WO2010050918A1

WO2010050918A1 - Branched chain detergents for membrane protein structural biology

Info

Publication number: WO2010050918A1
Application number: PCT/US2008/012358
Authority: WO
Inventors: Qinghai Zhang; Wen-Xu Hong
Original assignee: Qinghai Zhang; Wen-Xu Hong
Priority date: 2008-10-31
Filing date: 2008-10-31
Publication date: 2010-05-06

Abstract

The invention is directed to detergents adapted for sample preparation, including extraction, solubilization, purification and reconstitution, of lipophilic proteins such as intrinsic cell membrane proteins for biochemical and biophysical studies. Detergents are provided including polar head groups bonded to lipophilic tail moieties, optionally via a linker, wherein the tail moieties incorporate branches of short alkyl, cycloalkyl, or fluoroalkyl groups covalently attached to the main chain of the tail moiety at or near the junction of the tail moiety with the polar head group. The polar head group can include phosphocholine, carbohydrate, polyethyleneglycol, or other polar moieties. Micelles formed of the detergents are denser than micelles formed of straight chain detergents, and provide for improved performance in preparing samples of proteins such as intrinsic cell membrane proteins for structural characterization.

Description

BRANCHED CHAIN DETERGENTS FOR MEMBRANE PROTEIN

STRUCTURAL BIOLOGY

Background

Intrinsic membrane proteins tend to have exposed hydrophobic regions over a portion of the protein surface in the native conformation, and perhaps consequently, are well known in the art to be particularly difficult to handle, such as is needed to obtain protein X-ray crystal structures as well as a means of achieving high quality purification.

X-ray crystallography of integral membrane proteins (IMPs) is a forefront area in structural biology. IMPs, namely proteins embedded in the cell membrane, are encoded by about 20-30% of eukaryotic genomes and are pivotal to the signal conduction and molecule translocation across the membrane barrier. The determination of IMP structures is crucial for understanding many fundamental biological processes, and they could serve as a molecule basis for developing novel therapeutics since many of them are clinical drug targets. However, only a tiny fraction of IMP structures has been solved to date, mainly by X-ray crystallography, accounting for less than two percent of all structure entries in the protein data bank. This discontinuity has largely been due to the difficulty inherent in all phases of crystallogenesis of IMPs.

Once an IMP is overexpressed at an acceptable level, the biggest hurdle for further structural characterization is to solubilize the protein from its natively residing cell membranes in a stable, homogeneous, and natively fold state.

Detergents, structurally similar to cellular lipids with an alkyl chain at one end and a polar head group at the other, are indispensable in this process. IMPs must be stable in a detergent-bound, artificial membrane environment during the purification and crystallization process. Despite the advent and success of new techniques such as in- meso phase and bicelle crystallization, the use of micelle-forming detergents remains to be a primary solution for crystallography and many successful methods for reconstituting and crystallizing IMPs also rely on the unique behavior of detergents. Overall, the selection of detergents has been one of the major bottlenecks for the successful sample preparation and for the growth of high-quality IMP crystals.

Although numerous detergents have been used in membrane biochemistry, most of these molecules were originated from other research areas or from industrial applications. The availability of useful commercial detergents for membrane protein crystallographers is actually quite limited, and a majority of the IMP structures have been solved in only very few detergents based on sugar, polyethylene oxide and amine oxide head groups. On the other hand, X-ray diffraction of IMP crystals is often of lower quality (e.g. low resolution and anisotropic diffraction) and large solvent content. As a result, there are often the times that crystallographers will run out of the existing detergent pool and desperately find no desirable detergent to get high-quality crystals.

Summary The present invention is directed to compounds having detergent properties that are adapted for purification and crystallization of lipophilic proteins, such as intrinsic cell membrane proteins, to methods of preparation of these compounds, and to uses of these compounds in the purification, including the crystallization, of such types of proteins. In various embodiments, the invention provides a detergent adapted for use in crystallization of intrinsic cell membrane proteins, the detergent comprising a detergent adapted for use in crystallization of intrinsic cell membrane proteins, the detergent comprising a compound of formula (I):

(I) wherein P is a polar head group; L is a linker or is absent; R is (C|_-3)alkyl, monofluoro- or polyfluoro(Cι_-3)-alkyl, or (C_3-5)cycloalkyl; p is 0 to about 2; q is 0 to about 3; r is about 4 to about 14; and a carbon atom labelled A is present in chiral form or in racemic form.

In various embodiments, a micelle comprising a compound of the invention is provided. In various embodiments, the micelle further comprises a protein, such as an intrinsic cell membrane protein, or other lipophilic protein.

In various embodiments, a method of purification of a protein comprising extraction of the protein in a plurality of micelles of the invention, the micelles comprising a detergent comprising a compound of formula (I) of the invention. The method can comprise crystallization. In various embodiments, methods of preparation of a compound of the invention are provided.

Brief Description of the Figures

Figure 1 shows a schematic drawing of how a branched-chain detergent of the invention forms micelles in comparison to a straight-chain art detergent.

Figure 2 shows schematic and graphic representation of the effect of branch size on micellar properties.

Figure 3 shows graphs comparing the stability of proteins included in micelles of the invention compared to art micelles. Figure 4 shows schematic diagram of the molecular shape of straight and branched detergents, (a) monomer and micelle of classic detergent, (b) monomer and micelle of branched detergent.

Figure 5 shows a flow chart for micro-scale OmpX reconstitution in detergent micelles. Solid lines indicate the route to reconstituted OmpX for structural studies, and dashed lines indicate the salvage pathways for precipitated OmpX. The buffers are described in the Methods section. Asterisks indicate procedural steps that were decisively improved with the use of the new detergents (see Table 3).

Figure 6 shows (a) SDS-gel electrophoresis of OmpX samples at pH 8.5. Lane 1 : unfolded OmpX in 6 M urea; Lane 2: OmpX refolded in DDM; Lane 3: OmpX refolded in TPC. (b) pH effect on OmpX refolding. F is the fraction of the OmpX that was refolded. The protein (0.4 mg/mL), urea (0.2 M), and detergent (1% wt/vol) were incubated at room temperature for 3 hours, followed by quick analysis by SDS-PAGE. The fraction of folded OmpX was determined by densitometric analysis of the Coomassie stained gels (± 5% error).

Figure 7 shows examples of chemical structures of the detergents of the invention, categorized by the type of spacer groups between the phosphocholine head and branched alkyl tail.

Figure 8 shows 2D [¹⁵N₅ ¹H]-TROSY correlation NMR spectra of uniformly

[²H, ¹⁵N] -labeled OmpX reconstituted in mixed micelles with different detergents:

(a) DHPC. (b) 138-Fos. (c) 179-Fos, where the vertical band of peaks near 8.0 ppm represents ti-noise from the signal of the amide proton of 179-Fos. (d) 115-Fos. (e) TPC. (f) 34-Fos. (g) 185-Fos. The spectra were collected with the following parameters: data size 50 (J₁) x 1024 (t₂) complex points; ti_max = 25 ms; ^_max = 86 ms;

300 scans per t_\ increment, overall measurement time = 9 hours per experiment.

Before Fourier transformation, the data matrices were multiplied with an exponential window function in the acquisition dimension, and with a 75°-shifted sine bell window⁴⁶ in the indirect dimension.

Figure 9 is an electron micrograph of Cx26 in Mall 1-1, showing the expected donut shape indicating the presence of hexamers.

Figure 10 shows the thermal stability of Cx26 measured in DDM, UDM and MaI 11-1 detergents, with an average midpoint of the thermal transition of ~55°C. The midpoint of the thermal transition is approximately 2°C lower for UDM and MaI 11-1 compared to DDM.

Detailed Description

All chiral, diastereomeric, racemic forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds used in the present invention include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these are all within the scope of the invention.

By "chemically feasible" is meant a bonding arrangement or a compound where the generally understood rules of organic structure are not violated; for example a structure within a definition of a claim that would contain in certain situations a pentavalent carbon atom that would not exist in nature would be understood to not be within the claim.

Alkyl groups include straight chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso- butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. More particularly, alkyl groups can contain from 1 to 3 carbon atoms such as methyl, ethyl, n-propyl, or isopropyl groups.

The term "hydroxyl protecting group" or "O-protected" as used herein refers to those groups intended to protect an OH group against undesirable reactions during synthetic procedures and which can later be removed to reveal the amine. Commonly used hydroxyl protecting groups are disclosed in Protective Groups in Organic Synthesis, Greene, T. W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999). Hydroxyl protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifiuoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4- chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; acyloxy groups (which form urethanes with the protected amine) such as benzyloxycarbonyl (Cbz), p- chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4- dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4- dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5- dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1 -(p- biphenylyl)- 1 -methyl ethoxycarbonyl, α,α-dimethyl-3, 5- dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl, 2- trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl, fiuorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. It is well within the skill of the ordinary artisan to select and use the appropriate hydroxyl protecting group for the synthetic task at hand. Cycloalkyl groups are cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, and cyclopentyl.

A "fluoroalkyl" group includes mono-fluoro alkyl groups, and poly-fluoro alkyl groups. Examples of fluoroalkyl include trifluoromethyl, 1,1-difloroethyl, 1,2- difluoroethyl, and the like. A "polar" group is a group containing ionic and/or hydrophilic atoms or functional groups of atoms. A polar group can comprise organic, inorganic, or both, moieties. Examples include carboxylates, phosphates, ammonium groups, oxygen atoms, amine oxides, and the like, or any combination thereof.

