Acid-labile surfactants and methods for performing proteolysis or enzymatic digestion

Abstract

The present invention relates to acid-labile surfactants and methods for performing proteolysis or enzymatic digestion. The invention relates to a surfactant which is unstable to acid. In particular, the surfactants of the present invention include a dioxolane or dioxane functional group that enables the surfactant to hydrolyze in an acidic environment. The surfactant of this type can be used to enhance proteolysis / enzymatic digestion. Two components, namely a hydrophilic component and a hydrophobic component, are generally formed after a surfactant is destroyed by hydrolysis (the surfactant can cause a chromatographic problem). The polarity of the hydrophobic residues can be altered by altering the chemical properties of the hydrolyzable linkers, allowing them to be dissolved by significantly less organic solvents and minimizing the potential loss of the peptide material and expanding the available chromatographic conditions.

Description

[0001] This application is a divisional application of the invention application with patent application number 201980015629.4, application date February 26, 2019, and invention name “Acid-unstable surfactant and method for performing protein solubilization or enzymatic digestion”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 635,233, filed on February 26, 2018, entitled “ACID LABILE SURFACTANTS,” which is hereby incorporated by reference. Technical Field

[0004] The present invention relates to an acid-labile surfactant. This type of surfactant can be used to enhance protein solubilization / enzymatic digestion. After hydrolysis destroys the surfactant (which can cause chromatographic problems), two components are generally formed - a hydrophilic component and a hydrophobic component. By changing the chemical nature of the surfactant and / or the hydrolyzable linker, the hydrophobic residues can be made more polar, thereby allowing them to be dissolved by significantly less organic solvent and minimizing potential losses of peptide material. The surfactants disclosed in the present invention can expand the available chromatographic conditions; specifically, they allow the use of a larger gradient range. Background Art

[0005] Surfactants are used in a variety of applications. For example, surfactants are used commercially to clean manufactured products, remove coatings, in chemical processing, in emulsion polymerization, solubilize drugs, purify proteins, and in various bioanalytical applications.

[0006] A specific bioanalysis application using surfactants is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In the past three decades, SDS-PAGE has been widely used as a simple and relatively rapid tool for analyzing and purifying macromolecules such as proteins (UK Laemmli, Nature, Vol. 227, pp. 680-685, 1970). Sodium dodecyl sulfate (SDS) is an anionic surfactant that denatures proteins by forming stable complexes. During denaturation, SDS is bound to most proteins and peptides at a constant weight ratio of about 1.4:1. Therefore, SDS-protein complexes have almost the same charge density and therefore migrate in polyacrylamide gels according to molecular weight. If the gel has the correct porosity, the graph of log Mw to relative mobility Rf produces a linear relationship. The intensity of the swimming band after dyeing is a rough indicator of the amount present in the sample. When combined with another electrophoretic technique, isoelectric focusing, SDS-PAGE can separate complex mixtures into hundreds of discrete components.

[0007] The ability to estimate the size and amount of proteins has led to a variety of applications for SDS-PAGE. However, the technique has some disadvantages. For example, it is difficult to monitor and analyze samples separated by SDS-PAGE using mass spectrometry because SDS interferes with the sensitivity of mass spectrometry detection. In addition, it is difficult to separate SDS from SDS / protein complexes because the surfactant SDS forms an emulsion.

[0008] Several approaches have been tried to address these problems. Nonionic surfactants such as octyl β-pyranoglucoside have been used for mass spectrometry applications (P. Dainese Hatt, M. Quadroni, W. Staudenmann and P. James, Eur. J. Biochem., Vol. 246, pp. 336-343, 1997). However, electrophoretic separation still requires SDS and a time-consuming surfactant exchange step.

[0009] Another method is electroelution or electroblotting from polyacrylamide gels onto PVDF or nitrocellulose membranes. However, this method often results in a significant loss of protein recovery. Other methods that have been tried, which are also time consuming and can result in significant protein loss, are precipitation of proteins with guanidine hydrochloride (JE Schively, in Methods of protein microcharacterization; JE Schively, ed., Humana Press, 1996). Press, Clifton, NJ, 1986, p. 41), ion pair reagents (W. H. Koningsberg and L. H. Henderson, Methods Enzymol., vol. 91, p. 254, 1983), liquid-liquid extraction (P. Davidsson, A. Westman, M. Puchades, C. L. Nilsson and K. Blennow, Anal. Chem., vol. 71, pp. 642-647, 1999), and reversed-phase HPLC (H. Kawasaki and K. Suzuki, Anal. Biochem., vol. 186, p. 264, 1990).

