Acid-Labile Surfactants

Abstract

Provided herein are surfactant compounds that can be used, for example, to aid in the preparation of protein samples for analysis by mass spectrometry.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 883,249, filed August 6, 2019, the entire contents of which are hereby incorporated by reference in their entirety.

[0002] Provided herein are surfactant compounds that can be used, for example, to aid in the preparation of protein samples for analysis by mass spectrometry. [Background technology]

[0003] For example, preparation of protein samples for analysis by mass spectrometry typically involves three major steps: solubilization, digestion, and peptide recovery. Not all commonly used reagents are compatible with each step. For example, solubilizing agents such as detergents (e.g., sodium dodecyl sulfate (SDS)) or denaturants (e.g., acetonitrile, urea, or guanidine) may inhibit proteases. Even when used at concentrations that may be acceptable for protease activity, the presence of these detergents or denaturants interferes with subsequent analyses, such as liquid chromatography or mass spectrometry analysis. Thus, removal of detergents and organic solvents is typically required before further analysis of the sample is performed. The manipulations required for removal of these reagents complicate the sample preparation process and may lead to loss of sample material.

[0004] The digestion step can also present challenges in protein sample preparation. Typical protein digestion with trypsin requires overnight incubation to reach completion. Even after overnight incubation, some proteins that are resistant to digestion, such as membrane proteins, may remain intact. Current methods used in attempts to overcome these limitations and speed up the digestion process include the use of organic solvents (e.g., acetonitrile), high temperatures, denaturants (e.g., urea), and / or detergents (e.g., SDS), which improve protein solubilization and protein denaturation, and thus improve digestion. However, these alternative methods and additives often result in incomplete cleavage and poor reproducibility. The use of these reagents can also inhibit trypsin activity, impeding HPLC separation and suppressing peptide detection in mass spectrometry.

[0005] In-gel protein digestion can bring other challenges to protein sample preparation. The success of in-gel digestion depends not only on efficient protein digestion but also on efficient post-digestion peptide extraction from the gel, which can be time-consuming and often only moderately efficient in terms of peptide recovery. Recovered peptides are generally limited in size to about 2,500 Da, while longer peptides may remain trapped in the gel. Recovery of peptides with increased hydrophobicity can also be affected.

[0006] Another procedure related to protein sample preparation involves the analysis of post-translational protein modifications. Approximately 60% of all human proteins are glycosylated. To analyze glycosylation, the glycans should be separated from the protein. Deglycosylation is typically performed using glycosidases, which can be a time-consuming process. Reagents such as SDS can potentially improve deglycosylation by providing glycosidases with better access to the glycan binding sites. However, SDS can interfere with downstream sample preparation steps, mass spectrometry analysis, and HPLC analysis. Summary of the Invention

[0007] There is a need for improved methods for protein sample preparation, as well as methods or reagents compatible with protein solubilization, digestion, and peptide recovery. In particular, methods and reagents that do not inhibit protease activity and do not interfere with isolation and / or characterization techniques would be advantageous. There may be a particular need for in-gel digestion protocols to improve peptide recovery from gels, improved solubilization and digestion of pelleted proteins, more efficient protein extraction from cells and tissues, and increased compatibility with glycosylated proteins.

[0008] Provided herein are surfactant compounds that can have good water solubility and water stability even at higher temperatures and can be decomposed under acidic conditions. Such surfactants can be useful as protein solubilization and / or digestion reagents without interfering with protease or protein fragment mass spectrometry analysis.

[0009] An embodiment of the present disclosure is a compound of formula (I): [ka] or a salt thereof, wherein A is, [ka] is selected from R 1 is hydrogen, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C1-C 12 haloalkyl; R 2 is C4-C 20 Alkyl, C4-C 20 Alkenyl, C4-C 20 haloalkyl; Each R 3 are independent, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C1-C 12haloalkyl; m is 1, 2, or 3; R a and R b is independently selected at each occurrence from hydrogen and C1-C3 alkyl; Q is C1-C6-alkylene, C1-C6-hydroxyalkylene, and the formula: [ka] where n is 1, 2, 3, 4, 5, or 6; Each M is independently hydrogen, an alkali metal cation, or NR4 + wherein each R is independently selected from hydrogen and C-C 12 is selected from alkyl.

[0010] In some embodiments, A is [ka] It is.