A carboxylate, as the term is used herein, comprises a group of the structure -C(O)OH, or a salt thereof, as is well known in the art.

A phosphate, as the term is used herein, comprises a group of the structure -OP(O)(OH)2, or a salt thereof, as is well known in the art.

An ammonium group, as the term is used herein, comprises a nitrogen atom bearing a positive charge, such as a trimethylammonium group, with a suitable counterion such as a halide ion or a carboxylate ion.

An amine oxide, as the term is used herein, comprises a group of the formula -N(O)R2, wherein R is a carbon substituent such as an alkyl group. For example, a dimethylammonium N-oxide group is an amine oxide within the meaning herein.

A "detergent" or a "surfactant" is a substance comprising molecules having both a polar head group and a non-polar tail group, as is well known in the art. A detergent or surfactant, which is typically capable of forming micelles, can reduce interfacial surface tension between an aqueous medium and a non-polar or lipophilic molecule or domain of a molecule.

A "micelle" as the term is used herein refers to a non-covalent association of a plurality of detergent or surfactant molecules, typically roughly spherical in aqueous dispersion, with lipophilic cores and hydrophilic heads, the hydrophilic heads being on the exterior of the micelle and interacting with the aqueous medium surrounding the micelle. The lipophilic interior of the micelle can contain in non- covalent association lipophilic substances such as lipophilic proteins, for example, intrinsic cell membrane proteins.

A "head group" refers to the polar end of a detergent or surfactant molecule, which also possesses a hydrophobic (lipophilic) tail, as is well known in the art. The tail is a linear moiety composed of an alkyl group or other non-polar group, that in a compound of the invention includes a "branch" that is disposed relatively close to the polar head group.

By a "branch", as the term is used herein, is meant a relatively short alkyl, cycloalkyl, or fluoroalkyl group that is covalently attached to an atom disposed at or near the junction of the head group and the tail group of the molecule.

The polar head group and the non-polar tail group can be joined together by a linker. By the term "linker" is meant a moiety incorporated into a detergent or surfactant molecule adapted to covalently connect the polar head group and non- polar tail group in a chemically feasible manner. A "straight chain detergent" as the term is used herein refers to an art detergent substances wherein the molecular structure does not incorporate a branch on the main chain of the lipophilic tail moiety.

A "lipophilic protein" is a protein with non-polar, lipophilic groups such as alkyl groups exposed on the surface of the protein or having a relatively high abundance compared to polar groups such as carboxylate groups in the composition of the protein. An example is an intrinsic cell membrane protein, by which is meant a protein normally associated with a cell membrane in a living cell, wherein a portion of the protein is disposed within the lipophilic domain of a cell membrane formed by a lipid bilayer of cell membrane components. An intrinsic cell membrane protein can be a cell membrane spanning protein, as is well known in the art.

A "biological material" as the term is used herein refers to material derived from a living organism, itself being living or non-living, such as a biochemical preparation.

In various embodiments, the invention provides a detergent adapted for use in crystallization of intrinsic cell membrane proteins, the detergent comprising a compound of formula (I):

(I) wherein P is a polar head group; L is a linker or is absent; R is (C_1-3)alkyl, monofluoro- or polyfluoro(Ci_-3)-alkyl, or (C_3-s)cycloalkyl; p is 0 to about 2; q is 0 to about 3; r is about 4 to about 14; and, a carbon atom labelled A is present in chiral form or in racemic form.

For example, the detergent can comprise a compound of formula (I) wherein a polar head group comprises organic, inorganic, or both, moieties.

More specifically, the detergent can comprise a compound of formula (I) of claim 2a comprising a carboxylate, phosphate, amine oxide or ammonium group, or one or more oxygen atom, or any combination thereof.

For example, the detergent can comprise a compound of formula (I) wherein P comprises a carbohydrate moiety, a polyoxyethylene moiety, a phosphocholine moiety, or an amine-oxide moiety. The detergent can include a linker L, or the linker L can be absent. For example, the detergent can comprise a compound of formula (I) wherein L comprises a group of the formula -O-, -S-, -(O-CH2-CH2-O)m- wherein m = 1-3, - C(O)O-, -OC(O)-, -NR'-C(O)-, or -C(O)NR¹-, wherein R¹ is H or (C>.₃ alkyl).

More specifically, the detergent can comprise a compound of formula (I) of claim 2 wherein P-L comprises an oxygen-linked carbohydrate moiety. For instance, the detergent can comprise a compound of formula (I) wherein the carbohydrate moiety comprises a monosaccharide or a disaccharide or a sugar alcohol moiety, such as a β-glucosyloxy moiety. More specifically, when P-L comprises a monosaccharide or a disaccharide or a sugar alcohol moiety, p can be 0, q can be 0-2, and r can be 6-11 in the compound of formula (I).

In various other embodiments, the detergent can comprise a compound of formula (I) wherein P-L comprises a disaccharide moiety, which can comprise a β- (l-4)-glucosyl-β-glucosyloxy (cellobiosyl) moiety or an α-(l-4)-glucosyl-β- glucosyloxy (maltosyl) moiety. When P-L comprises a disaccharide moiety, p can be 0, q can be 0-2, and r can be 9-13 in the compound of formula (I).

In various embodiments, P-L can comprise a phosphocholine moiety, that is, a trimethylammoniumethylphosphoryloxy group that can be coupled via a phosphate oxygen atom to the tail incorporating a branch as defined herein of a detergent of the invention of formula (I). More specifically, when P-L comprises a phosphatidylcholine moiety, p can be 0, q can be 0-2, and r can be 8-12 in a compound of formula (I).

In various embodiments, P-L comprises an oxygen-linked polyoxyethylene moiety, that is, a group of the formula HO-(CH₂CH₂O)_n-O- wherein n is 1 to about 12. For example, P-L can comprise a diethyleneglycol moiety, wherein n=2.

In various embodiments, the detergent comprising a compound of formula (I) of claim 1 wherein P-L comprises a nitrogen-linked amine oxide moiety. For example, P-L can comprise a dimethylammonium-N-oxide group.

In various embodiments, the compound of formula (I) comprises: 195-Fos

— N® — N @ — N®

or any salt, hydrate, solvate, tautomer, or stereoisomer thereof.

In various embodiments, the invention provides a detergent comprising a compound of formula (I) wherein the compound of formula (I) comprises:

10+3 MaI or any salt, hydrate, solvate, tautomer, or stereoisomer thereof.

In various embodiment, the detergent comprises a compound of formula (I) wherein an angle formed by an envelope swept by rotation about a long axis of a molecule of the compound of any one of claims 1-13 is less than an angle formed by an envelope swept by rotation about a long axis of a molecule of a straight chain detergent. For instance, as shown in Figures 1 and 4, the density of packing of a detergent molecule in a micelle is related to a conical volume swept by rotation of the molecule on a long axis, that is, on the axis defined by the extended hydrophobic tail and the headgroup. The presence of the branch group of a detergent of the invention results in a smaller volume cone, and a narrower angle of the conical volume at its point, than is present in a similar cone related to a molecular volume in a molecule of an art detergent in a micelle.

In various embodiments, the invention provides a micelle comprising a detergent of the invention. A typical micelle is illustrated in Figures 1 and 4 herein. In a micelle of the invention, that is, a micelle comprising a detergent of the invention of formula (I), the micelle can be more densely packed than a micelle composed of straight chain detergents. A micelle of the invention can further comprise a protein extracted from biological material. For example, a micelle can include a lipophilic protein such as an intrinsic cell membrane protein.

Figure 2 shows a graph of micellar sizes as determined by dynamic light scattering of various compositions comprising detergents of the invention and art detergents. Micellar size is typically less in micelles comprising detergents of the invention.

In various embodiments, in a micelle of the invention comprising a non- covalently contained protein, the protein is more stable over a period of time compared to a micelle comprising straight chain detergents containing the protein. Figure 3 shows the stability over time of three different proteins included in micelles composed of detergents of the invention compared to art detergents. As can be seen, the inventive micelles provide a relatively high degree of protein stability over time compared to art micelles. In various embodiments, the invention provides a method of purification of a protein from biological material comprising extraction of the protein in a plurality of the micelles of the invention or of micelles comprising a detergent of the invention. For example, the method of purification can include crystallization of the protein.

In various embodiments, a method of preparation of a compound of formula (I) of the invention, the method comprising contacting a compound of formula (II)

(H) and a compound of formula P-L-Y wherein one of X or Y comprises a reactive atom suitable for coupling with the compound of formula (II) and the other of X or Y comprises a leaving group, wherein the reaction to product the compound of formula (I) takes place with elimination of X by Y or Y by X under conditions sufficient to bring about formation of the compound of formula (I). For example, X can include an oxygen atom, and P-L-Y a suitably protected cholinephosphorylchloride or other activate phosphate group, which mutually couple with elimination of chloride and formation of a new oxygen bridge linking the phosphocholine moiety to the tail plus branch moiety of the compound of formula (I). Or, X can include an oxygen atom and P-L-Y can be a suitably protected activated carbohydrate moiety, such as an O-protected protected glycosyl sulfonate ester or a O-protected glucosyl-glucosyl sulfonate ester.

Examples Example 1

Phosphocholine head groups

As shown in the process as depicted in Figure 5, micoscale NMR was used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized β- barrel E.coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with μg-amounts of uniformly ²H,^l5N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-F.