[0010] One way to solve these problems is to use anionic surfactants with dioxolane or dioxane functional groups that enable the surfactant to decompose under acidic conditions. This method has been described in U.S. Patent Application Nos. 11 / 811,696 and 10 / 169,002 (published as U.S. Patent No. 7,229,539), and these applications are incorporated herein by reference. However, for the materials described in U.S. Patent No. 7,229,539, after the surfactant decomposes under acidic conditions, the hydrophobic portion may be too hydrophobic under certain solution conditions and precipitate out from the solution. Therefore, it is necessary to modify the hydrophobicity of the hydrophobic portion. Summary of the invention

[0011] The invention features destructible surfactants and methods of using these surfactants to dissolve, analyze, separate, purify and / or characterize molecules. In one aspect, the invention provides surfactants that selectively decompose at relatively low pH. The resulting decomposition products of the surfactant can be relatively easily removed from the sample or will not interfere with further chromatography or analytical steps. The invention is applicable to a variety of techniques that benefit from the initial presence and ultimate removal of the surfactant.

[0012] Therefore, in one aspect, the present invention provides a surfactant represented by the following formula (Formula I):

[0013]

[0014] in

[0015] p and q are each independently 0, 1 or 2, and p+q is 1, 2 or 3;

[0016] R a and R b are each independently alkyl, alkenyl or alkynyl, and R a Straight or branched C5-C 30 , and R b It is a straight chain or branched chain C1-C5;

[0017] X is CR'2, O, NR' or S; wherein each R' is independently H, lower alkyl, lower alkenyl or lower alkynyl;

[0018] R1 and R2 are each independently hydrogen or a linear or branched C1-C5; and

[0019] R3 comprises an anionic, cationic, zwitterionic or nonionic hydrophilic group; in certain embodiments, R3 is selected from -R4SO3 - 、-R4OR5SO3 - 、-R4(OCH2CH2) n SO3 - 、-R4COO - ,

[0020] -R4OR5COO - 、-R4CONCH3CH2COO - 、-R4(OCH2CH2) n OP(OH)O2 - 、-R4OP(OH)O2 - ,

[0021] -R4OP(OH)2O - 、-R4OR5P(OH)2O - 、-R4OR5OP(OH)2O - 、-R4N(CH3)3 + 、-R4N(R6)3 + ,

[0022] -R4OR5N(R6)3 + 、-R4N + (CH3)2CH2COO - 、-R4N(CH3)3 + R5SO3 -; wherein R4, R5 and R6 are each independently lower alkyl, lower alkenyl, lower alkynyl, substituted lower alkyl, substituted lower alkenyl or substituted lower alkynyl; n is 1 to 6. In certain embodiments, R3 may comprise a phosphate, a phosphate diester, a phosphate monoester, phosphoric acid, a phosphonate or a phosphonic acid.

[0023] In one embodiment, R a is a straight chain alkyl group having 5 to 15 carbons, 7 to 13 carbons or 9 to 11 carbons, R b is CH2, and X is O.

[0024] In other aspects, this type of surfactant can be used to enhance protein dissolution / enzyme digestion. After hydrolysis destroys the surfactant, two components are generally formed-hydrophilic component and hydrophobic component. The hydrophobic component can cause chromatographic problems in subsequent steps after dissolution / enzyme digestion; for example, the hydrophobic component can precipitate out from the high aqueous solution (with the peptide and / or selectively take away more non-polar peptides), and change the peptide recovery rate and / or peptide ratio. By changing the chemical properties of the surfactant and / or the hydrolyzable linker, the hydrophobic residue can be made more polar, thereby allowing it to be dissolved by significantly less organic solvent, and minimize the potential loss of peptide material, and expand the available chromatographic conditions.

[0025] In one embodiment, the present invention relates to a method for analyzing a sample, comprising contacting the sample with a surfactant having a structure of Formula I. In another embodiment, the method comprises analyzing the sample by high performance liquid chromatography (HPLC). In yet another embodiment, the method comprises analyzing the sample by mass spectrometry (MS) or LC-MS. In yet another embodiment, the method comprises analyzing the sample by ion pair liquid chromatography.

[0026] In another embodiment, the invention provides a method for performing electrophoresis, the method comprising contacting a sample with a surfactant having a structure of Formula I. In another embodiment, the electrophoresis is gel electrophoresis or particularly polyacrylamide gel electrophoresis, including polyacrylamide gel electrophoresis in tubular, slab gel and capillary formats. In other embodiments, the electrophoresis is free zone electrophoresis or capillary electrophoresis. In yet other embodiments, the method includes a step of degrading the surfactant after electrophoresis. In another embodiment, the method includes using a relatively acidic solution to degrade the surfactant after electrophoresis. In yet another embodiment, the method includes a step of further purifying the sample after the degradation surfactant.

