteins were plotted against time using GraphPad Prism. For SEC analysis, test protein sam-ples of SQR were diluted (1:100) in SEC buffer (20 mM Tris (pH 7.5), 150 mM NaCl) containing either DDM or MNG-3 at 10× CMC. Aliquots (500 µl) of the diluted protein were either applied directly onto the column or heated at 40 °C for 120 min before being loaded onto a Superdex 200 column (GE Healthcare) pre-equilibrated in the respective SEC buffers. For the functional assay of SQR, DDM and MNG-3 at 10× or 50× CMC were used in activation and assay buffers. SQR was activated in buffer (30 mM K2PO4, 0.2 mM EDTA, 10 mM malonate) to remove bound oxaloacetate from the active site. The sample was diluted to 1 mg ml−1 in activation buffer and then a further 50 times in the same buffer. Enzyme activation was performed at 30 °C for 20 min, aliquots were removed and then the samples were heated at 40 °C for 60 min and 120 min. The amount of functional SQR was estimated based on the succinate-Q1-DCIP reductase activity of the samples. Functional activity was monitored at 600 nm as a decrease of absorbance of 2,6-dichloroindophenol (DCIP) (extinction coefficient = 21.8 mM−1 cm−1) when 50 µM DCIP, 10 mM succinate, varied amounts of coenzyme Q1 (CoQ1) including blank and 0.6 µl of activated and/or heated SQR enzyme samples were added to assay buffers. The slopes of the absorbance curves (up to 20 min) were measured for a range of concentra-tions of CoQ1 (0 to 30 µM) and converted to initial velocity (V0) values using the Beer-Lambert law. The V0 values were plot-ted against CoQ1 concentrations for each detergent condi-tion to fit the Michaelis-Menten equation. The saturation

concentration of CoQ1 was obtained for each condition as 20 µM. The Michaelis-Menten equation was used to calculate catalytic parameters for SQR-catalyzed succinate-Q1-DCIP reductase activity. The specific activity (kcat) for SQR was calculated from 1-ml assay as follows. On addition of the enzyme, the change in absorbance over the initial 3 min of the reaction at 20 µM CoQ1 was converted to rate of the reaction using the Beer-Lambert law. Because this rate of the reaction at 20 µM CoQ1 is also the Vmax, the kcat values were calculated as rate per amount of enzyme.

CMP-Sia solubilization. Expression and solubilization of CMP-Sia was performed as described previously35. In brief, the CMP-Sia was expressed as a fusion protein with a C-terminal GFP in FGY217 Saccharomyces cerevisiae cells. Membranes, generated as described previously31 were resuspended in 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.6 M sorbitol and the protein concentration measured. The membranes were adjusted to a concentration of 1 mg ml−1 and 1-ml aliquots were solubilized individually in DDM and MNG-3 at final detergent concentrations of 0.5 wt %, 1.0 wt %, and 2.0 wt % for 1 h at 4 °C. 100-µl aliquots were removed from each tube, and a fluorescence reading was taken for each sample before and after ultracentrifugation at 150,000g for 1 h to remove insoluble material. The solubilization efficiency (%) is the fluorescence reading of the soluble supernatant divided by the fluorescence reading of the total sample times 100. The remaining soluble fraction for each condition was submitted to fluorescent SEC (FSEC) as described previously36 using a Superose 6 column (GE Healthcare) equilibrated with buffer containing the appropriate agent (DDM or MNG-3).

LeuT solubilization, solubility and functionality assay. The wild-type version of the leucine transporter (LeuT) from Aquifex aeolicus in pET16b (pET16b-LeuT-His8) was expressed in Escherichia coli essentially as described22. The plasmid was kindly provided by E. Gouaux (Vollum Institute, Portland, Oregon, USA). LeuT was purified from isolated cell membranes solubilized with 1% DDM and was then subjected to Ni2+-affinity chroma-tography using Chelating Sepharose Fast Flow (GE Healthcare) and elution in buffer composed of 20 mM Tris-HCl (pH 8.0), 1 mM NaCl, 199 mM KCl, 0.05% DDM and 300 mM imidazole. For comparison, in the solubilization assay, LeuT was extracted and eluted using MNG-3 in the final concentration of 1% and 0.042%, respectively. In the solubility and functionality assay, selected LeuT fractions eluted in the presence of 0.05% DDM were pooled, divided into aliquots and diluted in the above-mentioned buffer without DDM but containing MNG amphiphiles or con-ventional detergents to the final concentration of 10× CMC or 0.026 wt % above their CMC values (OG was tested at 2× CMC). After incubation at the room temperature, samples were centri-fuged, and the protein concentration was determined at the indi-cated time points based on absorbance at 280 nm. Concomitantly, at the corresponding time points, [3H]leucine binding was assessed by scintillation proximity assay (SPA)24. Briefly, each SPA reaction mixture consisted of 6 µl from the respective samples, 33.3 nM [3H]leucine (PerkinElmer) and copper chelate (His-tag) YSi beads (GE Healthcare). SPA was performed in the presence or absence of 1 × 10−5 M leucine in 20 mM Tris-HCl (pH 8.0) sup-plemented with NaCl and the selected amphiphile or detergent to final concentrations of 200 mM and 3.3-fold dilution of the origi-nal concentrations, respectively. In the solubilization assay, the

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with DDM. Crystals were flash frozen in liquid nitrogen, and diffraction experiments were performed at the synchrotron SOLEIL (Saint-Aubin, France) on the beamline Proxima 1. Diffraction data were integrated with XDS37 and analyzed with the CCP4 package38. The model was refined with PHENIX39 and BUSTER40. Plastoquinone/plastocyanine oxidoreductase activity measurements were performed according to the literature41.

Cell-free expression of proteins in the presence of amphiphiles. Transcription and translation of genes cloned into the pEU-HIS cell-free expression vector was performed as previously reported42. Briefly, for each sample, a 50-µl transcription reaction containing 5 µg plasmid DNA, 80 mM HEPES, pH 7.5, 16 mM magnesium acetate, 2 mM spermidine, 10 mM dithiothreitol, 2.5 mM of each nucleotide triphosphate, 25 U RNasin (Promega) and 30 U Sp6 RNA polymerase was incubated at 37 °C for 3 h. The resultant mRNA was purified by ethanol precipitation and air-dried. The pellet was resuspended in 50 µl of the translation reaction solu-tion, which contained dialysis buffer (30 mM HEPES, pH 7.8, 100 mM potassium acetate, 2.7 mM magnesium acetate, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.4 mM sper-midine and 0.3 mM of each amino acid) supplemented with 35.6 µg creatine kinase, 24 U of RNasin and 15 µl of WEPRO 2240 (Cell Free Sciences). The reaction was transferred into a 12-kDa-MWCO dialysis cup (Cosmo Bio) suspended in a reser-voir of dialysis buffer and incubated for 16 h at 26 °C. Detergents and MNG amphiphiles were added when needed at 0.1 wt % or 0.2 wt % to both the translation reaction and dialysis buffer reservoir. After overnight incubation, the 50 µl translation reac-tion solution was transferred to a 1.7-ml centrifuge tube and spun at 15,000 r.p.m. in an Allegra 21R centrifuge (Beckman Coulter) and F2402H rotor at 20 °C for 10 min. The supernatant was removed and added to an equal volume of 2× sample buffer, while the pellet was resuspended in 100 µl 1× sample buffer. After being boiled for 5 min, the samples were loaded onto an SDS-PAGE gel, electrophoresed and visualized by Coomassie staining.

initial activity of LeuT was assessed by SPA performed in the pres-ence of 0.05% DDM or 0.042% MNG-3 and leucine (0–10−5 M, competition binding). SPA was performed with duplicate deter-mination of all individual data points. Samples were counted in a MicroBeta liquid scintillation counter (PerkinElmer). Normalized results are expressed as mean ± s.e.m.