Instrumentation. NMR spectra for characterization of intermediates and final products of detergent syntheses were recorded on Bruker DRX-500, AMX-400 or AMX-300 instruments. High-resolution mass spectra (HRMS) of the synthetic detergents were recorded on a VG ZAB-ZSE mass spectrometer using ESI (electrospray ionization).

Representative Synthetic Procedure

p.

2-Tridecanol ¹ 34-Fos

A mixture of 2-tridecanol (243 mg, 1.22 mmol) and triethylamine (215 mg, 2.13 mmol) in toluene (10 mL) was cooled to 0 °C. To this solution was added dropwise 2-chloro-l,3,2-dioxaphospholane (233 mg, 1.64 mmol) with stirring. The solution was kept at 0 ⁰C for 15 min before being raised to room temperature, then it was continued stirring for 4 h. The resulted ammonium salt precipitated and was removed by filtering, and the solvent was evaporated in vacuo. The oily residue 1 (318 mg) was directly dissolved into 10 mL of anhydrous acetonitrile in a pressure bottle. Into the solution was added 2 mL of trimethylamine while cooling by a dry ice-acetone bath. The reaction was heated to 70 °C for 2 days and then diluted with methanol. The mixture was concentrated under vacuum and the residue was subject to column chromatography on silica gel to give 34-Fos (360 mg, 80%) as a white solid. ¹H NMR (500 MHz, CD3OD) δ 4.30-4.23 (m, 3H), 3.62-3.60 (m, 2H), 3.21 (s, 9H), 1.64-1.28 (m, 20H), 1.25 (d, J= 6.5 Hz, 3H), 0.89 (t, J= 7.0 Hz, 3H) ppm; ¹³C NMR (125 MHz, CD3OD ): δ 73.4, 66.5, 59.2, 53.7, 38.2, 38.1, 32.1, 29.9, 29.8, 29.5, 25.5, 22.8, 21.1, 13.5 ppm; HR-MS: calcd for Ci₈H₄iNO₄P⁺ [M + H⁺]: 366.2768. found 366.2771. Detergent synthesis and purification. Phosphocholines with branched alkyl chains were synthesized directly from the respective commercially available secondary alcohols. Each detergent used for refolding and NMR experiments was prepared in 100 milligram batches, purified by reverse-phase HPLC (after silica-gel column chromatography) to > 99% purity, and its structure was confirmed by NMR and by mass spectrometry Determination of critical micelle concentration (CMC). CMC values of detergents were determined by monitoring the fluorescence of the ammonium salt of 8-anilino- 1 -naphtalenesulfonic acid (ANS), which becomes highly fluourescent (λ_ex = 405 nm; ^_em ⁼ 465 nm) when incorporated into the hydrophobic micellar environment.³⁵ Solutions containing 10 μM ANS and a range of concentrations for each detergent were examined on a DXT880 multiplate spectrofluorimeter (Beckman Coulter). The CMC is defined as the breakpoint in the plot of fluorescence intensity vs. concentration.

Expression and Purification ofOmpX in D₂O medium. OmpX was pre-cloned in the pET 3b plasmid and transformed into E. coli BL-21 (DE3) pLysS (Stratagene) competent cells for expression. One colony was used to inoculate a culture flask containing 20 mL of LB broth with the necessary antibiotics and shaken at 37 °C overnight. The cell culture was adapted to D₂O by inoculating 4 mL of standard M9 minimal medium containing 33% v/v D₂O with 40 μL of this LB culture and shaken at 37 ⁰C overnight. 100 μL of the 33% D₂O culture was then used to inoculate 10 mL of M9 minimal medium with 66% v/v D₂O and similarly shaken overnight. A uniformly ²H,¹⁵N-labeled OmpX sample was prepared by inoculating 1 L of standard M9 minimal medium containing 99% D₂O (Spectra Gases) and 1 g/L of [¹⁵N]-ammonium-chloride (CIL) with the resulting 10 mL of D₂O-adapted culture. This culture was shaken at 37 ⁰C until the cell density reached an OD₆₀O of approximately 1.0, and protein expression was then induced with 1 mM isopropyl-α- D-thiogalactopyranoside. Upon reaching the stationary growth phase after approximately 4 hours, the cells were harvested at 5,000 g for 10 minutes. TE buffer (20 mM Tris-HCl at pH = 8.0, 5 mM EDTA) was used to resuspend the cell pellet in a volume corresponding to approximately 10 times the wet cell mass in grams, and sonicated for 25 minutes using a Misonix 3000 sonicator. The solution was pelleted at 4,300 g for 1 hour and the supernatant was discarded. The remaining insoluble pellet was resuspended in the same volume of TE buffer plus 2% (v/v) Triton X-IOO at room temperature, pelleted at 4,300 g for 30 minutes, and the supernatant was discarded. Triton was then removed by repeating the same procedure with TE buffer. OmpX was recovered from the inclusion bodies by resuspending the cell debris in TE buffer plus 6 M urea for 2 hours at room temperature. Remaining cellular debris were then pelleted at 48,000 g for 20 minutes at 4 ⁰C, and the unfolded OmpX in the supernatant was recovered for ion exchange purification. Purification steps were performed using an AKTA purifier (Amersham/GE) with a 5 mL HiTrap Q HP anion exchange column (Amersham/GE). OmpX was bound to the column using buffer A (20 mM Tris-HCl at pH = 8.5, 6 M urea) and eluted with a linear NaCl gradient of 0 — 1 M at a rate of 33.3 mM per minute. All impure fractions were exchanged into buffer A and repurified, using the previously mentioned steps. The pooled pure OmpX fractions were concentrated to 1 mL using Vivaspin 2 (Sartorius) concentrators with a 3 kDa molecular weight cutoff. The concentrated sample was desalted with buffer A using a 5 mL HiTrap desalting column (Amersham/GE). Reconstitution of OmpX into Detergent Micelles for SDS gel Electrophoresis. Unfolded OmpX (1 1.9 mg/mL, 20 mM Tris at pH 8.5, 6 M urea) was diluted 30-fold with 1% (wt/vol) detergent solution at 25 °C (1% detergent concentration is well above the CMC for each of the commercial or newly synthesized detergents used). The detergents were pre-dissolved in either of two buffers, 50 mM citrate with 1 mM EDTA for the pH-range 3.4-6.0 or 50 mM Tris-HCl with 1 mM EDTA for the pH-range 6.0-9.8. The solution was incubated at 25 ⁰C for 3 hours before running the SDS-gel electrophoresis (using 15% polyacrylamide gels and without boiling the samples). The gels were stained with Coomassie blue and the fractions of refolded protein were estimated with densitometry.

Reconstitution ofOmpX into Detergent Micelles for NMR spectroscopy. OmpX reconstitution was carried out in DHPC (Avanti Polar Lipids) detergent micelles was modified for micro-scale experiments with a variety of widely different detergents, as shown in Figure 5. At 4 °C, 100 μL of unfolded OmpX at a concentration of 10 mg/mL was added to 600 μL of refolding buffer (20 mM Tris-HCl at pH = 8.5, 5 mM EDTA, 600 mM L-Arg, and 2% (w/v) detergent) over a period of 4 hours with vigorous stirring. The resulting OmpX solution was vigorously stirred at 4 ⁰C for about 16 hours. The detergent-refolded OmpX was then collected and exchanged into NMR buffer (20 mM sodium phosphate at pH = 6.8, 100 mM NaCl, 0.3% NaN₃, 10 % D₂O, detergent) by way of concentration and dilution using Vivaspin 500 concentrators (10,000 molecular weight cut-off), alternatively using NMR buffer with and without 2% (w/v) of detergent. The final sample was concentrated to 50 μL. NMR Spectroscopy. All NMR experiments were recorded at 25 °C on a Bruker DRX-700 spectrometer (BrukerBiospin, Billerica, MA) equipped with a 1 mm TXI microprobe. The 1 mm NMR capillaries were filled with 7 μL of the solution containing the mixed OmpX-detergent micelles, using a 10 μL Gilson Syringe (Gilson Co., Reno, NV). 2D [¹⁵N₅ ¹H]-TROSY correlation experiments were recorded. Data processing and analysis were carried out using TOPSPIN 1.3 (Bruker) and XEASY, respectively. Results

OmpX is a 148-residue, 16.5 kD outer membrane protein with an 8-stranded β- barrel structure. Twenty-three commercially available detergents (Table 1) were selected for OmpX reconstitution, which included the most popular representatives of the sugar (glucoside and maltoside), zwitterionic (Fos-choline series and lauryldimethylamine-N-oxide), cholate (sodium cholate, CHAPS, and CHAPSO), and amphipol (PMAL series: amphiphilic polymer detergents) classes; selected structures are shown in Supporting Information (Figure Sl). OmpX is resistant to denaturation by SDS (as are many /^-barrel membrane proteins), yet this detergent inhibits refolding of the protein once it is denatured. Thus, folded and unfolded OmpX can be easily distinguished by SDS gel electrophoresis, migrating at 18 and 16 kD, respectively, on the standard Laemmli gels (15% polyacrylamide, Figure 6a). The folded and unfolded OmpX migrated at a reversed order (12 and 19 kD respectively) on Bis-Tris gels (4-12% gradient polyacrylamide). The readout by SDS-PAGE was used to determine the OmpX folding efficiency in the presence of two well-known detergents, n-dodecyl-/?-D-maltopyranoside (DDM) and Fos- choline-13 (TPC), as a function of pH (Figure 6b). OmpX refolding was thus found to be more efficient in TPC than in DDM in general, and was increasingly favored at higher pH, with > 90% folded protein achieved at pH 8.0 in TPC. The addition of folding additives, such as L-arginine and trimethyl amine oxide, had no observable effect (data not shown). Standardized folding conditions (pH 8.5 and 1% detergent concentration) were then used to screen the 23 aforementioned commercial detergents. As summarized in Tables 1 and 2, the Fos-choline series (hydrophobic chain length 10-14 carbons) were the only candidates to support almost complete refolding of OmpX. In contrast, detergents with only glucose as the polar head (including octyl- and nonylglucoside), cholate-based detergents and amphipols were least efficient, with < 5% refolding in most of the cases. The larger maltose head group provided some improvement in refolding yield (40-60%), and lauryldimethylamine-N-oxide (LDAO) also gave a good yield (80%).