[0027] In another embodiment, the invention provides a method for performing enzymatic digestion of a protein using a surfactant having a structure of Formula I without any prior purification steps. In another embodiment, the method includes a step of degrading the surfactant after enzymatic digestion of the protein. In another embodiment, the method includes using a relatively acidic solution to degrade the surfactant after enzymatic digestion of the protein. In yet another embodiment, the method includes a step of further purifying the peptide after degrading the surfactant.

[0028] In yet another embodiment, the invention provides a kit for performing electrophoresis, the kit comprising a surfactant having a structure of Formula I. In another embodiment, the kit comprises a component for degrading the surfactant. In another embodiment, the kit comprises a molecular weight standard. In yet another embodiment, the kit comprises a staining reagent. In another embodiment, the surfactant of the invention is incorporated into a gel medium. DETAILED DESCRIPTION

[0029] Summary of the Invention

[0030] The present invention relates to surfactants, including the synthesis and use of surfactants. In particular, the surfactants of the present invention include dioxolane or dioxane functional groups that enable the surfactant to degrade in an acidic environment. The resulting degradation products can be removed from a matrix more easily than the original surfactant and / or minimize chromatographic problems in subsequent steps after the protein solubilization / enzyme digestion step.

[0031] In one aspect, the present invention relates to anionic surfactants, including the synthesis and use of anionic surfactants. Specifically, the present invention includes anionic surfactants having protein binding and electrophoretic properties similar to SDS. However, unlike SDS, the surfactants of the present invention include dioxolane or dioxane functional groups that enable the surfactant to degrade in an acidic environment. The resulting degradation products can be removed from the matrix more easily than the initial surfactant or will not interfere with further chromatography or analytical steps. In addition, even in the presence of these degradation products, the mass spectrometry sensitivity of the protein can be significantly and surprisingly greater than that of SDS in the presence of similar concentrations.

[0032] Destructible surfactants can be prepared as shown in the examples described in the present application and U.S. Patent No. 7,229,539. These surfactants have functions similar to SDS, but unlike SDS, they can be hydrolyzed in acidic aqueous solutions under mild conditions to produce two non-surfactant products: ions, water-soluble compounds and neutral, less water-soluble compounds. The surfactants of the present invention can be used for applications that benefit from the initial presence and final removal of surfactants. Specifically, the present invention can be used for the dissolution, analysis, separation, purification and / or characterization of macromolecules.

[0033] In addition, after the hydrolysis destroys the surfactant, two components are generally formed-a hydrophilic component and a hydrophobic component. The hydrophobic component can cause chromatographic problems in subsequent steps after dissolution / enzymatic digestion; for example, the hydrophobic component can precipitate out of the solution (taking the peptides with it and / or selectively taking more non-polar peptides), and changing the peptide recovery rate and / or peptide ratio. By changing the chemical properties and / or hydrolyzable linkers of the surfactant disclosed in U.S. Patent No. 7,229,539, the hydrophobic residues can be made more polar, thereby allowing them to be dissolved by significantly less organic solvents, and minimizing the potential loss of peptide materials, and expanding the available chromatographic conditions.

[0034] In order that the present invention may be more readily understood, a number of terms are first defined.

[0035] The term "alkyl" refers to a saturated aliphatic group, including straight chain alkyl, branched chain alkyl, cycloalkyl (alicyclic), alkyl substituted cycloalkyl and cycloalkyl substituted alkyl. In certain embodiments, a straight chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone (e.g., C1-C2 for a straight chain). 20 , for the branched chain C3-C 20 ).

[0036] Unless the number of carbon atoms is otherwise specified, the term "lower alkyl" refers to an alkyl group having from one to ten carbons. In certain embodiments, the lower alkyl group has from 2 to 6 carbon atoms or particularly 3 or 4 carbon atoms.

[0037] The term "alkenyl" refers to an unsaturated hydrocarbon group including a set of carbon-carbon double bonds, including straight-chain alkenyl, branched-chain alkenyl, cycloalkenyl (alicyclic), alkyl-substituted cycloalkenyl, and cycloalkyl-substituted alkenyl. In some embodiments, a straight-chain or branched alkenyl has 20 or fewer carbon atoms in its main chain (e.g., C2-C4 for a straight chain). 20 , for the branched chain C4-C 20 ).

[0038] Unless the number of carbon atoms is otherwise specified, the term "lower alkenyl" refers to alkenyl groups having two to ten carbons. In certain embodiments, lower alkenyl groups have 2 to 6 carbon atoms or particularly 3 or 4 carbon atoms.