R. capsulatus superassembly solubilization and stabilization assay. Specialized photosynthetic membranes from an engineered strain of R. capsulatus, U43[pUHTM86Bgl], lacking the LHII light-harvesting complex were used as the starting material26. The isolated flash-frozen membrane aliquots from this strain were thawed, homogenized and equilibrated to 32 °C for 30 min. The amphiphiles or detergents were added at the designated con-centration to 1-ml aliquots of the membranes. After incubation with the amphiphiles for 30 min at 32 °C, the solubilized material was separated from the membrane debris in an ultracentrifuge at 315,000g at 4 °C for 30 min. The supernatant was transferred into a new microcentrifuge tube containing Ni-NTA resin (Qiagen), and then the tube was incubated and inverted for 1 h at 4 °C. Once binding was complete, samples were loaded onto resin-retaining spin columns (for example, emptied His SpinTrap columns; GE Healthcare), which were then inserted into 2-ml microcentrifuge tubes. Samples were washed twice with 0.5 ml of amphiphile-containing binding buffer (a pH 7.8 Tris solution containing the amphiphile at 1× CMC, 0.017 wt % above CMC, 0.2 wt % or 1.0 wt %). Finally, protein was eluted with three 0.2-liter elution buffer aliquots (this buffer was identical to binding buffer with the addition to 1 M of imidazole with same pH). Purified protein was diluted with 0.4 ml of binding buffer to reach the final sample vol-ume to 1 ml. Small aliquots (0.2 ml) of the solutions were trans-ferred to 0.8 ml binding buffer, and UV-visible spectra of these solutions were measured as a function of time. Degradation of the material could be monitored with the 875 nm/680 nm absorbance ratio, which decreased with time.

Stabilization and crystallization of cytochrome b6 f /MNG-3 complex. Purification of the complex cytochrome b6 f from Chlamydomonas reinhardtii was carried out as described in the literature27 with slight modifications to exchange DDM for MNG-3. Briefly, after solubilization of thylakoid mem-branes with DDM and a first ion exchange chromatography in 0.4 mM DDM, the protein was loaded on a 1-ml HiTrap chelating Sepharose column (GE Healthcare), preloaded with nickel (Ni2+); subsequently, the column was extensively washed with 50 ml of 0.5 mM MNG-3 in 20 mM Tris-HCl, pH 8, 250 mM NaCl and eluted with 300 mM imidazole in the same buffer. The sample was desalted on a Sephadex G-25 column (GE Healthcare) with 10 mM Tris-HCl, pH 8, 0.5 mM MNG-3 and concentrated with a Vivaspin 500 (100,000 MWCO PES) concentrator (Sartorius) from 5 to 100 µM (that is, from 0.5 to 10 mg ml−1) before crystal-lization. All steps from purification until crystal handling were carried out at 4 °C in a cold room. Crystallization trials used the protocol involving vapor diffusion with the hanging-drop technique: the protein was mixed with an equal or half volume of reservoir solution devoid of surfactant27. The crystallization strategy was adapted from conditions suitable for crystallization

34. Pourcher, T., Leclercq, S., Brandolin, G. & Leblanc, G. Melibiose permease of Escherichia coli: large scale purification and evidence that H+, Na+, and Li+ sugar symport is catalyzed by a single polypeptide. Biochemistry 34, 4412–4420 (1995).

35. Drew, D. et al. GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat. Protoc. 3, 784–798 (2008).

36. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).

37. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

38. Collaborative Computational Project. Number 4. The CCP4 Suite: Programs for Protein Crystallography. Acta Crystallogr. d50, 760–763 (1994).

39. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

40. Bricogne, G. et al. BUSTER, version 2.8.0. (Global Phasing Ltd., Cambridge, UK, 2009).

41. Pierre, Y., Breyton, C., Kramers, D. & Popot, J.-L. Purification and characterization of the cytochrome b6f complex from Chlamydomonas reinhardtii. J. Biol. Chem. 270, 29342–29349 (1995).

42. Goren, M.A. & Fox, B.G. Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex. Protein Expr. Purif. 62, 171–178 (2008).

© 2

010

Nat

ure

Am

eric

a, In

c. A

ll ri

gh

ts r

eser

ved

.

Nature Methods

Maltose–neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins Pil Seok Chae, Søren G F Rasmussen, Rohini Rana, Kamil Gotfryd, Richa Chandra, Michael A Goren, Andrew C Kruse, Shailika Nurva, Claus J Loland, Yves Pierre, David Drew, Jean-Luc Popot, Daniel Picot, Brian G Fox, Lan Guan, Ulrik Gether, Bernadette Byrne, Brian Kobilka & Samuel H Gellman Supplementary Figure 1 Emission spectra of carazolol bound to β2AR-T4L.

Supplementary Figure 2 Melting temperatures (Tm) of the β2AR-T4L in MNG amphiphiles or conventional detergents.

Supplementary Figure 3 Thermal stability of Cytochrome bo3 and CMP-Sia in MNG amphiphiles or conventional detergents using CPM assay.

Supplementary Figure 4 Long-term solubility and activity of LeuT in MNG amphiphiles or conventional detergents.

Supplementary Figure 5 Absorption spectra of LHI-RC complex solubilized with MNG amphiphiles or conventional detergents.

Supplementary Figure 6 Absorption spectra of LHI-RC complex purified with MNG amphiphiles or conventional detergents.

Supplementary Figure 7 Long-term stability of LHI-RC complex in MNG amphiphiles or conventional detergents.

Supplementary Figure 8 Biochemical characterization of membrane proteins extracted with amphiphilic agents from the native membranes.

Supplementary Figure 9 SDS-PAGE of wheat germ CF translation of the proteins in the presence of MNG amphiphiles or conventional detergents.

Supplementary Table 1 Melting Temperatures (Tm) of carazolol bound β2 adrenergic receptor (β2AR-T4L) at various critical micelle concentrations (CMC) measured in °C.

Supplementary Table 2 Crystal data collection statistics for cytochrome b6f/MNG-3 complex.

Supplementary Note Synthetic scheme and detailed protocol for preparation of MNG amphiphiles and monopod amphiphiles (MPAs), and their physical characterization.

Nature Methods: doi:10.1038/nmeth.1526

1

Supplementary Figure 1 Emission spectra of carazolol bound to β2AR-T4L.

335 345 355 365 375

0.5

0.6

0.7

0.8

0.9

1.0

Flu

ore

sce

nce

inte

nsity

(no

rma

lize

d to

35

6 n

m p

ea

k)

Intact β2AR

(at 25oC)

Experimentalwindow

β2AR boundcarazolol

341 nmpeak

356 nmpeak

nm

335 345 355 365 375

0.5

0.6

0.7

0.8

0.9

1.0

All denatured

(at 85oC)

free unboundcarazolol

341 nmpeak

356 nmpeak

nm

a b

(a) Emission spectra of bound carazolol to the β2AR-T4L and (b) spectra of free carazolol in solution

following complete receptor denaturation. Thirteen continuous emission spectra were obtained

following 5 min incubations at temperatures ranging from 25 to 85 °C in 5 °C intervals. The 356 nm

peaks were all normalized and the height of the 341 nm peaks (indicated by the dotted lines) were

plotted as a function of temperature and normalized as illustrated in Supplementary figure 2a.

Nature Methods: doi:10.1038/nmeth.1526

2

Supplementary Figure 2 Melting temperatures (Tm) of the β2AR-T4L in MNG

ampihphiles or conventional detergents.