The following compounds of the invention, and comparative compounds, were prepared: 195-Fos

— N© — NΘ — N©

O=P P'-O^Θ 0=P P'-O^Θ

115-Fos 116-Fos 117-Fos 141-Fos 142-Fos 144-Fos 145-Fos176-Fos 178-Fos 175-Fos 177-Fos 174-Fos

180-Fos 179-Fos 165-Fos 172-Fos 173-Fos 170-Fos 171-Fos 169-Fos

Table 1. List of commercial detergents used (Anatrace, Inc.) and estimated OmpX refolding yields (± 5% error) by SDS-PAGE assay.

Detergent Yield (%) Detergent Yield (%) Detergent Yield (%)

«-Decyl-/^D-

40 Triton X-100 20 Sodium cholate <5 m al topyranosi de

/j-Undecyl-/?-D-

55 Fos-choline-10 >90 CHAPS <5 maltopyranoside n-Dodecyl-/?-D-

55 Fos-choline-1 1 >90 CHAPSO <5 maltopyranoside n-Tridecyl->3-D-

60 Fos-choline-12 >90 PMAL-ClO <5 maltopyranoside

CYMAL-6 55 Fos-choline-13 >90 PMAL-C 12 <5

CYMAL-7 60 Fos-choline-14 >90 PMAL-C 16 10

C-HEGA-I l 35 n-Octyl-β-D- <5 Lauryldimethylamine-TV-

80 gluopyranoside oxide n-Nony\-β-O-

MEGA-8 <5% 10 gluopyranoside

Table 2. List of detergents of the invention and estimated OmpX refolding yields (± 5% error) by SDS-PAGE assay.

Detergent Yield (%) Detergent Yield (%) Detergent Yield (%)

30-Fos > 90 168-Fos > 90 171-Fos 55

31-Fos > 90 167-Fos 84 142-Fos 52

34-Fos > 90 165-Fos 71 193-Fos 50

35-Fos > 90 140-Fos 70 173-Fos 49

38-Fos > 90 172-Fos 69 170-Fos 46

1 15-Fos > 90 1 17-Fos 67 175-Fos 45

1 16-Fos > 90 37-Fos __ 63 178-Fos 44

180-Fos > 90 190-Fos 60 194-Fos 44

179-Fos > 90 177-Fos 60 174-Fos 43

185-Fos > 90 192-Fos 58 195-Fos 30

228-Fos > 90 141-Fos 57 169-Fos 28

229-Fos > 90 137-Fos 56 144-Fos 28

182-Fos > 90 176-Fos 55 145-Fos 25

138-Fos > 90 33-Fos 55 41 -Fos < 5

Table 3. Micro-scale OmpX reconstitution with different detergents. Detergent structures can be found in Figures 3 and S2.

^a Recoverable OmpX loss due to precipitation during refolding. ^b Recoverable OmpX loss due to buffer exchange (Figure 1). ^c 2D [¹⁵N₅ ¹H]-TROSY spectra (Figure 4) rated "++" for spectra suitable for a structure determination, "+" for spectra with detergent artifacts, and "-" for spectra showing that OmpX was solubilized, but not reconstituted.

In preparation of compounds of the invention wherein the polar head group

comprises a phosphocholine moiety, we introduced two types of modifications to make the detergents more closely resemble natural phospholipids (see Figure 7). First, a spacer group to mimic the glycerol motif in lipids was inserted between the charged, potentially denaturing phosphocholine head group and the alkyl chain. Second, short branches were added to mimic the dialkyl chain structure of lipids while maintaining their solubility.

Similar refolding experimentations as described above yielded the data summarized in Table 2. Quite a number of the newly synthesized phosphocholine detergents could refold OmpX as efficiently as commercial ones. We have synthesized and evaluated other classes of new detergents, and the present study now corroborates that the phosphocholine series is among the top performers among all our new detergent structures for refolding OmpX. From the results shown in Table 2, it is apparent that detergents bearing dialkyl chains (branched through either an ester or amide linkage) are much less effective in refolding OmpX than analogues with single straight alkyl chains. Almost all the best candidates, with > 90% refolding, are either single alkyl chain detergents or those with a single methyl group on the branch (30-Fos, 31-Fos, 34-Fos and 38-Fos).

To extend the electrophoresis assay, we used uniform H- and N-labeling of the protein OmpX and TROSY-type 2D [¹⁵N, ¹H] -correlation NMR spectroscopy to assess the fold of OmpX in mixed micelles with different detergents. See Figure 8. A screening protocol using micro-coil NMR equipment was first tested with OmpX-DHPC mixed micelles (OmpX/DHPC). The 2D [¹⁵N₅ ¹H]-TROSY correlation spectrum of OmpX/DHPC measured with the 1 mm TXI microprobe shows a wide dispersion of cross peaks with roughly uniform intensities and line shapes. Comparison with the previously determined chemical shift list (BMRB accession code 4936) showed that all cross peaks were visible, with the sole exception of the four peaks belonging to the polypeptide segment E32-S35. This shows that our screening protocol, which required only about 10 μL of protein solution (active volume in the NMR microcoil = 7 μL), provided high-quality [¹⁵N,1H]-correlation maps that can be used as diagnostic fingerprints to assess the conformation of OmpX in mixed micelles with detergents.

Using the electrophoresis results to guide the selection of candidates, we examined [²H,¹⁵N]-labeled OmpX in mixed micelles with five newly synthesized detergents. The structures are representative of distinct classes (spacer groups) that fully refolded the protein (138-Fos, CMC 0.1%; 179-Fos, CMC 0.062%; 115-Fos, CMC 0.025%; 34-Fos, CMC 0.036%; 185-Fos, CMC 0.01%). A widely used commercial phosphocholine detergent (TPC, CMC 0.027%) was also used for comparison. OmpX was readily reconstituted into these detergent micelles at high concentrations (100 μL of 10 mg/mL unfolded OmpX was refolded into 600 μL detergent solution by slow addition); no protein precipitation was observed, and SDS-gel electrophoresis experiments confirmed complete solubilization.

The 2D [¹⁵N₁ ¹H]-TROSY correlation spectra of the mixed micelles with OmpX/115-Fos, OmpX/TPC, OmpX/34-Fos and OmpX/185-Fos all show a cluster of broad lines in the center of the spectrum and variable numbers of additional, resolved cross peaks of unequal shapes and intensities, which indicates that these protein samples are not homogeneously folded, and probably form non-specific soluble aggregates with these detergents.

Example 2 Carbohydrate polar head groups

Exemplary synthesis of branched alkyl β-D-pyranomaltoside detergents. A mixture of 2-dodecanol (5.0 g, 26.6 mmol), 1-thioethylhepta-O-benzoyl-β-D-maltose (29.7 g, 26.6 mmol) and 4 A molecular sieves (5.0 g) was stirred in anhydrous dichloromethane for 30 minutes. The solution was cooled to -15 ⁰C, to which was added N-iodosuccinimde (5.99 g, 26.6 mmol) and silver trifluoromethanesulfonate (2.04 g, 7.98 mmol) under N₂. The reaction mixture was slowly warmed up to room temperature and continued to stir for overnight. The reaction was quenched with saturated sodium bicarbonate solution and the aqueous phase was extracted with dichloromethane. The combined organic layers were washed with saturated sodium thiosulfate solution and brine, dried over sodium sulfate, filtered, and the solvent was evaporated in vacuo to afford the alkoxyl glycosylation product as yellow oil. The oil was subsequently dissolved in anhydrous MeOH (100 mL). To this solution was added catalytic amount of sodium methoxide (143 mg, 2.6 mmol) at room temperature. The reaction was stirred for 2 hours and the reaction mixture was neutralized with Dowex-50 (H⁺) to pH 6. The resulting mixture was filtered and the filtrate was concentrated in vacuo. The crude product was first roughly isolated by column chromatography over silica gel before further purification by Dowex l(OH) chromatography (methanol). 2-Dodecyl-β-D-maltopyranoside was obtained as a white powder (10.1 g 75% over 2 steps, > 99% purity), and no α-glycoside isomer was detected using this procedure. ¹H NMR (400 MHz, CD3OD) δ = 5.11 (d, J = 4.0 Hz, IH), 4.30 (d, J = 7.6 Hz, 0.5 H), 4.30 (d, J = 8.0 Hz, 0.5 Hz), 3.84-3.74 (m, 4 H), 3.66-3.54 (m, 4 H), 3.52-3.46 (m, IH), 3.41-3.38 (m, IH), 3.32-3.13 (m, 4 H), 1.61- 1.52 (m, 1 H), 1.40-1.25 (m, 20 H), 1.18 (d, J = 6.4 Hz, 1.5 H), 1.12 (d, J = 6.4 Hz, 1.5 H), 0.85 (t, J = 6.8 Hz, 3 H) ppm; ¹³C NMR (100 MHz, CD3OD ): δ = 102.6, 101.7, 100.9, 80.2, 80.1, 76.5, 75.3, 74.5, 73.8, 73.5, 73.0, 70.3, 61.5, 61.0, 37.2, 36.4, 31.9, 29.5, 29.2, 25.3, 25.2, 22.5, 20.7, 18.6, 13.2 ppm; IR (film) V_1118x = 3374, 2925, 2360, 1075, 774 cm^"1; HR-MS: calcd for C₂₄H₄₇O₁₁ ⁺ [M + H⁺]: 511.3113, found 511.3114.