[0039] The term "alkynyl" refers to an unsaturated hydrocarbon group including a set of carbon-carbon triple bonds, including straight chain alkynyl, branched chain alkynyl, cycloalkynyl, alkyl-substituted cycloalkynyl, and cycloalkyl-substituted alkynyl. In certain embodiments, a straight chain or branched chain alkynyl has 20 or fewer carbon atoms in its backbone (e.g., C2-C4 for a straight chain). 20 , for the branched chain C4-C 20 ).

[0040] Unless the number of carbon atoms is otherwise specified, the term "lower alkynyl" refers to alkynyl groups having two to ten carbons. In some embodiments, lower alkenyl groups have 2 to 6 carbon atoms or particularly 3 or 4 carbon atoms.

[0041] The term "sample / surfactant complex" refers to a complex formed by the surfactant of the present invention and the sample.

[0042] The term "sample" refers to any molecule that can be used in the present invention. Examples include, but are not limited to, macromolecules. Specific examples include proteins or peptides.

[0043] The term "lipophilic protein" refers to a relatively hydrophobic protein or peptide. Examples include, but are not limited to, proteins from myelin or central nervous system tissues and membrane-bound proteins such as receptors.

[0044] The term "receptor" is recognized in the art and generally refers to a membrane-bound molecule, including but not limited to a protein that binds a ligand and transmits a signal into a cell. Such receptors typically have an extracellular domain, a transmembrane domain, and an intracellular domain.

[0045] The term "inclusion body" is art-recognized and refers to intracellular structures, including but not limited to, intracellular structures that contain expressed proteins.

[0046] The term "solution for degrading surfactants" refers broadly to any relatively low pH solution. In certain embodiments, the pH of the solution is between 0 and 5. In still other embodiments, the pH of the solution is between 1 and 3. Generally speaking, the lower the pH of the solution for degrading surfactants, the less time required to degrade the surfactant. In addition, the compound used to prepare the solution for degrading surfactants is not particularly limited: any compound that provides a relatively low pH solution suitable for degrading the surfactant of the present invention without destroying the sample is sufficient. Therefore, for example, hydrochloric acid can be used as a solution for degrading surfactants. In specific embodiments, trifluoroacetic acid (TFA) can be used. In other embodiments, acetic acid or formic acid can be used as a solution for degrading surfactants.

[0047] The term "electrophoresis" refers to any of a variety of methods for analyzing molecules according to their rate of movement in an electric field (i.e., based on the molecule's charge-to-mass ratio). Examples include, but are not limited to, gel electrophoresis, polyacrylamide gel electrophoresis (including tube, slab gel, and capillary formats), free zone electrophoresis, and capillary electrophoresis.

[0048] The term "analysis" refers to any of a variety of methods for separating, purifying, solubilizing and / or characterizing macromolecules such as proteins or peptides. Examples include, but are not limited to, electrophoresis, mass spectrometry, high performance liquid chromatography, ion-pair liquid chromatography, liquid-liquid extraction, and ultraviolet detection.

[0049] The term "mass spectrometry detection" refers to any of a variety of methods such as spectroscopy. Examples include, but are not limited to, electrospray ionization ("ESI") and matrix-assisted laser desorption ionization ("MALDI").

[0050] Compounds of the present invention

[0051] In one aspect, the present invention provides a surfactant represented by the following formula (Formula I):

[0052]

[0053] in

[0054] p and q are each independently 0, 1 or 2, and p+q is 1, 2 or 3;

[0055] R a and R b are each independently alkyl, alkenyl or alkynyl, and R a Straight or branched C5-C 30 , and R b It is a straight chain or branched chain C1-C5;

[0056] X is CR'2, O, NR' or S; wherein each R' is independently H, lower alkyl, lower alkenyl or lower alkynyl;

[0057] R1 and R2 are each independently hydrogen or a linear or branched C1-C5; and

[0058] R3 comprises an anionic, cationic, zwitterionic or nonionic hydrophilic group; in certain embodiments, R3 is selected from -R4SO3 - 、-R4OR5SO3 - 、-R4(OCH2CH2) n SO3 - 、-R4COO - ,

[0059] -R4OR5COO - 、-R4CONCH3CH2COO - 、-R4(OCH2CH2) n OP(OH)O2 - 、-R4OP(OH)O2 - ,

[0060] -R4OP(OH)2O - 、-R4OR5P(OH)2O - 、-R4OR5OP(OH)2O - 、-R4N(CH3)3 + 、-R4N(R6)3 + ,

[0061] -R4OR5N(R6)3 + 、-R4N + (CH3)2CH2COO - 、-R4N(CH3)3 + R5SO3 -; wherein R4, R5 and R6 are each independently lower alkyl, lower alkenyl, lower alkynyl, substituted lower alkyl, substituted lower alkenyl or substituted lower alkynyl; n is 1 to 6. In certain embodiments, the substituents in the substituted lower alkyl and substituted lower alkenyl groups may include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, phosphino, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and urea), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, aralkyl or aromatic or heteroaromatic moieties. In specific embodiments, the substituents in the substituted lower alkyl and substituted lower alkenyl groups are fluorine substituents.