[0011] In some embodiments, R 1 is C6-C 12 is alkyl, R 2 is selected from hydrogen, methyl, and C4-C8 alkyl.

[0012] In some embodiments, A is [ka] is selected from.

[0013] In some embodiments, A is [ka] It is.

[0014] In some embodiments, R 3 Each instance of is the same. In some embodiments, each R 3is selected from C4-C8 alkyl and C4-C8 haloalkyl. 3 is selected from C4-C8 alkyl. In some embodiments, each R 3 is -(CH2) x -(CF2) y -CF3, where x is 1, 2, 3, or 4, and y is 1, 2, 3, 4, 5, 6, or 7. For example, in some embodiments, x is 2 and y is 3 or 5.

[0015] In some embodiments, A is [ka] is selected from.

[0016] In some embodiments, m is 1. In some embodiments, R a and R b are each hydrogen.

[0017] In some embodiments, Q is selected from C1-C6-alkylene and C1-C6-hydroxyalkylene. In some embodiments, Q is selected from -CH2CH2CH2- and -CH2CH(OH)CH2-. In some embodiments, Q is [ka] where n is 1, 2, 3, 4, 5, or 6.

[0018] In some embodiments, the group -Q-SO3M has the formula: [ka] has.

[0019] In some embodiments, each M is independently an alkali metal. In some embodiments, the alkali metal is sodium.

[0020] In some embodiments, the compound of formula (I) is [ka] [ka] [ka] [ka] is selected from.

[0021] An embodiment of the present disclosure also provides a method for digesting a protein, comprising: contacting a sample containing at least one protein with a protein digestion reagent and a compound of formula (I), or a salt thereof; Thereby, the method includes providing a sample comprising at least one digested protein.

[0022] In some embodiments, the sample is a gel, a solid support, or a solution. In some embodiments, the sample is a gel. In some embodiments, the sample is an aqueous solution. In some embodiments, the aqueous solution is a solution from in-solution protein digestion with a protease, protein pellet solubilization, protein extract from cells, protein extract from tissue, protein deglycosylation with a deglycosidase, or removal of non-specifically bound proteins from a protein immobilizing support.

[0023] In some embodiments, the protein digestion reagent comprises a protease, cyanogen bromide, hydroxylamine, or any combination thereof.

[0024] In some embodiments, the protein digestion reagent comprises a protease. In some embodiments, the protease is a serine protease. In some embodiments, the serine protease is trypsin, chymotrypsin, or Lys-C.

[0025] In some embodiments, the method further comprises decomposing the surfactant after the contacting step. In some embodiments, the decomposing step comprises contacting the surfactant with an acid. In some embodiments, the surfactant autolyzes after the contacting step.

[0026] In some embodiments, the method further comprises isolating at least one digested protein fragment. In some embodiments, the method further comprises analyzing at least one digested protein fragment. In some embodiments, the analyzing step comprises mass spectrometry, liquid chromatography, gel electrophoresis, or a combination thereof.

[0027] Embodiments of the present disclosure also include a composition comprising a gel and a compound of formula (I).

[0028] An embodiment of the present disclosure also provides a method for extracting peptides from a gel, comprising: contacting the gel with a compound according to any one of claims 1 to 16 and an aqueous solution; and separating the aqueous liquid from the gel, thereby providing a solution comprising the peptide. [Brief description of the drawings]

[0029] [Figure 1] 1 shows a representative SDS-PAGE gel of solubilized E. coli protein pellets following incubation with compounds described herein, as described in Example 31. [Diagram 2] 1 shows the results from a representative BCA assay of solubilized E. coli protein pellets following incubation with compounds described herein, as described in Example 31. [Diagram 3] 1 shows a representative SDS-PAGE gel of myoglobin denaturation and trypsin digestion products after incubation with compounds described herein as described in Example 31. [Figure 4]3 shows the results from a representative trypsin assay performed in the presence of compounds described herein, as described in Example 32. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Provided herein are surfactant compounds that can have good water solubility and water stability even at higher temperatures and can be decomposed under acidic conditions. Such surfactants can be useful as protein solubilization and / or digestion reagents without interfering with protease or protein fragment mass spectrometry analysis.