Table 4. Exemplary compounds of the invention with carbohydrate polar head groups

Compounds of the invention shown in Table 4 include compounds numbered 011, 012, 022, 022, 032, 040, 044, and 196-201-Glu, and 063-076, 106, 237, 241, 242, 243, and 253-Mal.

Synthetic Scheme

CH₃(CH₂)_ZCH₂Br 8 MgJ₂ CH₃(CH₂)₇CH₂MgBr 9 I) NIS₁ AgOTf, 2 2) NaOMe/MeOH

\^" CuI

11 OH

(S)J O THF, -78⁰C to O⁰C 2 hours

(S)-12

General Procedures. All reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions, unless otherwise noted. Dry tetrahydrofuran (THF), and methylene chloride (CH₂Cl₂) were obtained by passing commercially available pre-dried, oxygen-free formulations through activated alumina columns. Yields refer to chromatographically and spectroscopically (¹H NMR) homogeneous materials, unless otherwise stated. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60F-254) using UV light as visualizing agent and an ethanolic solution of phosphomolybdic acid and cerium sulfate, and heat as developing agents. E. Merck silica gel (60, particle size 0.040- 0.063 mm) was used for flash column chromatography. Preparative thin-layer chromatography (PTLC) separations were carried out on 0.25 or 0.50 mm E. Merck silica gel plates (60F-254). NMR spectra were recorded on Bruker DRX-500, AMX- 500 or AMX-400 instruments and calibrated using residual undeuterated solvent as an internal reference. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, quin = quintuplet, sext = sextet, sep = septet, br = broad. IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR spectrometer. Electrospray ionization (ESI) mass spectrometry (MS) experiments were performed on an API 100 Perkin Elmer SCIEX single quadrupole mass spectrometer at 4000V emitter voltage. High- resolution mass spectra (HRMS) were recorded on a VG ZAB-ZSE mass spectrometer using MALDI (matrix-assisted laser-desorption ionization) or ESI (electrospray ionization).

NaOMe/MβOH

Ethyl 2,3,6,-tri-0-(2,3,4,6-tetra-0-Benzoyl-β-D-Glucopyranosyl)-thio-β-D- glucopyranoside 2. To a solution of Octaacetyl-β-maltoside Sl (50 g, 73.7 mmol) in anhydrous dichloromethane (300 mL) at O⁰C was added thioethannol (4.57 g, 73.7 mmol) and boron trifiuoride diethyl etherate (10.46 g, 73.7 mmol). The reaction mixture was warmed to room temperature and stirred for 2 hours. Saturated aqueous NaHCO₃ solution was added to quench the reaction. The phases were separated and the aqueous phase was extracted with dichloromethane. The combined organic layers were washed with brine, dried over Na₂SO₄. The filtrate was concentrated to afford S2 (51.0 g) as a yellow solid which was used directly for the next step. The yellow residue was dissolved in anhydrous MeOH (250 mL) and sodium methoixde (398 mg, 7.37 mmol) was added to the solution. The reaction was stirred at room temperature overnight. Then reaction mixture was neutralized with Dowex- 50 (H⁺) to pH = 6. The resulting mixture was filtered and the filtrate was concentrated to provide S3 (27.7 g) as colorless oil, which was used directly for the next step. The oily residue and DMAP (500 mg, 4.10 mmol) was dissolved in pyridine (250 mL). After cooling by an ice bath, benzoate chloride (103.2 g, 737 mmol) was added dropwise to the mixture. The reaction mixture was warmed to room temperature and stirred overnight. The reaction was diluted with water and ethyl acetate. Excess Benzoate chloride was washed by saturated NaHCO₃ aqueous solution. The organic layers were concentrated and the resulting residue was purified by column chromatography over silica gel to give 4 (70.0 g, 85% over 3 steps) as a light yellow solid.

Data for Compound 2: [α]_D ²⁵ = +60.56 (CHCl₃, c = 0.5), ¹H NMR (400 MHz,

CHCl₃) δ = 8.11-7.17 (m, 35H), 6.21-5.24 (m, 4 H), 5.01-3.88 (m, 10H), 2.81-2.58 (m, 2H), 1.22 (t, J = 7.6Hz, 3H) ppm; ¹³C NMR (100 MHz, CHCl₃ ): δ = 166.3-

165.2, 133.6-133.1, 130.2-128.3, 96.5, 83.6, 76.3, 73.3, 71.2, 70.1, 69.3, 63.9, 62.7, 60.6, 24.4, 15.1 ppm;HR-MS: calcd for C₃₆H₅₄O_nSNa⁺ [M + Na⁺]: 1137.2974, found 1137.2961

Ma U CH₃(CH₂)₇CH₂Br __∑_1 „ CH₃(CH₂)₇CH2MgBr 8 ⁹

Nonylmagnesium bromide 9. To a suspension of Mg (1.74 g, 72.5 mmol) in THF

(50 mL) in argon protect flask at O⁰C was added catalytic I₂ (50 mg) to activate Mg, and a solution of nonyl bromide 8 (1Og, 48.3 mmol) in THF (50 mL) was added dropwise with a gentle heating. After the addition, the mixture was warmed to 45⁰C for 30 min and cooled to room temperature. The resulting supernatant was used directly for the next step.

(R)-13 OH Data of (R)-13: [α]_D ²⁵ - - 6.3 (CHCl₃, c = 0.5) ; ¹H NMR (400 MHz, CD3OD) δ - 3.82-3.78 (m, IH), 1.46-1.27 (m, 20H), 1.19 (d, J = 6.4 Hz, 3H), 0.87 (t, J = 6.8 Hz, 3H) ppm

9+1

2-decyl-β-D-maltopyranoside 15: ¹H NMR (400 MHz, CD3OD) δ = 5.15 (d, J = 3.6 Hz, IH), 4.33 (d, J = 7.6 Hz, 0.5 H), 4.33 (d, J = 7.6 Hz, 0.5H), 3.87-3.78 (m, 4H), 3.70-3.50 (m, 5.0 H), 3.45-3.42 (m, IH), 3.36-3.2 (m, 4H), 1.63-1.57 (m, IH), 1.43-1.28 (m, 14H), 1.21 (d, J = 6.0 Hz, 1.5 H), 1.15 (d, J = 6.0 Hz, 1.5 H), 0.88 (t, J = 6.4 Hz, 3.0 H) ppm; ¹³C NMR (100 MHz, CD3OD ): δ = 102.6, 101.7, 100.9, 80.2, 80.1, 76.7, 76.7, 76.5, 75.3, 75.3, 74.6, 73.9, 73.6, 73.0, 61.6, 61.1, 37.3, 36.5, 31.9, 29.5, 25.4, 25.2, 22.5, 20.8, 18.6, 13.3 ppm; IR (film) V_max= 3383, 2925, 2855, 1150, 1076, 1023 cm^"1;

9+2

3-undecyl-β-D-maltopyranoside 16: ¹H NMR (500 MHz, CD3OD) δ = 5.17 (d, J = 3.5 Hz, IH), 4.34 (d, J = 8.0 Hz, 0.5 H), 4.33 (d, J = 8.0 Hz, 0.5H), 3.89-3.80 (m,

3H), 3.72-3.60 (m, 5.0 H), 3.58-3.53 (m, IH), 3.47-3.44 (m, IH), 3.37-3.21 (m, 3H), 1.61-1.52 (m, 4H), 1.35-1.31 (m, 12H), 0.96-0.89 (m, 6 H) ppm; ¹³C NMR (125 MHz, CD3OD ): δ = 102.6, 102.3, 101.9, 81.1, 80.6, 80.4, 77.0, 75.5, 74.1, 73.9, 73.8, 73.2, 70.6, 61.8, 61.4, 34.3, 33.5, 32.1, 30.1, 29.7, 27.7, 26.3, 25.4, 25.1, 22.8, 13.5, 9.1, 8.6 ppm; IR (film) V_max = 3349, 2924, 2854, 1150, 1074, 1026, 777 cm^"1; HR-MS: calcd for C₂₃H₄₅O_n ⁺ [M + H⁺]: 497.2956, found 597.2951

9+3 4-dodecyl-β-D-maltopyranoside 17: ¹H NMR (400 MHz, CD3OD) δ = 5.16 (d, J = 3.6 Hz, IH), 4.31 (d, J = 7.6 Hz, 1 H), 3.88-3.78 (m, 3H), 3.71-3.51 (m, 6 H), 3.45- 3.42 (m, IH), 3.35-3.18 (m, 4H), 1.58-1.29 (m, 18H), 0.94-0.8 (m, 6 H) ppm; ¹³C NMR (100 MHz, CD3OD ): δ = 102.3, 102.1, 101.7, 80.2, 79.3, 76.7, 75.3, 73.9, 73.5, 73.0, 70.3, 61.6, 61.1, 36.1, 33.8, 29.8, 29.7, 29.5, 29.2, 24.9, 22.5, 18.1, 13.2 ppm; IR (film) V_max= 3394, 2924, 2853, 1145, 1071, 1027 cm-1; HR-MS: calcd for C₂₄H₄₇Ol IN⁺ [M + H⁺]: 511.3119, found 511.3120