[0062] In some embodiments, q is 1, and p is 0 or 1. In some other embodiments, R a is an alkyl group having six to twenty carbon atoms, more specifically eight to eighteen carbon atoms or ten to sixteen carbon atoms. b is an alkyl group having one to three carbon atoms, more specifically a CH2 group. In a certain embodiment, X is O. In certain embodiments, R3 is -R4SO3 - 、-R4OR5SO3 - or -R4(OCH2CH2) n SO3 - ; especially R3 is –CH2O(CH2)3SO3 - or –CH2O(CH2)4SO3 - In certain embodiments, R4 and R5 are each independently an alkyl, alkenyl, or alkynyl group having one to eight carbons, more specifically two to six carbon atoms, and more specifically three or four carbon atoms.

[0063] In one embodiment, R a is a straight chain alkyl group having 5 to 15 carbons, 7 to 13 carbons or 9 to 11 carbons, R b is CH2, and X is O.

[0064] In another embodiment, the surfactant of the present invention has the following general formula (Formula II):

[0065]

[0066] in

[0067] Ra and R b are each independently alkyl, alkenyl or alkynyl, and R a Straight or branched C5-C 30 , and R b It is a straight chain or branched chain C1-C5;

[0068] X is CR'2, O, NR' or S; wherein each R' is independently H, lower alkyl, lower alkenyl or lower alkynyl;

[0069] R1 and R2 are each independently hydrogen or a linear or branched C1-C5; and

[0070] R3 comprises an anionic, cationic, zwitterionic or nonionic hydrophilic group; in certain embodiments, R3 is selected from -R4SO3 - 、-R4OR5SO3 - 、-R4(OCH2CH2) n SO3 - 、-R4COO - ,

[0071] -R4OR5COO - 、-R4CONCH3CH2COO - 、-R4(OCH2CH2) n OP(OH)O2 - 、-R4OP(OH)O2 - ,

[0072] -R4OP(OH)2O - 、-R4OR5P(OH)2O - 、-R4OR5OP(OH)2O - 、-R4N(CH3)3 + 、-R4N(R6)3 + ,

[0073] -R4OR5N(R6)3 + 、-R4N + (CH3)2CH2COO - 、-R4N(CH3)3 + R5SO3 -; wherein R4, R5 and R6 are each independently lower alkyl, lower alkenyl, lower alkynyl, substituted lower alkyl, substituted lower alkenyl or substituted lower alkynyl; n is 1 to 6. In certain embodiments, the substituents in the substituted lower alkyl and substituted lower alkenyl groups may include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, phosphino, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and urea), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, aralkyl or aromatic or heteroaromatic moieties. In specific embodiments, the substituents in the substituted lower alkyl and substituted lower alkenyl groups are fluorine substituents.

[0074] In one embodiment, R a is a straight chain alkyl group having 5 to 15 carbons, 7 to 13 carbons or 9 to 11 carbons, R b is CH2, and X is O.

[0075] In other embodiments, R a is an alkyl group having six to twenty carbon atoms, more specifically eight to eighteen carbon atoms or ten to sixteen carbon atoms. b is an alkyl group having one to three carbon atoms, more specifically a CH2 group. In a certain embodiment, X is O. In certain embodiments, R3 comprises an anionic surfactant group selected from the group consisting of: -R4SO3 - 、-R4OR5SO3 - or -R4(OCH2CH2) n SO3 - In certain embodiments, R3 is -CH2O(CH2)3SO3 - or –CH2O(CH2)4SO3 - In certain embodiments, R4 and R5 are each independently an alkyl group having one to eight carbons, more specifically two to six carbon atoms, and more specifically three or four carbon atoms.

[0076] In certain embodiments, the surfactants of the present invention have the following chemical structure:

[0077]

[0078] wherein X is independently O, NR', NH, S or CH2; and R' is H, lower alkyl, lower alkenyl or lower alkynyl.

[0079] In some embodiments, the surfactants of the present invention have the following chemical structures.

[0080]

[0081] In certain embodiments, the surfactants of the present invention have the following chemical structure:

[0082]

[0083] wherein X is independently O, NR', NH, S or CH2; and R' is H, lower alkyl, lower alkenyl or lower alkynyl.

[0084] In some embodiments, the surfactants of the present invention have the following chemical structures.