[0031] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0032] 1.Definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, any nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are well known and commonly used in the art. The meaning and scope of the terms should be clear, however, in case of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or foreign definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0033] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, chemical elements are defined according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Sorrell, Organic Chemistry, 2001, pp. 111-115, 1997. nd edition,University Science Books,Sausalito,2006,Smith,March's Advanced Organic Chemistry:Reactions,Mechanism,and Structure,7 th Edition, John Wiley & Sons, Inc., New York, 2013, Larock, Comprehensive Organic Transformations, 3 rd Edition, John Wiley & Sons, Inc., New York, 2018, Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are incorporated herein by reference.

[0034] The term "alkyl" as used herein refers to an alkyl group having 1 to 30 carbon atoms, e.g., 1 to 16 carbon atoms (C1-C 16 Alkyl), 1 to 14 carbon atoms (C1-C 14 Alkyl), 1 to 12 carbon atoms (C1-C 12 Alkyl), 1 to 10 carbon atoms (C1-C 10"alkyl" refers to a straight or branched saturated hydrocarbon chain containing 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), or 1 to 4 carbon atoms (C1-C4 alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0035] The term "alkenyl" as used herein means a straight or branched hydrocarbon chain containing 2 to 30 carbon atoms and at least one carbon-carbon double bond. For example, an alkenyl group can be an alkyl group having 2 to 16 carbon atoms (C2-C 16 Alkenyl, 2 to 14 carbon atoms (C2-C 14 Alkenyl, 2 to 12 carbon atoms (C2-C 12 Alkenyl, 2 to 10 carbon atoms (C2-C 10 alkenyl), 2 to 8 carbon atoms (C2-C8 alkenyl), 2 to 6 carbon atoms (C2-C6 alkenyl), or 2 to 4 carbon atoms (C2-C4 alkenyl). Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0036] The term "alkylene," as used herein, refers to an alkylene group having 1 to 10 carbon atoms (C1-C 10alkylene), e.g., a divalent group derived from a straight or branched chain hydrocarbon of 1 to 6 carbon atoms (C1-C6 alkylene). Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2CH2-, and -CH(CH3)CH2CH2CH2CH2-.

[0037] The term “halogen” or “halo” as used herein means F, Cl, Br, or I.

[0038] The term "haloalkyl" as used herein refers to an alkyl group as defined herein, in which one or more hydrogen atoms are replaced by halogen.For example, 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms can be replaced by halogen, or all hydrogen atoms can be replaced by halogen.Representative examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, 2-fluoroethyl, 2,2-difluoroethyl, and 2,2,2-trifluoroethyl.

[0039] The term "hydroxy" or "hydroxyl" as used herein means an --OH group.

[0040] The term "hydroxyalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with at least one hydroxy group. Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 2,3-dihydroxypentyl, 4-hydroxybutyl, 2-ethyl-4-hydroxyheptyl, 3,4-dihydroxybutyl, and 5-hydroxypentyl.

[0041] The term "hydroxyalkylene," as used herein, refers to an alkylene group, as defined herein, substituted with at least one hydroxy group. Representative examples of hydroxyalkylene include, but are not limited to, hydroxymethylene (-CH(OH)-), 2-hydroxyethylene (-CH-CH(OH)-), 2-hydroxypropylene (-CH-CH(OH)-CH-), and 3-hydroxypropylene (-CH-CH-CH(OH)-).

[0042] In some cases, the number of carbon atoms in a group (e.g., alkyl, haloalkyl, or cycloalkyl) is indicated by the prefix "C x -C y -", where x is the minimum and y is the maximum number of carbon atoms in the group. Thus, for example, "C1-C3-alkyl" refers to an alkyl group containing from 1 to 3 carbon atoms, and "C1-C6-haloalkyl" refers to a haloalkyl group containing from 1 to 6 carbon atoms.

[0043] The term "substituents" refers to a group substituted on an atom of the indicated group. When a group or moiety can be substituted, the term "substituted" indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6, in some embodiments 1, 2, or 3, and in other embodiments 1 or 2) hydrogens of the indicated group in the phrase using "substituted" can be replaced with a selection of the listed indicated group or a suitable group known to one of ordinary skill in the art (e.g., one or more of the groups listed below). Substituents include, but are not limited to, halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0044] The terms "analysis" and "analyzing" refer to any of a variety of methods for solubilizing, separating, detecting, isolating, purifying, and / or characterizing molecules, such as, for example, proteins, peptides, and fragments thereof. Examples include, but are not limited to, solid-phase extraction; solid-phase microextraction; electrophoresis; mass spectrometry, such as matrix-assisted laser desorption / ionization-mass spectrometry (MALDI-MS) or electrospray ionization-mass spectrometry (ESI-MS); liquid chromatography; for example, high performance, such as reversed-phase, normal-phase, or size-exclusion chromatography; ion-pair liquid chromatography; liquid-liquid extraction, such as accelerated fluid extraction, supercritical fluid extraction, microwave-assisted extraction, membrane extraction, or 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.