1 0+1 2-undecyl-β-D-maltopyranoside 18 ¹H NMR (500 MHz, CD3OD) δ = 5.17 (d, J = 4.0 Hz, IH), 4.36 (d, J = 8.0 Hz, 0.5 H), 4.35 (d, J = 8.0 Hz, 0.5 Hz), 3.89-3.79 (m, 4H), 3.72-3.60 (m, 4 H), 3.57-3.52 (m, IH), 3.47-3.44 (m, IH), 3.38-3.19 (m, 4H), 1.66-1.60 (m, IH), 1.47-1.31 (m, 16H), 1.24 (d, J = 6.0 Hz, 1.5 Hz, 1.18 (d, J = 6.0 Hz), 0.91 (d, J = 6.5 Hz, 3H) ppm; ¹³C NMR (125 MHz, CD3OD ): δ = 102.9, 101.9, 101.2, 76.9, 76.7, 75.6, 74.9, 74.1, 73.8, 73.2, 70.6, 61.8, 61.3, 37.4, 36.7, 32.1, 29.8, 29.5, 25.6, 25.4, 22.8, 21.0, 18.8, 13.5 ppm; IR (film) W_109x= 3363, 2922, 2852, 1146, 1072, 1036 cm-1 HR-MS: calcd for C₂₃H₄₄Ol INa⁺ [M + Na⁺]: 519.2776, found 519.2786

3-dodecyl-β-D-maltopyranoside 19 ¹H NMR (500 MHz, CD3OD) δ = 5.17 (d, J = 4.0 Hz, IH), 4.34 (d, J = 7.5 Hz, 0.5 H), 4.33 (d, J = 7.5 Hz, 0.5 Hz), 3.89-3.80 (m, 3H), 3.72-3.60 (m, 5 H), 3.57-3.52 (m, IH), 3.46-3.44 (m, IH), 3.36-3.21 (m, 4H), 1.61-1.31 (m, 20H), 0.96-0.90 (m, 6H) ppm; ¹³C NMR (125 MHz, CD3OD ): δ = 102.6, 102.3, 101.9, 81.1, 80.6, 80.4, 77.0, 75.5, 74.1, 73.9, 73.8, 73.2, 70.6, 61.8, 61.4, 34.3, 33.5, 32.1, 29.8, 29.5, 27.7, 26.3, 25.4, 25.1, 22.8, 13.5, 9.1, 8.6 ppm; IR (film) V_max= 3353, 2923, 2854, 1147, 1073, 1022 HR-MS: calcd for C₂₄H₄₆Ol INa⁺ [M + Na⁺]: 533.2932, found 533.2938

10+3

4-tridecyl-β-D-maltopyranoside 20 ¹H NMR (500 MHz, CD3OD) δ = 5.24 (d, J = 3.5 Hz, IH), 4.40 (d, J = 8.0 Hz, 0.5 H), 4.40 (d, J = 8.0 Hz, 0.5 Hz), 3.96-3.87 (m, 3H), 3.78-3.60 (m, 6H), 3.53-3.50 (m, IH), 3.43-3.27 (m, 4H), 1.63-1.38 (m, 18 H), 1.02-0.96 (m, 6H) ppm; ¹³C NMR (125 MHz, CD3OD ): δ = 102.5, 102.4, 101.9, 80.4, 79.6, 79.4, 77.0, 75.5, 74.1, 73.9, 73.8, 73.2, 70.6, 61.8, 61.4, 37.2, 36.4, 35.0, 34.1, 32.1, 29.8, 29.5, 25.1, 22.8, 18.5, 18.3, 13.5 ppm; IR (film) V_max = 3370, 2924, 2854, 1148, 1074, 1026

3-tridecyl-β-D-maltopyranoside 22: ¹H NMR (500 MHz, CD3OD) δ = 5.25 (d, J = 4.0 Hz, IH), 4.41 (d, J = 8.0 Hz, 0.5 H), 4.40 (d, J = 8.0 Hz, 0.5 Hz), 3.97-3.87 (m, 3 H), 3.79-3.68 (m, 5 H), 3.65-3.60 (m, IH), 3.54-3.51 (m, IH), 3.44-3.28 (m, 3 H), 1.68-1.37 (m, 20 H), 1.03-0.96 (m, 6 H) ppm; ¹³C NMR (125 MHz, CD3OD ): δ - 102.6, 102.3, 101.9, 81.1, 80.4, 76.9, 75.5, 74.1, 73.9, 73.8, 73.2, 70.5, 61.8, 61.4, 34.3, 33.5, 32.1, 29.8, 29.5, 27.7, 26.3, 25.4, 25.2, 22.8, 13.5, 9.1, 8.7 ppm, IR (film) V_max= 3366, 2922, 2853, 2359, 1147, 1073, 1022, 759 cm^"1;

4-tridecyl-β-D-maltopyranoside 23 ¹H NMR (500 MHz, CD3OD) δ = 5.24 (d, J = 3.5 Hz, IH), 4.40 (d, J = 8.0 Hz, 0.5 H), 4.40 (d, J = 8.0 Hz, 0.5 Hz), 3.95-3.87 (m, 3 H), 3.79-3.67 (m, 5 H), 3.64-3.60 (m, IH), 3.53-3.51 (m, IH), 3.43-3.27 (m, 4 H), 1.63-1.37 (m, 22 H), 1.02-0.96 (m, 6 H) ppm; ¹³C NMR (125 MHz, CD3OD ): δ = 102.5, 102.4, 101.9, 80.4, 79.6, 76.9, 75.5, 74.1, 73.7, 73.1, 70.6, 61.8, 61.4, 37.2, .36.4, 35.0, 34.1, 32.1, 30.1, 29.8, 29.5, 25.1, 22.8, 18.5, 18.3, 13.6, 13.5 ppm

12+1

2-tridecyl-β-D-maltopyranoside 24 ¹H NMR (400 MHz, CD3OD) δ = 5.14 (d, J = 3.6 Hz, IH), 4.32 (d, J - 7.6 Hz, 0.5 H), 4.32 (d, J = 7.6 Hz, 0.5 Hz), 3.87-3.77 (m, 4 H), 3.69-3.57 (m, 4 H), 3.54-3.49 (m, IH), 3.44-3.41 (m, IH), 3.35-3.16 (m, 3 H), 1.66-1.55 (m, 1 H), 1.42-1.27 (m, 20 H), 1.21 (d, J = 6.0 Hz, 1.5 H), 1.15 (d, J = 6.0 Hz, 1.5 H), 0.88 (t, J = 6.8 Hz, 3 H) ppm; ¹³C NMR (100 MHz, CD3OD ): δ = 102.6, 101.7, 100.9, 80.2, 80.1, 76.7, 76.5, 75.3, 75.2, 74.6, 73.9, 73.5, 73.0, 70.3, 61.5, 61.1, 37.2, 36.5, 31.9, 29.7, 29.6, 29.6, 25.4, 25.2, 22.5, 20.8, 18.6, 13.3 ppm; IR (film) V_m8x = 3359, 2924, 2853, 1145, 1070, 1027 HR-MS: calcd for C₂₅H₄₈O₁₁Na⁺ [M + Na⁺]: 547.3089, found 547.3090

12+2

3-tetradecyl-β-D-maltopyranoside 25 ¹H NMR (500 MHz, CD3OD) δ = 5.24 (d, J = 4.0 Hz, IH), 4.41 (d, J = 8.0 Hz, 0.5 H), 4.40 (d, J = 8.0 Hz, 0.5 H)₅ 3.96-3.86 (m, 3 H), 3.79-3.68 (m, 5 H), 3.65-3.60 (m, IH), 3.53-3.51 (m, IH), 3.43-3.28 (m, 3 H), 1.68-1.37 (m, 22 H), 1.03-0.96 (m, 6 H) ppm; ¹³C NMR (125 MHz, CD3OD ): δ = 102.6, 102.3, 101.9, 91.1, 80.6, 80.4, 76.9, 75.5, 73.9, 73.8, 73.2, 70.5, 61.8, 61.4, 34.3, 33.5, 32.1, 30.1, 29.8, 29.5, 27.7, 26.3, 25.4, 25.1, 22.8, 13.5, 9.1, 8.6 ppm; IR (film) V_max= 3322, 2924, 2853, 1145, 1071, 1027 cm-1

12+3

4-pentadecyl-β-D-maItopyranoside 26 ¹H NMR (400 MHz, CD3OD) δ = 5.15 (d, J = 4.0 Hz, IH), 4.31 (d, J = 7.6 Hz, 1 H), 3.86-3.78 (m, 3 H), 3.68-3.53 (m, 6 H), 3.44-3.41 (m, IH), 3.33-3.18 (m, 3H), 1.51-1.29 (m, 26 H), 0.90-0.87 (m, 6 H) ppm; ¹³C NMR (100 MHz, CD3OD ): δ = 102.2, 101.7, 80.2, 79.5, 76.7, 75.3, 73.9, 73.7, 73.6, 73.0, 70.3, 61.6, 61.1, 34.7, 33.7, 31.9, 31.9, 29.8, 29.7, 29.2, 25.1, 24.9, 22.6, 13.3 ppm IR (film) V_max = 3360, 2925, 1148, 1074, 1024 cm^"1;