25 35 45 55 65 75 85

0

25

50

75

100

Line indicating

melting temperature (Tm)

No

rma

lize

d 3

41

nm

/3

56

nm

pe

ak r

atio

(p

erc

en

t)

Temperature (Celsius)

1 10 100 100050

55

60

65

70

Tm

(C

els

ius)

Log fold above CMC

a b

DDMMNG-1 MNG-2 MNG-3 MPA-1 MPA-3 TDM DM

(a) Temperature dependence of fluorescence peak ratio (341 nm/356 nm) of β2AR-T4L in the

presence of detergents or amphiphiles at 10 × CMC. (b) Tm values of the receptor were plotted in

terms of CMC of the MNG amphiphiles (MNG-1, MNG-2, and MNG-3) or conventional detergents

(MPA-1, MPA-3, DM, DDM, and TDM). β2AR-T4L with bound carazolol (an inverse agonist) was

incubated with various agents at the various concentrations at indicated temperatures for 5 min prior to

fluorescence emission measurement as described in the Online Methods and in Supplementary

Figure 1. Normalized results are expressed as mean ± s.e.m. (n = 3, 4, or 5).

Nature Methods: doi:10.1038/nmeth.1526

3

Supplementary Figure 3 Thermal stability of Cytochrome bo3 and CMP-Sia in MNG

amphiphiles or conventional detergents using CPM assay.

0 25 50 75 100 125

0

20

40

60

80

100Cyctochrome bo3

Rela

tive a

mount

of

fold

ed p

rote

in (

perc

ent)

Time (min)

0 25 50 75 100 125

0

20

40

60

80

100

Rela

tive a

moun

t of

fold

ed p

rote

in (

perc

en

t)

Time (min)

CMP-sia

a b

DMMNG-1 MNG-2 MNG-3 MPA-4 DDM SDS

CPM assays for (a) Cytochrome bo3, and (b) CMP-Sia with MNG amphiphiles (MNG-1, MNG-2, and

MNG-3) or conventional detergents (MPA-4, DDM, DM and SDS). All agents were used at 10 ×

CMC. The unfolding of the each protein in various amphiphiles was monitored at 40 °C for 130 min

using a microplate spectrofluorometer set at an excitation wavelength of 387 nm and an emission

wavelength of 463 nm. Measurements were taken every 5 min after automatic agitation of the plate.

Nature Methods: doi:10.1038/nmeth.1526

4

Supplementary Figure 4 Long-term solubility and activity of LeuT in MNG

amphiphiles or conventional detergents.

0 2 4 6 8 10 12

0

20

40

60

80

100

120

Time (day)

Pro

tein

co

nce

ntr

atio

n (

pe

rce

nt)

0 2 4 6 8 10 12

0

20

40

60

80

100

120

cpm

[3H

]-L

eu

, p

erc

ent of co

ntr

ol

Time (day)

0 2 4 6 8 10 12

0

20

40

60

80

100

120

Time (day)

Pro

tein

co

nce

ntr

atio

n (

pe

rce

nt)

a b

c

MNG-1

MNG-2

MNG-3

MPA-1

MPA-3

DDM

LDAO

OG

CMC + 0.026 wt %

10 x CMC 10 x CMC

Long-term (a) solubility and (b) activity ([3H]-Leu binding) assay for LeuT solubilized with MNG

amphiphiles (MNG-1, MNG-2, and MNG-3) and conventional detergents (MPA-1, MPA-3, DDM,

laurydimethylamine-N-oxide (LDAO) and n-octyl-β-D-glucopyranoside (OG)) at 10 × CMC except

OG, which was tested at 2 × CMC. (c) LeuT solubility in MNG amphiphiles or DDM at 0.026 wt %

above each CMC. LeuT concentrations and activities were monitored at indicated time points for

protein stored at the room temperature. Protein concentration was measured by UV-visible

spectroscopy and activity was evaluated using a scintillation proximity assay (SPA). Results are

expressed as % concentration or % activity relative to the appropriate day 0 measurement.

Normalized results are expressed as mean ± s.e.m. (n = 2).

Nature Methods: doi:10.1038/nmeth.1526

5

Supplementary Figure 5 Absorption spectra of LHI-RC complex solubilized with

MNG amphiphiles or conventional detergents.

650 750 850 9500

2

4

6

8

Ab

so

rba

nce

Wavelength (nm)

650 750 850 9500

2

4

6

8

Ab

so

rba

nce

Wavelength (nm)

a b

LDAOMNG-1 MNG-2 MNG-3 MPA-3 DDM OG

Spectroscopic comparison of LHI-RC complex from Rhodobacter Capsulatus solubilized with (a)

MNG amphiphiles (MNG-1, MNG-2, and MNG-3) and (b) conventional detergents (MPA-3, DDM,

laurydimethylamine-N-oxide (LDAO), and n-octyl-β-D-glucopyranoside (OG)). All amphiphiles or

detergents were used at 1.0 wt % except OG, which was tested at 2.0 wt %.

Nature Methods: doi:10.1038/nmeth.1526

6

Supplementary Figure 6 Absorption spectra of LHI-RC complex purified with MNG

amphiphiles or conventional detergents.

650 750 850 9500.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nce

Wavelength (nm)

650 750 850 9500.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nce

Wavelength (nm)

a b

LDAOMNG-1 MNG-2 MNG-3 MPA-3 DDM OG

Spectroscopic comparison of LHI-RC complex from Rhodobacter Capsulatus purified with (a) MNG

amphiphiles (MNG-1, MNG-2, and MNG-3) and (b) conventional detergents (MPA-3, DDM, LDAO,

and OG). The complex was purified with the amphiphiles or detergents at 1 × CMC at room

temperature.

Nature Methods: doi:10.1038/nmeth.1526

7

Supplementary Figure 7 Long-term stability of LHI-RC complex in MNG

amphiphiles or conventional detergents.

0 5 10 15 200

5

10

15

20

25

Ab

so

rba

nce

ra

tio, A

87

5/A

68

0

Time (day)

0 5 10 15 200

5

10

15

20

25

Ab

so

rba

nce

ra

tio, A

87

5/A

68

0

Time (day)

a b

1.0 wt %

0 5 10 15 200

5

10

15

20

25

Ab

so

rba

nce

ra

tio

, A

87

5/A

68

0

Time (day)

c

MNG-1 MNG-2 MNG-3 MPA-3 DDM

0.2 wt %

CMC + 0.34 mM

0 5 10 15 200

5

10

15

20

25A

bso

rba

nce

ra

tio, A

87

5/A

68

0

Time (day)

CMC+ 0.017 wt %

d

All agents were tested at (a) CMC + 0.017 wt %, (b) 0.2 wt %, (c) 1.0 wt % and (d) CMC + 0.34 mM.

The absorption ratios (A875/A680) of the protein samples were followed as a function of time at room

temperature.

Nature Methods: doi:10.1038/nmeth.1526

8

Supplementary Figure 8 Biochemical characterization of membrane proteins

extracted with amphiphilic agents from the native membranes.

DDM

(1%

)

TDM

(1%

)

MNG

-3 (1

%)

DDM

(2%

)

TDM

(2%

)

MNG-3

(2%

)0

2

4

6

Sp

ecifi

cβ

2A

R a

ctiv

ity (

pm

ol/m

g)

0 20 40 60 800

10

20

30

40 MNG-3 (1%)

DDM (1%)

RF

U (

arb

.)

Fraction number

a b

c

-9 -8 -7 -6 -50

0

20

40

60

80

100

120

//

DDM (0.05 wt %)

MNG-3 (0.04 wt %)

cp

m [

3H

]-L

eu

, p

erc

en

t o

f co

ntr

ol

log([Leu]), (M)

(a) Activity of wild-type β2AR extracted with MNG-3, DDM, or TDM at 1% or 2% from an insect

cell membrane. Results are expressed as mean ± s.d. (n = 3). (b) Competition [3H]-Leu/Leu binding

curves for LeuT extracted and purified with MNG-3 and DDM from a bacterial membrane.