[0045] The term "digestion" refers to the process of breaking down molecules, e.g., biomolecules, e.g., proteins, into simpler chemical compounds (fragments). Digestion is carried out using digestive reagents, such as enzymes, e.g., proteases, or by reaction with chemical cleavage reagents. Digestion can result in the destruction of amide bonds. In certain cases, chemicals can destroy certain amide bonds.

[0046] The term "sample" as used herein refers to any specimen containing protein that can be used in the disclosed methods. Examples include, without limitation, protein extracts from cells, protein extracts from tissues, pellets containing proteins, solutions from protein digestion with proteases, solid supports containing at least one protein, gels containing proteins, and the like. Samples can include crude or purified, e.g., isolated or commercially obtained samples. Further examples include, but are not limited to, inclusion bodies, biological fluids, biological tissues, biological matrices, embedded tissue samples, and cell culture supernatants.

[0047] For the compounds described herein, groups and substituents thereof may be selected according to the allowed valences of atoms and substituents such that selection and substitution result in stable compounds that do not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like.

[0048] The terms "comprise(s), "include(s), "having", "having", "can", "containing(s)", and "variations thereof", as used herein, are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a", "and", and "the" include plural references unless the context clearly indicates otherwise. Many embodiments herein are described using open "comprising" language. Such embodiments encompass multiple closed "consisting of" and / or "consisting essentially of" embodiments that may alternatively be claimed or described using such language. The present disclosure also contemplates other embodiments that "comprise", "consist of", and "consist essentially of" the embodiments or elements presented herein, whether or not expressly defined.

[0049] For the recitation of numerical ranges herein, each intermediate number therebetween with the same degree of precision is expressly contemplated. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0050] 2.Compound Compounds of formula (I): [ka] or a salt thereof, A is, [ka] is selected from R1 is hydrogen, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C1-C 12 haloalkyl; R 2 is C4-C 20 Alkyl, C4-C 20 Alkenyl, C4-C 20 haloalkyl; Each R 3 are independent, C1-C 12 Alkyl, C2-C 12 Alkenyl, and C1-C 12 haloalkyl; m is 1, 2, or 3; R a and R b is independently selected at each occurrence from hydrogen and C1-C3 alkyl; Q is C1-C6-alkylene, C1-C6-hydroxyalkylene, and the formula: [ka] where n is 1, 2, 3, 4, 5, or 6; Each M is independently hydrogen, an alkali metal cation, or NR4 + wherein each R is independently selected from hydrogen and C-C 12 is selected from alkyl.

[0051] In some embodiments, A is [ka] It is.

[0052] In some embodiments, R 1 is C1-C 12 In some embodiments, R 1 is C5-alkyl. In some embodiments, R 1 is C-alkyl. In some embodiments, R 1is C-alkyl. In some embodiments, R 1 is C-alkyl. In some embodiments, R 1 is C-alkyl. In some embodiments, R 1 is C 10 -alkyl. In some embodiments, R 1 is C 11 -alkyl. In some embodiments, R 1 is C 12 -alkyl.

[0053] In some embodiments, R 2 is selected from hydrogen, methyl, and C4-C8 alkyl. In some embodiments, R 2 is hydrogen. In some embodiments, R 2 is methyl. In some embodiments, R 2 is C4-C8 alkyl. In some embodiments, R 2 is C5-alkyl. In some embodiments, R 2 is C-alkyl. In some embodiments, R 2 is a C7-alkyl.

[0054] In some embodiments, R 1 is C6-C 12 is alkyl, R 2 is selected from hydrogen, methyl, and C4-C8 alkyl. In some embodiments, R 1 is C6-C 12 is alkyl, R 2 is hydrogen. In some embodiments, R 1 is C6-C 12 is alkyl, R 2 is methyl. In some embodiments, R 1 is C6-C 12 is alkyl, R 2 is a C4-C8 alkyl.

[0055] In some embodiments, A is [ka] is selected from.

[0056] In some embodiments, A is [ka] It is.