[0085]

[0086] In certain embodiments, the surfactants of the present invention have the following chemical structure:

[0087]

[0088] wherein X is independently O, NR', NH, S or CH2; and R' is H, lower alkyl, lower alkenyl or lower alkynyl.

[0089] In some embodiments, the surfactants of the present invention have the following chemical structures.

[0090]

[0091] In one embodiment, the surfactant of the present invention has the following chemical structure.

[0092]

[0093] In certain embodiments, the surfactants of the present invention have the following chemical structure:

[0094]

[0095] wherein X is O, NR', NH, S or CH2; and R' is H, lower alkyl, lower alkenyl or lower alkynyl.

[0096] In some embodiments, the surfactants of the present invention have the following chemical structures.

[0097]

[0098] As indicated in more detail in the examples, the synthetic methods of the present invention produce isomers. Although the methods using the surfactants of the present invention do not require separation of these isomers, such separation can be accomplished by methods known in the art if desired. For example, preparative high performance liquid chromatography can be used for isomer purification.

[0099] Method of the present invention

[0100] The surfactants of the present invention can be used in applications that benefit from the initial presence and eventual removal of the surfactant. Specifically, the present invention can be used to dissolve, analyze, separate, isolate, purify, detect and / or characterize molecules in biological samples, such as biological fluids, biological tissues, biological matrices, embedded tissue samples, and cell culture supernatants.

[0101] In one embodiment, the invention provides a method for analyzing a molecule, the method comprising contacting a sample comprising at least one molecule with a surfactant of the invention, thereby analyzing the molecule. In certain embodiments, the sample may be heated before or after contacting the sample with the surfactant of the invention. In certain embodiments, the step of analyzing the sample comprises electrophoresis. In specific embodiments, the electrophoresis is free zone electrophoresis or capillary electrophoresis.

[0102] Analysis of the sample may include, but is not limited to, solid phase extraction, solid phase microextraction, electrophoresis, mass spectrometry (e.g., MALDI-MS or ESI), liquid chromatography (e.g., high performance, such as reverse phase, normal phase or size exclusion, ion pair liquid chromatography), liquid-liquid extraction (e.g., accelerated fluid extraction, supercritical fluid extraction, microwave-assisted extraction, membrane extraction, Soxhlet extraction), precipitation, clarification, electrochemical detection, staining, elemental analysis, Edmund degradation, nuclear magnetic resonance, infrared analysis, flow injection analysis, capillary electrochromatography, ultraviolet detection, and combinations thereof.

[0103] Another embodiment of the present invention provides a method for performing cell lysis, the method comprising contacting a cell comprising at least one molecule with a surfactant of the present invention, thereby lysing the cell. In certain embodiments, the molecule is analyzed after cell lysis, such as mass spectrometry or electrophoresis. In certain embodiments, the surfactant is degraded after electrophoresis. The degradation of the surfactant can be performed by contacting the surfactant with an acidic solution. In specific embodiments, the molecule is purified, for example, by solid phase extraction or HPLC after the degradation of the surfactant.

[0104] In another embodiment, the present invention provides a kit for performing cell lysis on a sample containing at least one molecule to separate the molecule, the kit comprising a surfactant of the present invention and instructions for use. In certain embodiments, the kit may further comprise a solution or solid phase extraction device for degrading the surfactant.

[0105] Additional embodiments of the present invention use the destructible surfactant of the present invention to compound with a mixture (e.g., biological sample, e.g., cell culture) comprising at least one molecule to perform electrophoresis. After electrophoretic separation, the components separated, such as small molecules and proteins, are released from the surfactant of the present invention by treating with an acid solution. The molecules separated can be further purified by conventional separation methods such as liquid-liquid extraction, solid phase extraction, or liquid chromatography. This ability to be easy to release molecules from the surfactant after electrophoresis can be used for various applications, and has significant benefits to separation science.

[0106] In other embodiments, the step of analyzing the sample after protein solubilization / enzyme digestion comprises electrophoresis. In certain embodiments, the electrophoresis is gel electrophoresis, free zone electrophoresis, or capillary electrophoresis. In a specific embodiment, the electrophoresis is polyacrylamide gel electrophoresis, including polyacrylamide gel electrophoresis in tube, slab gel, and capillary formats.

[0107] In still other embodiments, the step of analyzing the sample comprises mass spectrometry, high performance liquid chromatography, ion-pair liquid chromatography, liquid-liquid extraction, or ultraviolet detection.

[0108] In one aspect, the present invention uses a destructible surfactant to compound with a protein mixture so as to perform polyacrylamide gel electrophoresis. After electrophoretic separation, the protein is released from the surfactant by treating the gel with an acid solution. The protein mixture can be further purified by conventional separation methods such as liquid-liquid extraction, solid phase extraction or liquid chromatography. This ability to be easy to release protein from the surfactant after polyacrylamide gel electrophoresis can be used for various applications and has significant benefits to separation science.