Normalized results are expressed as mean ± s.e.m. (n = 2). (c) Fluorescent size exclusion

chromatography (FSEC) analysis of CMP-Sia-GFP fusion protein after solubilization with 1% MNG-3

or 1% DDM from a yeast membrane.

Nature Methods: doi:10.1038/nmeth.1526

9

Supplementary Figure 9 SDS-PAGE of wheat germ CF translation of the proteins in

the presence of MNG amphiphiles or conventional detergents.

a

b

a

b

S P

S P S P S P S P S P S P S P

S P S P S P S P S P S P S P

MNG-1 MNG-2 MNG-3 MPA-10.1% 0.2% 0.1% 0.2% 0.1% 0.2% 0.1% 0.2%

MPA-3 DDM DM -0.1% 0.2% 0.1% 0.2% 0.1% 0.2%

SDS-PAGE of wheat germ CF translation of (a) green fluorescent protein (GFP) and (b) bacterio-

opsin (BO) in the presence of MNG amphiphiles (MNG-1, MNG-2, and MNG-3) and conventional

detergents (MPA-1, MPA-3, DDM, and DM). The agents were used at 0.1 wt % or 0.2 wt %. Arrows

indicate the location of GFP and BO protein bands in SDS-PAGE. “S” and “P” represent supernatant

and pellet fractions of protein samples after centrifugation. GFP study showed that CF expression is

not inhibited by any of the conventional detergents at 0.1 wt %, but significant inhibition is observed

at 0.2 wt % of each detergent. The three MNG amphiphiles did not inhibit GFP expression even at 0.2

wt %. CF expression of a membrane protein, BO, showed that expression of the protein was inhibited

in the presence of the conventional detergents at 0.2 wt % (DDM is the best of the conventional

detergents). These detergents at 0.1 wt % did not effectively solubilize the BO, most of which was

found in an aggregated form. In contrast, 0.2 wt % MNG-1 or MNG-2 did not cause any inhibition of

BO expression, as seen with GFP, and these amphiphiles efficiently solubilized the BO that was

produced. The behavior of MNG-3 was slightly different. Even at 0.1 wt %, this MNG amphiphile was

reasonably effective at solubilizing the BO produced, but 0.2 wt % MNG-3 seemed to reduce BO

expression slightly (all expressed protein was soluble).

Nature Methods: doi:10.1038/nmeth.1526

10

Supplementary Table 1 Melting Temperatures (Tm) of carazolol bound β2 adrenergic

receptor (β2AR-T4L) at various critical micelle concentrations (CMC) measured in °C.

Melting temperatures (Tm) of carazolol bound β2AR-T4L at various critical micelle concentrations (CMC) measured in °C

1.5x CMC

Tm ± SEM

3.5x CMC

Tm ± SEM

6.5x CMC

Tm ± SEM

10x CMC

Tm ± SEM

50x CMC

Tm ± SEM

250x CMC

Tm ± SEM

∆Tm

DDM 62.0 ± 0.23 (n=5) 64.2 ± 0.06 (n=4) 64.4 ± 0.14 (n=4) 63.5 ± 0.16 (n=4) 63.0 ± 0.08 (n=4) 62.2 ± 0.22 (n=4)

TDM n.d. n.d. n.d. 63.2 ± 0.28 (n=3) 63.5 ± 0.34 (n=3) 63.2 ± 0.24 (n=3) -0.9

DM 58.2 ± 0.29 (n=3) 56.0 ± 0.33 (n=3) n.d. 55.4 ± 0.63 (n=4) 56.0 ± 0.64 (n=3) n.d. -6.2

MPA-1 n.d. n.d. n.d. 51.4 ± 0.21 (n=3) n.d. n.d. -13.0

MPA-3 n.d. n.d. n.d. 58.8 ± 0.14 (n=3) 58.3 ± 0.10 (n=3) n.d. -5.6

MNG-1 n.d. n.d. n.d. 65.1 ± 0.25 (n=3) 65.1 ± 0.14 (n=3) 64.2 ± 0.21 (n=3) +0.7

MNG-2 n.d. n.d. n.d. 64.6 ± 0.37 (n=3) 67.1 ± 0.10 (n=3) 67.0 ± 0.31 (n=3) +2.7

MNG-3 n.d. n.d. n.d. 67.4 ± 0.06 (n=3) 68.4 ± 0.09 (n=3) 66.8 ± 0.15 (n=3) +4.0

The highest Tm value obtained for each amphiphiles is shown in bold and used to calculate ∆Tm relative to DDM.

The highest Tm value obtained for each amphiphile is shown in bold and used to calculated ∆Tm

relative to DDM. n.d. = not determined; n = number of replicates.

Nature Methods: doi:10.1038/nmeth.1526

11

Supplementary Table 2 Crystal data collection statistics for cytochrome b6f/MNG-3

complex.

Space group I222 Unit cell dimension (Å) 102.1 169.3 352.6 Number of reflections 195299 Number of unique reflections 39941 Resolution limits (Å) 44.03 - 3.47 Higher resolution shell (Å) (3.68 - 3.47) Completness 99.6% (99.2%) Redundancy 4.9 Rmerged 8.69% (59.9%) Rmeasured 9.7% (67.1%) I/σ(I) 13.8 (2.6) Crystallographic refinement statistics Resolution range (Å) 39.97 - 3.47 Number of reflections 39850 R / FreeR 0.1937 / 0.2334 Number of atoms (total) 7821 Mean B value (Å2 ) 112.60 rms deviation from ideal values: bond length (Å) 0.010 bond angle (degree) 1.20

Nature Methods: doi:10.1038/nmeth.1526

12

Supplementary Note Synthetic scheme and detailed protocol for preparation of MNG

amphiphiles and monopod amphiphiles (MPAs), and their physical characterization.

O

O O

O

MNG-2

HN

NH

O

O

OOH

OOH

HO OHO HO OH

OH

O

OH

O

OH

HO

OH

OHO

OH

OH

MNG-1

MNG-3

Br

Br O

O

H2N

H2NOH

OH

O

O

O

O

HN

NH

OH

OH

O

O OH

OH

a b,c

b,c

b,c

O

O

O

O

OOH

OOH

HO OHO HO OH

OH

O

OH

O

OH

HO

OH

OHO

OH

OH

d,e

f,g

1 2

3 4

5 6

O

O

OOH

OOH

HO OHO HO OH

OH

O

OH

O

OH

HO

OH

OHO

OH

OH

OH

OH

Synthetic route for MNG amphiphiles (MNG-1, MNG-2, and MNG-3): (a) undecanoic acid, EDC • HCl, HOBt, room

temperature; (b) perbenzoylated maltosylbromide (2.1 equiv.), AgOTf, CH2Cl2, − 45 °C � room temperature; (c) NaOMe,

MeOH, room temperature; (d) decanol, NaH, DMF, 120 °C; (e) p-TSA, MeOH, room temperature; (f) NaH, decyl iodide,

THF, room temperature; (g) LiAlH4, THF, room temperature.

General procedure for glycosylation reactions

This reaction was performed according to a literature method1 with slight modification. A mixture of

alcohol derivative, AgOTf (2.4 equiv.), 2,4,6-collidine (1.8 equiv.) in anhydrous CH2Cl2 (40 mL) was

stirred at − 45 °C. A solution of perbenzoylated maltosylbromide (2.4 equiv.) in CH2Cl2 (40 mL) was

added dropwise over 0.5 h to this suspension. Stirring was continued for 0.5 h at -45 °C, and then the

reaction mixture was allowed to warm to 0 °C and left stirring for 1.5 h. After completion of reaction

(as detected by TLC), pyridine was added to the reaction mixture, and it was diluted with CH2Cl2 (40

mL) before being filtered over celite. The filtrate was washed successively with a 1 M aqueous

Na2S2O3 solution (40 mL), a 0.1 M aqueous HCl solution (40 mL), and brine (2 × 40 mL). Then the

organic layer was dried with anhydrous Na2SO4 and the solvents were removed by rotary evaporation.