[0057] In some embodiments, R 3 Each instance of is the same. In some embodiments, each R 3 is C1-C 12 Alkyl and C1-C 12 In some embodiments, each R 3 is selected from C4-C8 alkyl and C4-C8 haloalkyl. 3 is C1-C 12 In some embodiments, each R 3 is C4-C8 alkyl. In some embodiments, each R 3 is C5-alkyl. In some embodiments, each R 3 is C-alkyl. In some embodiments, each R 3 is C1-C 12 In some embodiments, each R 3 is C-C haloalkyl. In some embodiments, each R 3 is -(CH2) x -(CF2) y -CF3, where x is 1, 2, 3, or 4, and y is 1, 2, 3, 4, 5, 6, or 7. For example, in some embodiments, x is 2 and y is 3 or 5. In some embodiments, x is 2 and y is 3. In some embodiments, x is 2 and y is 5.

[0058] In some embodiments, A is [ka] is selected from.

[0059] In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, R a and R b is independently selected from hydrogen and methyl. In some embodiments, R a and R b is hydrogen. In some embodiments, m is 1 and R a and R b are each hydrogen.

[0060] In some embodiments, Q is selected from C1-C6-alkylene and C1-C6-hydroxyalkylene. In some embodiments, Q is selected from -CH2CH2CH2- and -CH2CH(OH)CH2-. In some embodiments, Q is -CH2CH2CH2-. In some embodiments, Q is -CH2CH(OH)CH2-.

[0061] In some embodiments, Q is [ka] where n is 1, 2, 3, 4, 5, or 6.

[0062] In some embodiments, the group -Q-SO3M has the formula: [ka] has.

[0063] In some embodiments, each M is independently an alkali metal. In some embodiments, the alkali metal is sodium.

[0064] In some embodiments, the compound of formula (I) is [ka] [ka] [ka] [ka] is selected from.

[0065] The compounds described herein, such as the compound of formula (I), can be in the form of salt.The neutral form of the compound can be regenerated by contacting the salt with base or acid and isolating the parent compound in conventional manner.The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvent, but otherwise the salt is equivalent to the parent form of the compound for the purposes of this disclosure.

[0066] For example, a compound may be anionic or may be anionic (e.g., -COOH may be -COO - and -SO3H can be -SO3 - In the case of a cation having a functional group, salts may be formed with suitable cations. Examples of suitable inorganic cations are Na + and K + Alkali metal ions such as Ca 2- and Mg 2+ Examples of suitable organic cations include, but are not limited to, alkaline earth cations such as ammonium ion (i.e., NH + ) and substituted ammonium ions (e.g., NH3R1 + , NH2R2 + , NHR3 + , NR4 +Some examples of suitable substituted ammonium ions include, but are not limited to, ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine.

[0067] The compound is or can be cationic (e.g., -NH2 becomes -NH3 + If the compound has a functional group, salts may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyloxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, trifluoroacetic acid, and valeric acid.

[0068] Unless otherwise specified, a reference herein to a particular compound of formula (I) also includes the salt forms thereof.

[0069] Certain compounds of formula (I) may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including, but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+)- and (-)-forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial- and equatorial-, boat-, chair-, twisted-, envelope-, and half-chair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

[0070] In some embodiments, the compounds described herein can be enantiomerically enriched isomers of the stereoisomers described herein.For example, the compounds can have at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% enantiomeric excess.As used herein, enantiomer refers to any of a pair of chemical compounds whose molecular structures are mirror images of each other.

[0071] In some embodiments, preparations of the compounds disclosed herein are enriched for an isomer of the compound having a selected stereochemistry, e.g., R or S, corresponding to a selected stereocenter. For example, the compound has a purity corresponding to a compound having the selected stereochemistry at a selected stereocenter of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0072] In some embodiments, the compositions described herein include preparations of compounds disclosed herein that are enriched for structures having a selected stereochemistry, e.g., R or S, at a selected stereocenter. Exemplary R / S configurations can be those provided in the examples described herein.

[0073] An "enriched preparation," as used herein, is enriched for a selected stereoconfiguration of one, two, three, or more selected stereocenters in a target compound. Exemplary selected stereocenters and their exemplary stereoconfigurations can be selected from those provided herein, for example, in the examples described herein. "Enriched" means, for example, that at least 60% of the molecules of the compound in the preparation have the selected stereochemistry of the selected stereocenter. In one embodiment, it is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. "Enriched" refers to the level of the target molecule(s) and does not imply a process limitation unless expressly stated.