13+1

2-tetradecyl-β-D-maltopyranoside 27 ¹H NMR (500 MHz, CD3OD) δ = 5.24 (d, J = 4.0 Hz, IH), 4.42 (d, J = 8.0 Hz, 0.5 H), 4.42 (d, J = 7.5 Hz, 0.5 H), 3.96-3.87 (m, 4 H), 3.77-3.67 (m, 4 H), 3.64-3.59 (m, IH), 3.56-3.51 (m, IH), 3.45-3.26 (m, 3H), 1.72-1.67 (m, IH), 1.52-1.37 (m, 20H), 1.31 (d, J = 6.0Hz, 1.5 H), 1.24 (d, J = 6.0 Hz, 1.5 H), 0.98 (t, J - 7.0 Hz, 3H) ppm; ¹³C NMR (125 MHz, CD3OD): δ = 102.2, 101.9, 101.1, 80.4, 80.3, 76.9, 75.8, 75.6, 75.5, 74.9, 74.1, 73.8, 70.6, 61.8, 61.3, 37.4, 36.7, 32.1, 30.0, 29.9, 29.8, 29.8, 29.5, 25.6, 25.4, 22.8, 21.0, 18.8, 13.5 ppm; IR (film) V_H18X = 3351, 2922, 2853, 1145, 1073, 1023 cm-1

14+1

2-pentadecyl-β-D-maltopyranoside 28 ¹H NMR (400 MHz, CD3OD) δ = 5.15 (d, J = 4.0 Hz, IH), 4.33 (d, J = 7.6 Hz, 0.5 H), 4.32 (d, J = 8.0 Hz, 0.5 H), 3.87-3.78 (m, 4 H), 3.68-3.57 (m, 4 H), 3.55-3.49 (m, IH), 3.44-3.41 (m, IH), 3.35-3.16 (m, 3H), 1.63-1.57 (m, IH), 1.43-1.27 (m, 26H), 1.21 (d, J = 6.0Hz, 1.5 H), 1.15 (d, J = 6.0 Hz, 1.5 H), 0.89 (t, J = 6.4 Hz, 3H) ppm; ¹³C NMR (100 MHz, CD3OD ): δ = 102.6, 101.7, 100.9, 80.2, 76.7, 75.4, 74.6, 73.9, 73.6, 73.0, 70.3, 61.6, 61.1, 37.2, 36.5, 31.9, 29.6, 29.6, 29.3, 25.4, 25.2, 22.5, 20.8, 18.6, 13.3 ppm; IR (film) V_max= 3361, 2924, 2853, 1145, 1075, 1029 cm-1

1 1 +1

(S)-2-dodecyl-β-D-maltopyranoside 12 [α]_D ²⁵ = 41.8 (MeOH, c = 0.5), ¹H NMR (400 MHz, CD3OD) δ = 5.14 (d, J = 3.6 Hz, IH), 4.32 (d, J = 7.6 Hz, 1 H), 3.89- 3.77 (m, 4 H), 3.69-3.57 (m, 4 H), 3.53-3.49 (m, 1 H), 3.44-3.40 (m, IH), 3.34-3.32 (m, IH), 3.27-3.16 (m, 2H), 1.63-1.55 (m, IH), 1.38-1.28 (m, 18H), 1.21 (d, J = 6.0Hz, 3 H), 0.88 (t, J = 6.8 Hz, 3H) ppm; ¹³C NMR (100 MHz, CD3OD ): δ = ppm; IR (film) V_max = 3365, 2924, 2853, 1145, 1071, 1029 cm^"1;

11 +1

(R)-2-dodecyl-β-D-maltopyranoside 14 [α]_D ²⁵ = 43.8 (MeOH, c = 0.5), ¹H NMR (400 MHz, CD3OD) δ = 5.14 (d, J = 3.6 Hz, IH), 4.32 (d, J = 7.6 Hz, 1 H), 3.86- 3.77 (m, 4 H), 3.69-3.57 (m, 4 H), 3.52 (t, J = 9.6 Hz, 1 H), 3.42 (dd, J = 4.0, 9.6 Hz, IH), 3.35-3.16 (m, 3H), 1.63-1.54 (m, IH), 1.42-1.27 (m, 18H), 1.14 (d, J = 6.4 Hz, 3 H), 0.87 (t, J = 6.8 Hz, 3H) ppm; ¹³C NMR (100 MHz, CD3OD): δ = ppm; IR (film) V_max= 3414, 2924, 2853, 1144, 1071, 1029 cm-1

Table 2 Summary of CMC values and HPLC retention

255 MaI

256 MaI Determination of Critical Micelle Concentration

CMC values of detergents were determined by monitoring the fluorescence of the ammonium salt of 8-anilino-l-naphtalenesulfonic acid (ANS), which becomes highly fluourescent (λex= 388 run; λem = 465 nm) when incorporated into the hydrophobic micellar environment.31 Solutions containing lOμM ANS and a range of concentration for each detergent were examined on a DXT880 multiplate spectrofluorimeter (Beckman Coulter). The CMC is defined as the breakpoint in the plot of fluorescence intensity vs concentration.

The Critical Micelle Concentration (CMC) assay and the relationship between classic detergents and branched detergents, each branched carbon was added as 0.5 carbon in the straight alkyl is shown in the graph below.

8.5 9.5 10.5 11.5 12.5 13.5 14.5

Alkyl length

Measurement of Micelle Size by Dynamic Light Scattering (DLS).

The hydrodynamic radius (R_h) of detergent micelles was determined on DynaPro Titan instrument (Wyatt Technology Corporation, CA) equipped with a plate reader and a laser operating at 830 nm. The scattered light was measured at an angle of 158° relative to the primary beam. 50 μL of various concentrations of detergents in dl water was placed in a 384-well plate, and all measurements were carried out at 20 ⁰C with triplicate samples, with 5 acquisitions for each well. All the detergent stock solutions were carefully filtered through 0.2 μm membrane and diluted with dl water which was filtered through 0.02 μm filter beforehand. The data were analyzed using the integrated Dynamics software with the instrument that analyzes the time scale of the scattered light intensity fluctuations by an autocorrelation function that gives R_h. The viscosity value of pure water (1.0 centipoise at 20 ⁰C) is used for all the analyses, with the assumption that low concentrations of detergents (< 0.8%) have little effect.

Micelle size by dynamic light scattering of different detergents in different concentrations

O)/ O)/ Oj/ Oj/ ^) / _NQ / _No / ^ / _NN ' _N\ / _N\ / _SN / $,' jy/ ^y £>/ £>s

Detergents

Preparation ofConnexin 26 for Stability Measurements and X-ray Crystallizations.

Connexin 26 (Cx26) with the two carboxy-terminal cysteines mutated to serine (C21 IS, C218S) and C-terminal hexahistadine tag added is expressed in Tn5 insect cells using a baculovirus vector. It is then extracted from membranes and purified by nickel affinity chromatography using each single detergent. Once purified the protein is exchanged into a buffer containing 20 mM MES, 1.0 M NaCl, protease inhibitors and the same detergent (2x CMC) using a PDlO desalting column.

For thermal stability measurements an aliquot (10 μg) of concentrated protein was diluted into 50 mM phosphate buffer (pH7.5) containing 1.0 M NaCl, 2x CMC of detergent and the CPM fluorescent dye [7-diethylamino-3-(4'- maleimidylphenyl)-4-methylcoumarin] (xx μM). Samples were then heated at 2 °C per minute in a Cary Eclipse spectrofluorimeter. Dye fluorescence was monitored using excitation and emission wavelengths of 387 and 463 ran, respectively.

For crystallization Cx26 was concentrated to approximately 6 mg/ml using ultrafiltration. Crystallization screens were set using an Innovadyne Screenmaker 96+8 robot. Crystals of Cx26 using both DDM and MaI 11-1 were grown at 20 ⁰C with a well solution containing 0.1 M Hepes, 0.2-0.3M LiSO₄ and 25-30% polyethylene glycol 400 at pH 7.0. Stereoselective syntheses of anomerically pure branched-chain maltoside detergents. Acetobromomaltose has been employed as the glycosyl donor in the literature syntheses of β-maltoside detergents. In the procedure, the glycosyl donor was activated by stoichiometric amounts of silver salt (Ag₂CO₃) and a trace of iodine. The formation of undesired α-anomer could be suppressed, albeit incomplete, by using a large excess (10 fold) of primary alkyl alcohol. However, the reaction of acetobromomaltose and 2-dodecanol under similar conditions was unsatisfactory, which was sluggish, possibly due to the steric effect of the secondary alcohol, and yielded only 30% of final product in a ratio of α:β = 1 :4. The low yield and stereoselectivity resulted in problematic purification, especially in large scale synthesis. Final success was achieved by using perbenzoylated ethylthiomaltoside. The reaction of 2-dodecanol and this thioglycoside was complete within 2 hours in the presence of 7V-iodosuccinimide (NIS) together with a catalytic amount of silver trifluoromethanesulfonate, and subsequent removal of the benzoyl protective groups using catalytic amounts of sodium methoxide in methanol afforded 2-dodecyl β- maltoside as the single anomer. Other branched alkyl β-maltosides were prepared in a similar way.