Nature Methods: doi:10.1038/nmeth.1526

13

The residue was purified by silica gel column chromatography (EtOAc/hexane) providing desired

product as a glassy solid.

General Procedure for the de-O-benzoylations under Zemplén’s conditions1

The O-benzoylated compounds were dissolved in MeOH and then treated with the required amount

of a methanolic solution of 0.5 M NaOMe such that the final concentration of NaOMe was 0.05 M.

The reaction mixture was left stirring for 6 h at room temperature, and then neutralized with Amberlite

IR-120 (H+ form) resin. The resin was removed by filtration and washed with MeOH and solvent was

removed from the combined filtrate in vacuo. The residue was purified by silica gel column

chromatography (MeOH/CH2Cl2). Further purification carried out by recrystallization using

CH2Cl2/MeOH/diethyl ether afforded fully de-O-benzoylated product as a white solid.

Synthesis and characterization of MNG amphiphiles

Scheme 1

(a) undecanoic acid, EDC • HCl, HOBt, room temperature, 91%; (b) (g) perbenzoylated maltosylbromide (2.4 equiv.), AgOTf, CH2Cl2, − 45 °C � room temperature, 92% ; (c) NaOMe, MeOH, room temperature, 95%.

Undecanoic acid (2,2-bis-hydroxymethyl-3-undecanolylamino-propyl)-amide (2)

This compound was synthesized according to scheme 1. 2,2-bis-aminomethyl-propane-1,3-diol (1)2

(0.51 g, 3.8 mmol), undecanoic acid (1.42 g, 7.6 mmol), 1-hydroxybenzotriazole monohydrate (HOBt)

(1.2 g, 9.1 mmol) was dissolved in anhydrous DMF (30 mL). 1-(3-(dimethylamino)propyl)-3-

ethylcarbodiimide hydrochoride (EDC • HC1) (1.7 g, 0.91 mmol) was added in small portions at 0 °C

and the resulting solution left stirring at room temperature for 20 hr. The solution was taken up with

Nature Methods: doi:10.1038/nmeth.1526

14

EtOAc (100 mL) and was washed successively with a 1 M aqueous NaHCO3 solution (100 mL), a 0.1

M aqueous HCl solution (100 mL) and brine (2 × 100 mL). Then the organic layer was dried with

anhydrous Na2SO4 and the solvent was removed by rotary evaporation. The reaction mixture was

precipitated with ether (100 mL) and the resulting solid was collected and dried in vacuo to afford

amide-containing diol (2) as a white solid (1.63 g, 91%). This product was used for next reaction

without further purification. 1H NMR (300 MHz, CDCl3): δ 6.97 (t, J = 6.8 Hz, 2H), 4.65 (t, J = 6.6

Hz, 2H), 3.27 (d, J = 7.0 Hz, 4H), 3.01 (d, J = 7.0 Hz, 4H), 2.25 (t, J = 7.4 Hz, 2H), 1.64 (quin, 4H),

1.30-1.23 (m, 28H), 1.38-1.21 (m, 6H), 0.88 (t, J = 7.0 Hz, 6H); 13C NMR (75 MHz, CDCl3): δ 176.1,

61.0, 46.6, 38.3, 36.9, 32.1, 29.8, 29.7, 29.5, 26.2, 22.9, 14.3; HRMS (ESI): calcd. for C27H54N2O4

[M]+ 471.4157, found 471.4154.

MNG-1a was synthesized according to the general procedure for glycosylation. 1H NMR (300 MHz,

CDCl3): δ 8.07 (d, J = 8.4 Hz, 4H), 8.02-7.85 (m, 12H), 7.87 (d, J = 8.4 Hz, 4H), 7.80 (d, J = 8.4 Hz,

4H), 7.74 (d, J = 8.4 Hz, 4H), 7.65-7.20 (m, 42H), 6.22-6.15 (m, 2H), 6.12 (t, J = 10.0 Hz, 2H), 5.70

(d, J = 4.1 Hz, 2H), 5.66 (t, J = 10.0 Hz, 2H), 5.32 (t, J = 9.4 Hz, 2H), 5.17 (dd, J = 10.0, 3.5 Hz, 2H),

5.06 (dd, J = 10.0, 8.0 Hz, 2H), 4.75 (d, J = 10.0 Hz, 2H), 4.57 (dd, J = 12.7, 3.2 Hz, 2H), 4.40-4.27

(m, 5H), 4.18 (dd, J = 13.1, 4.4 Hz, 2H), 3.53 (d, J = 9.6 Hz, 2H), 3.39 (m, 2H), 3.21 (d, J = 8.0 Hz,

2H), 3.06 (m, 4H), 2.91 (dd, J = 13.6, 3.0 Hz, 2H), 2.26 (t, J = 7.5 Hz, 4H), 1.6 (br s, 4H), 1.35-1.15

(br s, 28H), 0.86 (t, J = 6.9 Hz, 6H); 13C NMR (75 MHz, CDCl3): δ 174.1, 166.2, 166.0, 165.9, 165.6,

165.4, 165.2, 165.1, 134.3, 133.8, 133.6, 133.5, 133.4, 133.3, 130.2, 130.1, 129.9, 129.8, 129.7, 129.5,

129.4, 129.2, 129.1, 129.0, 128.9, 128.8, 128.6, 128.5, 101.3, 95.9, 74.3, 72.8, 72.1, 71.4, 69.9, 69.3,

69.1, 62.7, 42.2, 42.0, 36.6, 32.0, 29.8, 29.7, 29.5, 26.0, 22.9, 14.3; MS (MALDI-TOF): calcd. for

C149H150N2O38Na [M+Na]+ 2597.9759, found 2597.9653.

MNG-1 was synthesized according to the general procedure for de-O-benzoylation. 1H NMR (300

MHz, CD3OD): δ 5.20 (d, J = 3.8 Hz, 2H), 4.34 (d, J = 7.9 Hz, 2H), 3.96-3.80 (m, 8H), 3.73-3.63 (m,

9H), 3.60-3.39 (m, 10H), 3.38-3.22 (m, 7H), 2.27 (t, J = 6.6 Hz, 4H), 1.65 (br t, 4H), 1.42-1.25 (br s,

28H), 0.93 (t, J = 6.8 Hz, 6H); 13C NMR (75 MHz, CD3OD): δ 177.1, 104.8, 103.1, 81.4, 77.9, 76.8,

75.2, 74.9, 74.8, 74.3, 71.7, 71.3, 62.9, 62.2, 45.9, 41.0, 37.5, 33.2, 30.9, 30.8, 30.6, 30.5, 27.2, 23.9,

14.6; HRMS (ESI): calcd. for C51H94N2O24Na [M+Na]+ 1141.6089, found 1141.6071.

Scheme 2

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(d) decanol, NaH, DMF, 120 °C; (e) p-TSA, MeOH, room temperature, 92% (in two steps); (b) perbenzoylated maltosylbromide (2.4

equiv.), AgOTf, CH2Cl2, − 45 °C � room temperature, 93%; (c) NaOMe, MeOH, room temperature, 96%.