[0074] The compounds can be prepared in racemic form or as individual enantiomers or diastereomers, either by stereospecific synthesis or resolution. The compounds can be resolved into their component enantiomers or diastereomers by standard techniques, such as, for example, the formation of stereoisomeric pairs by salt formation with optically active bases, followed by fractional crystallization and regeneration of the free acid. The compounds can also be resolved by the formation of stereoisomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds can be resolved using chiral HPLC columns. Enantiomers can also be obtained from the dynamic resolution of the corresponding racemates of esters using lipase enzymes.

[0075] Except as tautomeric forms are discussed below, specifically excluded from the term "isomer" as used herein are structural (or constitutional) isomers (i.e., isomers that differ in the bonds between atoms rather than simply by the position of the atoms in space). For example, a reference to a methoxy group, -OCH3, should not be construed as a reference to its structural isomer, a hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl should not be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may include isomeric forms that are structurally within that class (e.g., C3-alkyl or propyl includes n-propyl and iso-propyl, C4-alkyl or butyl includes n-, iso-, sec-, and tert-butyl, and methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl). The above exclusion does not pertain to tautomeric forms, e.g., keto, enol, and enolate forms, as in, for example, the following tautomeric pairs: keto / enol, imine / enamine, amide / iminoalcohol, amidine / amidine, nitroso / oxime, thioketone / enethiol, N-nitroso / hydroxyazo, and nitro / aci-nitro.

[0076] Note that specifically included within the term "isomer" are compounds that have one or more isotopic substitutions. For example, H is 1 H, 2 H(D), and 3 H(T) and C can be in any isotopic form, including 12 C. 13 C, and 14 It can be in any isotopic form, including C, and O is 16 O and 18 It can be in any isotopic form, including O, etc.

[0077] The compounds can be prepared by various methods. For example, the compounds can be prepared as shown in Schemes 1-3. The abbreviations used in the schemes are as follows: ACN is acetonitrile, DIPEA is N,N-diisopropylethylamine, DMF is N,N-dimethylformamide, TFA is trifluoroacetic acid, THF is tetrahydrofuran, rt is room temperature, and TsOH is p-toluenesulfonic acid. Scheme 1. Exemplary Compound Synthesis [ka] Scheme 2. Exemplary Compound Synthesis [ka] Scheme 3. Exemplary Compound Synthesis [ka]

[0078] The compounds and intermediates herein can be isolated and purified by methods well known to those skilled in the art of organic synthesis. Examples of conventional methods for isolating and purifying compounds can include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, by recrystallization at high or low temperatures, with optional pretreatment with activated carbon, thin layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, as described, for example, in "Vogel's Textbook of Practical Organic Chemistry," 5th edition (1989), by Furniss, Hannaford, Smith, and Tatchell, pub. Longman Scientific & Technical, Essex CM20 2JE, England.

[0079] The reaction conditions and reaction times for each individual step may vary depending on the specific reactants employed and the substituents present in the reactants used. Specific procedures are provided in the Examples section. The reactions are carried out in a conventional manner, for example by removing the solvent from the residue, which may be further purified according to methodologies generally known in the art, such as, but not limited to, crystallization, distillation, extraction, trituration, and chromatography. Unless otherwise stated, starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature. If not commercially available, the starting materials can be prepared by procedures selected from standard organic chemistry techniques, techniques analogous to the synthesis of known structurally similar compounds, or techniques analogous to the schemes described above, or procedures described in the Synthetic Examples section.

[0080] Routine experimentation, including appropriate manipulation of the reaction conditions, reagents, and sequences of the synthetic route, protection of any chemical functionality that may not be compatible with the reaction conditions, and deprotection at suitable points in the reaction sequence of the method, are within the scope of the present invention. Suitable protecting groups and methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art, and examples thereof can be found in the monograph by PGM Wuts entitled "Greene's Protective Groups in Organic Synthesis" (5th ed.), John Wiley & Sons, Inc. (2014), which is incorporated herein by reference in its entirety. The synthesis of the compounds of the present invention can be achieved by methods similar to those described in the above synthetic schemes and specific examples.