To probe the relationship between stereocenter coming from racemic alcohol and the performance of branched detergents in protein crystallization. Two chiral compounds of the typical branched detergent, 1 l+l_Mal were made. Commercially available nonyl bromide was transformed into the corresponding Grignard reagent. Copper-catalyzed epoxide ring-opening of (-)-propylene oxide with nonyl magnesium bromide afforded chiral alcohol in 88% yield. Further glycosidation and debenzoylation were achieved in the similar procedures to obtain final S and R enantiomers of product. Physico-chemical Properties of the branched detergents:

To investigate the influence of adding branches to the hydrophobic tail moeity with respect to detergent hydrophobicity, we measured their retention time by reverse phase HPLC. It was found that adding carbon atoms to the main chain increases the detergent's hydrophobicity while adding a carbon atom to the branch does not in as great an increase in hydrophobicity as measured by HPLC retention time. It was found that adding two carbons on branch makes the product more hydrophobic than adding one carbon to the main chain of the hydrophobic tail, as shown in the graph below.

Hydrophobicity test of classic detergents and branched detergents in reserved phase HPLC.

alkyl

Connexin Purification

Gap junction channels allow direct cell-to-cell movement of ions and signaling molecules to control the metabolic and electrical activities within tissues. The channel is formed by the end-to-end docking of two hemi-channels, each comprised of 6 connexin subunits. Each subunit contains 4 transmembrane domains (Ml to M4), and electron crystallography revealed that the hemichannel is formed by an annular bundle of 24 alpha-helices. Tight packing of the extracellular loops bridges the gap between cells and prevents exchange of molecules with the extracellular environment. Connexin 26 (Cx26) is one of the smallest family members, mutations of which are a common cause of nonsyndromic deafness.

While the relative positions of the transmembrane helices have been determined by cryoelectron 2D crystallography a high resolution X-Ray diffraction structure has yet to be determined (Unger et al. 1999, Fleishman et al. 2004, Oshima et al. 2007). In this study a novel branched chain maltoside detergent has been evaluated for its potential usefulness in generating 3D crystals of Cx26. Thermal stability measurements (Alexandrov et al, 2008, below) and crystallization screening of Cx26 using a branched chain maltoside detergent have been completed and compared to two commercially available maltoside detergents.

The gap junction protein Connxin 26 can be readily extracted and purified in MaI 11-1 with yields and purity similar to those obtained with UDM or DDM. The protein is well behaved and can be concentrated to levels suitable for 3-D crystallization. The solubility of Cx26 in short chain detergents such as OG or DM is insufficient for structural work. Electron micrographs (Fig. 9) of Cx26 in Mall 1- 1 show the expected donut shape indicating the presence of hexamers.

Upon thermal denaturation a cysteine in the forth transmembrane domain (C202) becomes accessible for labeling by a thio-reactive fluorescent dye which upon binding undergoes a large increase in quantum yield. This allows the thermal stability of the protein to be measured in a number of different detergents using only microgram quantities. Stability is good and did not differ greatly in DDM, UDM and MaI 11-1 detergents, with an average midpoint of the thermal transition of ~55°C (figure 10). The midpoint of the thermal transition is approximately 2°C lower for UDM and MaI 11-1 compared to DDM. This might indicate that the stability is either dominated by the protein itself or by the longer of the aliphatic chains with the branch have little effect upon protein stability.

Crystals of Cx26 were obtained by vapor diffusion under similar conditions using both DDM and Mall 1-1. However, the crystals differ in space group and unit cell dimension between the two detergents. Crystals obtained with DDM are of the monoclinic space group C2 with unit cell dimensions of x=171.7A, y=l 14.8A, z=153.5A, α=90°, β=l 12°, γ=90°. While crystals obtained from Mall 1-1 are of the hexagonal space group P3 or higher with unit cell dimensions x=y=102.5A, z=151.4A, α=β=90°, γ=120°. In both cases crystals grow to beyond 50um size over the course of about 1 week. Both crystal forms diffract anisotropically. Diffraction in the weakest direction is limited to approximately 7.5A for both detergents while diffraction in the strongest direction extends to 4.5A for the DDM crystals and 3.5A for the Mall 1-1 crystals. Monoclinic crystals were obtained from UDM along while hexagonal crystals could be generated from UDM by supplementing the crystallization drop with shorter chained detergents (OG or NG). In each case crystals grown from UDM were of lower quality than those with DDM or MaI 11-1. This shift in crystal symmetry upon introducing a branch in or mixture of the aliphatic chain is striking and is likely the result of micelle size and or shape since protein stability is similar in each.

Claims

WHAT IS CLAIMED IS:

1. A detergent adapted for use in crystallization of intrinsic cell membrane proteins, the detergent comprising a compound of formula (I):

(I) wherein

P is a polar head group;

L is a linker or is absent;

R is (Ci_-3)alkyl, monofiuoro- or polyfluoro(Ci.₃)-alkyl, or (C_3-5)cycloalkyl, p is 0 to about 2; q is O to about 3; r is about 4 to about 14; and a carbon atom labelled A is present in chiral form or in racemic form.

2. The detergent comprising a compound of formula (I) of claim 1 wherein a polar head group comprises organic, inorganic, or both, moieties.

3. The detergent comprising a compound of formula (I) of claim 1 wherein P comprises a carbohydrate moiety, a polyoxyethylene moiety, a phosphocholine moiety, or an amine-oxide moiety.

4. The detergent comprising a compound of formula (I) of claim 1 comprising a carboxylate, phosphate, amine oxide or ammonium group, or one or more oxygen atom, or any combination thereof.

5. The detergent comprising a compound of formula (I) of claim 1 wherein L comprises a group of the formula -O-, -S-, -C(O)O-, -OC(O)-, -NR^C(O)-, or- C(O)NR¹-, wherein R¹ is H or (C_1-3 alkyl) or -(O-CH2-CH2-O)m- wherein m = 1-3.

6. The detergent comprising a compound of formula (I) of claim 1 wherein P-L comprises an oxygen-linked carbohydrate moiety.

7. The detergent comprising a compound of formula (I) of claim 6 wherein the carbohydrate moiety comprises a monosaccharide or a disaccharide or a sugar alcohol moiety.

8. The detergent comprising a compound of formula (I) of claim 7 wherein the monosaccharide moiety comprises a β-glucosyloxy moiety.

9. The detergent comprising a compound of formula (I) of claim 8 wherein p is O, q is 0-2, and r is 6-11.

10. The detergent comprising a compound of formula (I) of claim 7 wherein the disaccharide moiety comprises a β-(l-4)-glucosyl-β-glucosyloxy (cellobiosyl) moiety or an α-(l-4)-glucosyl-β-glucosyloxy (maltosyl) moiety.

11. The detergent comprising a compound of formula (I) of claim 10 wherein p is O, q is 0-2, and r is 9-13.

12. The detergent comprising a compound of formula (I) of claim 1 wherein P-L comprises a phosphatidylcholine moiety.

13. The detergent comprising a compound of formula (I) of claim 12 wherein p is O, q is 0-2, and r is 8-12.

14. The detergent comprising a compound of formula (I) of claim 1 wherein P-L comprises an oxygen-linked polyoxyethylene moiety.

15. The detergent of claim 14 wherein P-L comprises a group of the formula HO-(CH₂CH₂O)_n-O- wherein n is 1 to about 12.

16. The detergent of claim 14 wherein P-L comprises a di ethyl eneglycol moiety.

17. The detergent comprising a compound of formula (I) of claim 1 wherein P-L comprises a nitrogen-linked amine oxide moiety.

18. The detergent of claim 17, wherein P-L comprises a dimethylammonium-N- oxide group.

19. The detergent comprising a compound of formula (I) of claim 1 wherein the compound of formula (I) comprises:

30-Fos 31-Fos 34-Fos 35-Fos 38-Fos 37-Fos 192-Fos 190-Fos 33-Fos 194-Fos 193-Fos 195-Fos

or any salt, hydrate, solvate, tautomer, or stereoisomer thereof.

20. The detergent comprising a compound of formula (I) of claim 1 wherein the compound of formula (I) comprises:

or any salt, hydrate, solvate, tautomer, or stereoisomer thereof.

21. The detergent comprising a compound of formula (I) of claim 1 wherein an angle formed by an envelope swept by rotation about a long axis of a molecule of the compound of any one of claims 1-13 is less than an angle formed by an envelope swept by rotation about a long axis of a molecule of a straight chain detergent.

22. A micelle comprising a detergent of claim 1.

23. The micelle of claim 22 wherein the micelle is more densely packed than a micelle composed of straight chain detergents.

24. The micelle of claim 22 further comprising an protein extracted from biological material.

25. The micelle of claims 24 wherein the protein comprises an intrinsic cell membrane protein.

26. The micelle of claim 24 wherein the protein is more stable over a period of time compared to a micelle comprising straight chain detergents containing the protein.

27. A method of purification of a protein from biological material comprising use of the micelles of any one of claims 22-26 or of micelles comprising a detergent of any one of claims 1-21.

28. The method of purification of claims 27 comprising crystallization.

29. A method of preparation of a compound of formula (I) of claim 1 , the method comprising contacting a compound of formula (II)

(H) and a compound of formula P-L-Y wherein one of X or Y comprises a reactive atom suitable for coupling with the compound of formula (II) and the other of X or Y comprises a leaving group, wherein the reaction to product the compound of formula (I) takes place with elimination of X by Y or Y by X under conditions sufficient to bring about formation of the compound of formula (I).