2,2-Bis-decyloxymethyl-propane-1,3-diol (4)

This compound was synthesized according to scheme 2. To a solution of decanol (3.3 g, 17 mmol) in

DMF (40 mL) was added NaH (0.69 g, 0.17 mmol, 60%) at 0 °C. The mixture was stirred at room

temperature under N2 atmosphere for 0.5 h. After addition of 5,5-bis-bromomethyl-2,2-dimethyl-

[1,3]dioxane (3)3 (1.3 g, 4.3 mmol), the reaction mixture was warmed to 120 °C and stirred further for

15 hr. After cooling to room temperature, the reaction was quenched with ice-cold H2O (100 mL) and

extracted with ether (3 × 80 mL). The combined organic layer washed with brine (2 × 100 mL), dried

with anhydrous Na2SO4 and then concentrated by rotary evaporation. To the residue dissolved in 1:1

mixture of CH2Cl2 and MeOH (120 mL) was added p-toluenesulfonic acid (p-TSA) monohydrate (300

mg) and left stirring at room temperature for 2 hr. After the neutralization of the reaction mixture with

a saturated aqueous NaHCO3 solution, the volume of solvent was reduced by rotary evaporation. The

residue was partitioned between CH2Cl2 and water. The separated organic layer was washed with brine,

dried with anhydrous Na2SO4, and then concentrated in vacuo. Flash column chromatography

(EtOAc/hexane) affords ether-containing diol (4) as a white solid (1.89 g, 92% (two steps)). 1H NMR

(300 MHz, CDCl3): δ 3.64 (d, J = 6.4 Hz, 4H), 3.51 (s, 4H), 3.42 (t, J = 6.3 Hz, 4H), 2.87 (t, J = 6.3

Hz, 2H), 1.56 (quin, J = 6.7 Hz, 4H), 1.26 (br s, 28H), 1.38-1.21 (m, 28H), 0.88 (t, J = 6.8 Hz, 6H); 13C NMR (75 MHz, CDCl3): δ 73.4, 72.3, 71.1, 65.7, 44.7, 32.1, 29.8, 29.7, 29.6, 29.5, 26.4, 22.9,

14.3; HRMS (ESI): calcd. for C25H52O4Na [M+Na]+ 439.3758, found 439.3778.

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MNG-2a was synthesized according to the general procedure for glycosylation. 1H NMR (300 MHz,

CDCl3): δ 8.07 (d, J = 8.4 Hz, 4H), 8.02-7.95 (m, 8H), 7.91 (d, J = 8.4 Hz, 4H), 7.87 (d, J = 8.4 Hz,

4H), 7.80 (d, J = 8.4 Hz, 4H), 7.74 (d J = 8.4 Hz, 4H), 7.65-7.20 (m, 42H), 6.12 (t, J = 9.8 Hz, 2H),

5.68 (d, J = 4.5 Hz, 2H), 5.65 (t, J = 9.4 Hz, 2H), 5.40 (t, J = 9.8 Hz, 2H), 5.22-5.10 (m, 4H), 4.65-

4.55 (m, 4H), 4.38-4.13 (m, 8H), 3.70 (d, J = 9.2 Hz, 2H), 3.46 (d, J = 8.0 Hz, 2H), 3.35-3.15 (m, 8H),

2.97 (t, J = 9.4 Hz, 4H), 1.40 (br s, 4H), 1.33-1.12 (br s, 30H), 0.87 (t, J = 7.0 Hz, 6H); 13C NMR (75

MHz, CDCl3): δ 166.3, 166.0, 165.9, 165.7, 165.2, 165.0, 133.9, 133.6, 133.5, 133.4, 133.3, 130.3,

130.2, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.5, 129.3, 129.2, 129.1, 129.0, 128.9, 128.8, 128.7,

128.6, 128.5, 128.4, 101.1, 95.9, 74.8, 72.4, 72.3, 71.7, 71.5, 69.9, 69.2, 69.1, 68.9, 68.6, 63.5, 62.7,

45.0, 32.1, 29.8, 29.7, 29.5, 26.2, 22.9, 14.3; MS (MALDI-TOF): calcd. for C147H148O38Na [M+Na]+

2543.9541, found 2543.9468.

MNG-2 was synthesized according to the general procedure for de-O-benzoylation. 1H NMR (300

MHz, CD3OD): δ 5.19 (d, J = 3.8 Hz, 2H), 4.36 (d, J = 7.9 Hz, 2H), 3.98-3.80 (m, 8H), 3.77-3.67 (m,

12H), 3.55-3.23 (m, 20H), 1.58 (br m, 4H), 1.45-1.30 (br s, 28H), 0.94 (t, J = 6.8 Hz, 6H); 13C NMR

(75 MHz, CD3OD): δ 105.2, 103.1, 81.5, 78.0, 76.7, 75.2, 75.0, 74.9, 74.3, 72.8, 71.6, 62.9, 46.7, 33.2,

31.0, 30.9, 30.8, 30.7, 27.6, 23.9, 14.6; HRMS (ESI): calcd. for C49H92O24Na [M+Na]+ 1087.5871,

found 1087.5876.

Scheme 3

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(f) NaH, decyl iodide, THF, room temperature; (g) LiAlH4, THF, room temperature, 93% (in two steps); (b) perbenzoylated

maltosylbromide (2.4 equiv.), AgOTf, 2,4,6-Collidine, CH2Cl2, − 45 °C � room temperature, 90%; (c) NaOMe, MeOH, room

temperature, 94%.

2,2-Bis-decyl-propane-1,3-diol (6)

This compound was synthesized according to a literature method4 (scheme 3) with slight modification.

To a solution of diethyl malonate (5) (1.04 mL, 6.9 mmol) in THF (40 mL) was added dropwise a

solution of NaH (0.82g, 21 mmol) in THF at 0 °C and left stirring for 20 min. After addition of

iododecane (3.8 mL, 18 mmol), the reaction mixture was stirred at room temperature for 24 hr,

quenched by adding ice-cold saturated NH4Cl (100 mL) and then extracted with diethyl ether (2 × 50

mL). The organic layer was washed with brine and dried with anhydrous Na2SO4. After complete

evaporation of solvent, LiAlH4 (0.52 g, 14.0 mmol) was added slowly to the residue dissolved in THF

(50 mL) at 0 °C. The mixture was stirred at room temperature for 4 hr, quenched with MeOH, water, a

1 N aqueous HCl solution successively at 0 °C and then extracted with diethyl ether (2 × 50 mL). The

combined organic layer was washed with brine and dried with anhydrous Na2SO4. The residue was

purified by silica gel column chromatography (EtOAc/hexane) providing alkyl-containing diol (6) as a

white solid (2.3 g, 93% (two steps)). 1H NMR (300 MHz, CDCl3): δ 3.55 (s, 4H), 2.55 (s, 2H), 1.38-

1.08 (m, 36H), 0.88 (t, J = 6.8 Hz, 6H); 13C NMR (75 MHz, CDCl3): δ 69.6, 41.2, 32.1, 31.0, 30.8,

29.9, 29.8, 29.6, 23.1, 22.9, 14.3; HRMS (ESI): calcd. for C23H48O2Na [M+Na]+ 379.3547, found

379.3546.

MNG-3a was synthesized according to the general procedure for glycosylation. 1H NMR (300 MHz,

CDCl3): δ 8.05 (d, J = 8.4 Hz, 4H), 8.02-7.95 (m, 8H), 7.92 (d, J = 8.4 Hz, 4H), 7.86 (d, J = 8.4 Hz,

4H), 7.86 (d, J = 8.4 Hz, 4H), 7.80 (d, J = 8.4 Hz, 4H), 7.75-7.18 (m, 42H), 6.12 (t, J = 9.8 Hz, 2H),

5.68–5.58 (m, 4H), 5.34 (t, J = 10.2 Hz, 2H), 5.18-5.06 (m, 4H), 4.68-4.52 (m, 4H), 4.38-4.16 (m, 8H),

3.32 (d, J = 7.6 Hz, 2H), 2.94-2.86 (m, 2H), 2.70 (d, J = 8.6 Hz, 2H), 1.35-0.98 (m, 34H), 0.87 (t, J =

6.9 Hz, 6H); 13C NMR (75 MHz, CDCl3): δ 166.3, 166.0, 165.7, 165.3, 165.2, 165.0, 133.9, 133.7,

133.6, 133.4, 133.3, 130.3, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.4, 129.2, 129.0, 128.9, 128.8,

128.6, 128.5, 95.9, 74.5, 72.3, 72.2, 71.5, 69.2, 62.8, 40.4, 32.1, 30.6, 30.3, 29.9, 29.8, 29.7, 29.6, 22.9,

22.3, 14.3; MS (MALDI-TOF): calcd. for C145H144O36Na [M+Na]+ 2483.9330, found 2483.928.