[0109] As demonstrated by the PAGE results herein, proteins treated with the acid-labile surfactants of the present invention migrated in a pattern similar to that of proteins treated with SDS. Additionally, the gel staining procedure was similar in the presence of ALS or SDS.

[0110] The present invention also provides methods for solubilizing various substances, such as inclusion bodies, lipophilic proteins (such as receptors and other membrane-bound proteins) and biological tissues. In a specific embodiment, the present invention provides a method for obtaining a protein expressed in a cell by treating the inclusion bodies of the cell with a surfactant having the structure of Formula I.

[0111] In yet another embodiment, the present invention provides a method for cleaning and / or regenerating an HPLC column. In another embodiment, a surfactant having a structure of Formula I is contacted with an adsorbent of an HPLC column such that proteins bound to the column are removed.

[0112] Example

[0113] The present invention is further illustrated by the following examples, which should not be construed as limiting.

[0114] The present invention utilizes a change in the chemical bonding sequence of the reagents so that upon hydrolysis the solubility of the hydrophobic group in a standard buffer solution is significantly greater than the equivalent group in the surfactant identified as Compound 1 below disclosed in U.S. Patent No. 7,229,539. The chemical characteristics of the hydrophobic group may also be altered to enhance this solubility. This includes the use of embedded polar groups and side chains.

[0115] For investigation, two exemplary surfactants 4 and 7 with different alkyl chain lengths were generated. Both have the same hydrophilic hydrolysis product 5. The main difference from compound 1 is that upon hydrolysis of the cyclic acetal, the diol functionality will be located on the alkyl chain instead of on the polar molecule. In addition, in contrast to surfactant 1 as previously reported, the alkyl chain will now have an optional embedded ether functionality.

[0116]

[0117] The hydrophilic hydrolysis products of these agents have logP coefficients calculated to be approximately 0.9-1.4 orders of magnitude smaller than those of compound 3, indicating that they are more soluble in water than the latter, ie, the hydrophobic component of compound 1.

[0118] Table 1. Calculated logP of selected hydrolysates of surfactant compounds 1, 4 and 7

[0119]

[0120] Example 1 :

[0121] Preparation of the selected hydrophobic moiety

[0122] This example describes the preparation of certain hydrophobic moieties of the invention. Various modifications of the following procedures will be routine to one of ordinary skill in the art in light of the teachings herein.

[0123] 1. Synthesis of 3-(Undecyloxy)-1,2-propanediol (Compound 6)

[0124]

[0125] 3.3 g of DL-1,2-O-isopropylidene glycerol was added to a freshly prepared 50% aqueous NaOH solution (50 mL). Stirring was started and the mixture was heated to 80° C. and maintained at this temperature for 1 hour. After this time, 1-bromodecane (15.8 g) and tert-butylammonium bromide (1.3 g) were added in sequence and heating was maintained for 7 hours.

[0126] After cooling to room temperature, the mixture was poured into water (150 mL) and extracted with CH2Cl2 (50 mL×3). The combined organic extracts were washed with water (25 mL×2), dried (Na2SO4) and concentrated to provide a crude product. The crude product was dissolved in a mixture of ethanol (20 mL) and 2M HCl aqueous solution (30 mL) and refluxed for 1 h. Ethanol was removed using a rotary evaporator, and the remaining aqueous phase was neutralized with a saturated NaHCO3 solution and extracted with CH2Cl2. The organic extracts were first dried and concentrated, and then purified by column chromatography (3:1 ethyl acetate: petroleum ether) to obtain pure 3-(undecyloxy)-1,2-propylene glycol (compound 6) (4.6 g, 75% yield).

[0127] 2. Synthesis of 3-nonyloxypropane-1,2-diol (Compound 8)

[0128]

[0129] 3.3 g of DL-1,2-O-isopropylidene glycerol was added to a freshly prepared 50% aqueous NaOH solution (50 mL). Stirring was started and the mixture was heated to 80° C. and maintained at this temperature for 1 hour. After this time, 1-bromononane (13.9 g) and tert-butylammonium bromide (1.3 g) were added in sequence and heating was maintained for 7 hours.