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MNG-3 was synthesized according to the general procedure for de-O-benzoylation. 1H NMR (300

MHz, CD3OD): δ 5.18 (d, J = 3.8 Hz, 2H), 4.39 (d, J = 7.9 Hz, 2H), 3.98-3.78 (m, 4H), 3.77-3.60 (m,

6H), 3.58-3.23 (m, 20H), 1.58 (br m, 6H), 1.42-1.16 (br s, 34H), 0.93 (t, J = 6.8 Hz, 6H); 13C NMR

(75 MHz, CD3OD): δ 105.1, 103.0, 78.0, 76.6, 74.9, 74.8, 71.6, 42.2, 33.2, 31.7, 30.9, 30.8, 30.6, 23.9,

14.6; HRMS (ESI): calcd. for C47H88O22Na [M+Na]+ 1027.5660, found 1027.5653.

Synthesis and characterization of monopod amphiphiles (MPAs)

Scheme 4

(b) perbenzoylated maltosylbromide (1.2 equiv.), AgOTf, CH2Cl2, − 45 °C � room temperature, 95% (MPA-1a), 95% (MPA-2a),

94% (MPA-3a), 93% (MPA-4a); (c) NaOMe, MeOH, room temperature, 96% (MPA-1 and MPA-2), 95 % (MPA-3 and MPA-4).

Undecanonic acid (2-hydroxy-ethyl)-amide (7) & undecanonic acid (2-hydroxy-propyl)-amide (8)

These compounds were synthesized according to the synthetic protocol for the preparation of amide-

containing diol (2) using 1.1 equivalent of 2-aminoethanol and 3-amino-propan-1-ol with undecanoic

acid, respectively.

Undecanonic acid (2-hydroxy-ethyl)-amide (7), Yield: 95%; 1H NMR (300 MHz, CDCl3): δ 6.14

(br s, 1H), 3.72 (q, J = 5.0 Hz, 2H), 3.42 (q, J = 5.4 Hz, 2H), 3.16 (t, J = 5.1 Hz, 1H), 2.21 (t, J = 7.6

Hz, 2H), 1.63 (quin, J = 7.0 Hz, 2H), 1.38-1.20 (m, 14H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz,

CDCl3): δ 174.8, 62.7, 42.6, 36.9, 32.1, 29.8, 29.7, 29.5, 25.9, 22.9, 14.3; HRMS (ESI): calcd. for

C13H27NO2 [M]+ 229.2037, found 229.2043.

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Undecanonic acid (2-hydroxy-propyl)-amide (8), Yield: 93%; 1H NMR (300 MHz, CDCl3): δ 6.25

(br s, 1H), 3.67 (quin, J = 6.0 Hz, 4H), 3.62 (q, J = 5.6 Hz, 2H), 3.40 (q, J = 6.2 Hz, 2H), 2.19 (t, J =

7.4 Hz, 2H), 1.72-1.56 (m, 4H), 1.36-1.20 (m, 14H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz,

CDCl3): δ 174.9, 59.3, 36.9, 36.3, 32.5, 32.1, 29.7, 29.6, 29.5, 29.4, 26.0, 22.8, 14.3; HRMS (ESI):

calcd. for C14H29NO2 [M]+ 243.2193, found 243.2188.

MPA-1a was synthesized according to the general procedure for glycosylation. 1H NMR (300 MHz,

CDCl3): δ 8.09 (d, J = 8.3 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.92-7.83 (m, 4H), 7.78-7.67 (m, 4H),

7.65-7.17 (m, 23H), 6.10 (t, J = 10.2 Hz, 1H), 5.85-5.73 (m, 3H), 5.67 (t, J = 9.8 Hz, 1H), 5.35-5.22

(m, 2H), 4.97 (dd, J = 12.3, 2.5 Hz, 1H), 4.79 (dd, J = 12.2, 3.2 Hz, 2H), 4.55-4.45 (m, 3H), 4.34-4.25

(dd, J = 12.9, 3.8 Hz, 1H), 4.15-4.07 (m, 1H), 3.92-3.83 (m, 1H), 3.66 (td, J = 10.4, 4.0 Hz, 1H), 3.55-

3.43 (m, 1H), 3.36-3.23 (m, 1H), 1.91-1.81 (m, 2H), 1.42 (quin, J = 7.2 Hz, 2H), 1.35-1.05 (m, 14H),

0.88 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ 173.3, 166.3, 166.0, 165.8, 165.6, 165.5,

165.3, 165.2, 133.8, 133.6, 133.5, 133.3, 130.2, 130.1, 130.0, 129.9, 129.8, 129.7, 129.5, 129.2, 128.9,

128.8, 128.6, 128.4, 128.3, 101.2, 96.7, 74.8, 73.4, 72.6, 71.1, 69.6, 69.4, 69.3, 63.5, 62.7, 39.1, 36.6,

32.1, 29.8, 29.7, 29.5, 29.4, 29.3, 25.7, 22.9, 14.3; HRMS (ESI): calcd. for C74H75NO19Na [M+Na]+

1304.4826, found 1304.4805.

MPA-2a was synthesized according to the general procedure for glycosylation. 1H NMR (300 MHz,

CDCl3): δ 8.09 (d, J = 8.3 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.92-7.83 (m, 4H), 7.78-7.67 (m, 4H),

7.65-7.17 (m, 23H), 6.11 (t, J = 10.2 Hz, 1H), 5.95 (t, J = 5.6 Hz, 1H), 5.85-5.75 (m, 2H), 5.68 (t, J =

9.8 Hz, 1H), 5.34-5.24 (m, 2H), 5.00 (dd, J = 12.3, 2.5 Hz, 1H), 4.80-4.71 (m, 2H), 4.56-4.45 (m, 3H),

4.31 (dd, J = 12.9, 3.8 Hz, 1H), 4.15-4.05 (m, 1H), 4.01-3.90 (m, 1H), 3.62-3.53 (m, 1H), 3.35-3.23

(m, 1H), 3.21-3.08 (m, 1H), 2.10 (t, J = 7.0 Hz, 2H), 1.82-1.63 (m, 2H), 1.62-1.50 (m, 2H), 1.35-1.18

(m, 14H), 0.86 (t, J = 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ 173.4, 166.3, 166.0, 165.8, 165.6,

165.5, 165.2, 165.1, 133.7, 133.6, 133.5, 133.4, 133.2, 130.1, 130.0, 129.9, 129.8, 129.7, 129.6, 129.1,

129.0, 128.9, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2, 101.0, 96.7, 74.8, 73.5, 72.6, 71.1, 69.4, 69.3,

68.8, 63.4, 62.7, 37.1, 36.7, 32.0, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 26.0, 22.8, 14.3; HRMS (ESI):

calcd. for C75H77NO19Na [M+H]+ 1296.5163, found 1296.5222.

MPA-1 was synthesized according to the general procedure for de-O-benzoylation. 1H NMR (300

MHz, CD3OD): δ 5.15 (d, J = 3.4 Hz, 1H), 4.29 (d, J = 7.8 Hz, 1H), 3.97-3.75 (m, 4H), 3.73-3.54 (m,

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5H), 3.54-3.20 (m, 7H), 2.19 (t, J = 7.5 Hz, 2H), 1.59 (br quin, 2H), 1.29 (br s, 14H), 0.89 (t, J = 6.8

Hz, 3H); 13C NMR (75 MHz, CD3OD): δ 176.6, 104.6, 103.1, 81.5, 77.9, 76.8, 75.2, 74.9, 74.8, 74.3,

71.6, 69.9, 62.9, 40.7, 37.3, 33.2, 30.9, 30.8, 30.6, 30.5, 27.2, 23.9, 14.6; HRMS (ESI): calcd. for

C25H47NO12Na [M+Na]+ 576.2991, found 576.2964.

MPA-2 was synthesized according to the general procedure for de-O-benzoylation. 1H NMR (300

MHz, CD3OD): δ 5.21 (d, J = 3.8 Hz, 1H), 4.32 (d, J = 7.7 Hz, 1H), 3.99-3.76 (m, 4H), 3.75-3.56 (m,

5H), 3.56-3.23 (m, 7H), 2.22 (t, J = 7.0 Hz, 2H), 1.83 (quin, J = 6.2 Hz, 2H), 1.64 (br quin, 2H), 1.33

(br s, 14H), 0.94 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CD3OD): δ 176.5, 104.4, 103.0, 81.5, 77.9,

76.7, 75.2, 74.9, 74.9, 74.8, 74.3, 71.6, 68.5, 62.9, 62.3, 37.6, 37.3, 33.2, 30.8, 30.7, 30.6, 30.5, 30.4,

27.2, 23.9, 14.6; HRMS (ESI): calcd. for C26H49NO12Na [M+Na]+ 568.3328, found 568.3345.

2-decyloxy-ethanol (9) & 3-decyloxy-propan-1-ol (10)

These were synthesized according to a literature method5.

2-decyloxy-ethanol (9); 1H and 13C NMR spectra of 9 are in good agreement with the previously

reported spectra; HRMS (ESI): calcd. for C12H26O2Na [M+H]+ 203.2007, found 203.2011.

3-decyloxy-propan-1-ol (10); 1H and 13C NMR spectra of 10 are in good agreement with the

previously reported spectra; HRMS (ESI): calcd. for C13H28O2Na [M+H]+ 217.2163, found 217.2171.

MPA-3a was synthesized according to the general procedure for glycosylation. 1H NMR (300 MHz,

CDCl3): δ 8.10 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.90-7.83 (m, 4H), 7.74 (d, J = 8.4 Hz,

4H), 7.65 (d, J = 8.4 Hz, 2H), 7.61-7.10 (m, 2H), 6.10 (t, J = 10.0 Hz, 1H), 5.82-5.70 (m, 2H), 5.66 (t,

J = 9.9 Hz, 1H), 5.36-5.23 (m, 2H), 5.00-4.88 (m, 2H), 4.75 (dd, J = 12.0, 4.1 Hz, 1H), 4.55-4.37 (m,

3H), 4.28 (dd, J = 12.4, 3.6 Hz, 1H), 4.15-4.07 (m, 1H), 4.00-3.89 (m, 1H), 3.56-3.42 (m, 2H), 3.25 (t,

J = 6.8 Hz, 2H), 1.42-1.06 (m, 14H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ 166.3,

166.0, 165.8, 165.6, 165.5, 165.4, 165.2, 133.7, 133.6, 133.5, 133.4, 133.3, 133.2, 130.1, 130.0, 129.9,

129.8, 129.7, 129.6, 129.5, 129.1, 128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 101.0, 96.6, 75.2,

73.5, 73.1, 72.5, 71.8, 71.1, 70.1, 69.9, 69.4, 69.3, 63.8, 62.7, 32.1, 29.8, 29.7, 29.6, 29.5, 26.1, 22.9,

14.3; HRMS (ESI): calcd. for C73H74O19Na [M+Na]+ 1277.4717, found 1277.4775.

MPA-4a was synthesized according to the general procedure for glycosylation. 1H NMR (300 MHz,

CDCl3): δ 8.10 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.90-7.83 (m, 4H), 7.74 (d, J = 8.4 Hz,

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4H), 7.65 (d, J = 8.4 Hz, 2H), 7.61-7.10 (m, 2H), 6.10 (t, J = 10.0 Hz, 1H), 5.82-5.70 (m, 2H), 5.66 (t,

J = 9.9 Hz, 1H), 5.36-5.23 (m, 2H), 4.92 (dd, J = 12.0, 2.6 Hz, 1H), 4.81-4.70 (m, 2H), 4.55-4.37 (m,

3H), 4.27 (dd, J = 12.4, 3.6 Hz, 1H), 4.15-4.05 (m, 1H), 4.00-3.92 (m, 1H), 3.67-3.56 (m, 1H), 3.27 (t,

J = 6.0 Hz, 2H), 3.18-3.03 (m, 2H), 1.82-1.68 (m, 2H), 1.39 (quin, J = 6.8 Hz, 2H), 1.34-1.10 (m,

12H), 0.88 (t, J = 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ 166.4, 166.0, 165.8, 165.6, 165.3, 165.2,

133.7, 133.6, 133.5, 133.4, 133.3, 133.2, 130.2, 130.1, 130.0, 129.9, 129.7, 129.6, 129. 5, 129.1, 129.0,

128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 101.0, 96.6, 75.1, 73.4, 73.1, 72.5, 71.2, 71.1, 70.1,

69.3, 67.4, 67.1, 63.7, 62.7, 32.1, 30.0, 29.8, 29.7, 29.6, 29.5, 26.2, 22.9, 14.3; HRMS (ESI): calcd.

for C74H76O19Na [M+Na]+ 1291.4874, found 1291.4838.

MPA-3 was synthesized according to the general procedure for de-O-benzoylation. 1H NMR (300

MHz, CD3OD): δ 5.20 (d, J = 3.7 Hz, 1H), 4.36 (d, J = 8.0 Hz, 1H), 4.18-4.00 (m, 1H), 3.97-3.82 (m,

3H), 3.82-3.60 (m, 8H), 3.58-3.47 (m, 4H), 3.45-3.38 (m, 1H), 3.37-3.26 (m, 2H), 1.62 (br quin, 2H),

1.34 (br s, 14H), 0.94 (t, J = 7.0 Hz, 3H); 13C NMR (75 MHz, CD3OD): δ 104.5, 103.1, 81.4, 77.8,

76.8, 75.2, 74.9, 74.8, 74.3, 72.6, 71.6, 71.2, 69.8, 62.9, 62.3, 40.7, 33.2, 30.9, 30.8, 30.7, 30.6, 27.3,

23.9, 14.6; HRMS (ESI): calcd. for C21H41NO7Na [M+Na]+ 442.2781, found 442.2776.

MPA-4 was synthesized according to the general procedure for de-O-benzoylation. 1H NMR (300

MHz, CD3OD): δ 5.20 (d, J = 3.7 Hz, 1H), 4.31 (d, J = 7.5 Hz, 1H), 4.04-3.94 (m, 1H), 3.93-3.81 (m,

3H), 3.76-3.54 (m, 9H), 3.52-3.43 (m, 3H), 3.43-3.37 (m, 1H), 3.36-3.23 (m, 2H), 1.91 (quin, J = 6.4

Hz, 2H), 1.60 (br quin, 2H), 1.34 (br s, 14H), 0.94 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CD3OD): δ

104.6, 103.1, 81.4, 77.9, 76.7, 75.2, 74.9, 74.8, 74.3, 72.2, 71.6, 68.9, 68.1, 62.9, 62.3, 33.2, 31.2, 30.9,

30.8, 30.7, 30.6, 27.4, 23.9, 14.6; HRMS (ESI): calcd. for C24H46O12Na [M+Na]+ 549.2882, found

549.2874.

Nature Methods: doi:10.1038/nmeth.1526

22

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Nature Methods: doi:10.1038/nmeth.1526