[0130] After cooling to room temperature, the mixture was poured into water (150 mL) and extracted with CH2Cl2 (50 mL × 3). The combined organic extracts were washed with water (25 mL × 2), dried (Na2SO4) and concentrated to provide a crude product. The crude product was dissolved in a mixture of ethanol (20 mL) and 2M HCl aqueous solution (30 mL) and refluxed for 1 h. Ethanol was removed using a rotary evaporator, and the remaining aqueous phase was neutralized with a saturated NaHCO3 solution and extracted with CH2Cl2. The organic extracts were first dried and concentrated, and then purified by column chromatography (3:1 ethyl acetate: petroleum ether) to obtain pure 3-(nonyloxy)-1,2-propylene glycol (compound 8) (4.1 g, 67% yield).

[0131] Example 2 :

[0132] Solubility studies of selected hydrophobic moieties

[0133] Compounds 3 (commercially available - Sigma Aldrich P / N 17283999%), 6 and 8 (synthesized above) were subjected to dissolution studies in different buffer solutions containing a mixture of standard chromatography solvents (e.g., acetonitrile and 20 mM aqueous ammonium carbonate) at 21°C. A 20 mM aqueous ammonium bicarbonate solution was prepared by dissolving 474 mg ammonium bicarbonate in 300 mL MiliQ water. The results are given in Tables 2 and 3.

[0134] Table 2. Buffer solutions of selected hydrophobic moieties in 30 mL acetonitrile and 70 mL 20 mM ammonium bicarbonate (30% acetonitrile) Solubility in

[0135]

[0136] Table 3. Buffer solutions of selected hydrophobic moieties in 10 mL acetonitrile and 90 mL 20 mM ammonium bicarbonate (10% acetonitrile) Solubility in

[0137]

[0138] Incorporated by Reference

[0139] All patents, published patent applications, and other references cited herein are hereby expressly incorporated by reference in their entirety.

[0140] Equivalent form

[0141] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed within the scope of the following claims.

Claims

1. A method for performing protein solubilization or enzymatic digestion, the method comprising contacting a sample with a surfactant represented by the following formula I: in p and q are each independently 0, 1 or 2, and p+q is 1, 2 or 3; R a and R b are each independently alkyl, alkenyl or alkynyl, and R a Straight or branched C5-C 30 , and R b It is a straight chain or branched chain C1-C5; X is O, NR' or S; wherein each R' is independently H, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 Alkynyl; R1 and R2 are each independently hydrogen or a linear or branched C1-C5; and R3 is a group selected from the following: -R4SO3 - ,-R4OR5SO3 - ,-R4(OCH2CH2) n SO3 - ,-R4OR5COO - ,-R4CONCH3CH2COO - ,-R4(OCH2CH2) n OP(OH)O2 - ,-R4OP(OH)O2 - ,-R4OP(OH)2O - , -R4OR5P(OH)2O - ,-R4OR5OP(OH)2O - ,-R4OR5N(R6)3 + ,-R4N + (CH3)2CH2COO - , or -R4N(CH3)3 + R5SO3 - ; wherein R4 is a C1 alkyl group, and R5 and R6 are each independently C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, substituted C1-C 10 Alkyl, substituted C2-C 10 Alkenyl or substituted C2-C 10 alkynyl; and n is 1 to 6.

2. The method according to claim 1, wherein R3 is selected from -R4SO 3- 、-R4OR5SO 3- 、-R4(OCH2CH2) n SO3 - .

3. A method according to any one of claims 1 to 2, comprising the additional step of degrading the surfactant after protein solubilization or enzyme digestion.

4. The method of claim 3, wherein the step of degrading the surfactant after protein solubilization or enzyme digestion comprises contacting the surfactant with an acidic solution.

5. The method of claim 3, comprising the additional step of purifying the sample.

6. A method according to any one of claims 1 to 5, comprising the additional step of degrading the surfactant after protein solubilization or enzyme digestion.

7. The method of claim 6, wherein the step of degrading the surfactant after protein solubilization or enzyme digestion comprises contacting the surfactant with an acidic solution.

8. The method according to claim 7, comprising the further step of purifying the sample.

9. A method for performing protein solubilization or enzymatic digestion, the method comprising contacting a sample with a surfactant selected from the following chemical structures:

10. The method according to claim 9, comprising the additional step of degrading the surfactant after protein solubilization or enzyme digestion.

11. The method of claim 10, wherein the step of degrading the surfactant after protein solubilization or enzyme digestion comprises contacting the surfactant with an acidic solution.

12. The method according to claim 11, comprising the further step of purifying the sample.

13. A surfactant selected from the following chemical structures:

14. A method for analyzing a sample, the method comprising first contacting the sample with the surfactant according to claim 16.

15. The method of claim 14, wherein the step of analyzing the sample comprises high performance liquid chromatography.

16. The method of claim 14, wherein the step of analyzing the sample comprises mass spectrometry detection.

17. The method of claim 14, wherein the step of analyzing the sample comprises ion-pair liquid chromatography.

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