[0081] The synthetic schemes and specific examples described are illustrative and should not be construed as limiting the scope of the invention, which is defined in the claims. All alternatives, modifications, and equivalents of the synthetic methods and specific examples are included within the scope of the claims.

[0082] 3. Method The embodiments of the present disclosure include methods for digesting proteins. The use of compounds of formula (I) in such methods may improve protein digestion, for example, by solubilizing hydrophobic or other insoluble compounds and / or by aiding in protein unfolding, facilitating protein digestion. Compounds of formula (I) may also aid in the recovery of generated protein fragments by improving extraction from gels and / or preventing protein fragment loss by absorption or precipitation. For example, compounds of formula (I) may be easily degraded during or after protein digestion, and fragments may be easily removed from samples or may not substantially interfere with any subsequent analysis steps.

[0083] The method includes contacting a sample containing at least one protein with a protein digestion reagent and a compound of formula (I), or a salt thereof, thereby providing a sample containing at least one digested protein.

[0084] The method includes the use of a protein digestion reagent. The protein digestion reagent cleaves peptide bonds in proteins to produce smaller peptide fragments. The protein digestion reagent may include a protease. In some embodiments, the protease may be a serine protease, such as trypsin, chymotrypsin, Lys-C, Glu-C, elastase, or protease K. In some embodiments, the protease may be a zinc metalloprotease, such as Lys-N or Asp-N. In some embodiments, the protease may be a cysteine ​​protease, such as papain or Arg-C. In some embodiments, the protease may be an aspartic acid protease, such as pepsin. The protease may be specific or non-specific. In some embodiments, the protein digestion reagent is a non-enzymatic digestion reagent, such as cyanogen bromide or hydroxylamine. Any suitable combination of protein digestion reagents may also be used. In some embodiments, the protein digestion reagent is trypsin.

[0085] The method also includes the use of compounds of formula (I). Without wishing to be limited by theory, compounds of formula (I) may aid in protein digestion by solubilizing and / or denaturing the protein to be digested. For example, the compounds may promote protein unfolding to provide digestion reagents access to internal protein sites.

[0086] The method can be used with a variety of protein samples. In some embodiments, the sample is a gel, a solid support, or a solution. In some embodiments, the sample is a gel. In some embodiments, the sample is an aqueous solution. In some embodiments, the aqueous solution is a solution from in-solution protein digestion with a protease, protein pellet solubilization, protein extract from cells, protein extract from tissue, protein deglycosylation with a deglycosidase, or removal of non-specifically bound proteins from a protein immobilizing support.

[0087] In some embodiments, the method further comprises decomposing the surfactant after the contacting step. In some embodiments, the decomposition step comprises contacting the surfactant with an acid. For example, the addition of an acid may decompose the compound into polar and lipophilic components, which may be removed from the sample prior to analysis (e.g., by passing the sample through a silica plug, such as a C18 derivatized silica plug). Inorganic acids (e.g., hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, or phosphorous acid), or organic acids (e.g., 2-acetyloxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxymaleic acid, hydroxyphenylsulfonic ... Any suitable acid may be used, including naphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, trifluoroacetic acid, and valeric acid.

[0088] In some embodiments, the surfactant auto-degrades after the contacting step, in other words, in certain embodiments, the surfactant may degrade on its own during the course of the method without the addition of a reagent or other stimulus to initiate decomposition.

[0089] In some embodiments, the method further comprises isolating at least one digested protein fragment. The digested protein may be isolated from the sample according to established techniques such as extraction from gels, chromatographic separation, centrifugation, precipitation, affinity purification, and the like.

[0090] For example, protein fragments (e.g., peptides) can be extracted from a gel by contacting the gel with a compound of formula (I) and an aqueous solution, and then separating the aqueous liquid from the gel to provide a solution containing the protein fragments.

[0091] In some embodiments, the method further comprises analyzing at least one digested protein fragment or peptide. In some embodiments, the analyzing step comprises mass spectrometry, liquid chromatography (e.g., high performance liquid chromatography, reverse phase chromatography, normal phase chromatography, size exclusion chromatography, etc.), gel electrophoresis (e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or native polyacrylamide gel electrophoresis (native PAGE)), or any combination thereof.

[0092] Embodiments of the present disclosure also include a composition comprising a gel and a compound of formula (I).

[0093] 4. Working Example It will be apparent to those skilled in the art that other suitable modifications and adaptations of the disclosed methods described herein are readily applicable and understandable, and may be made using suitable equivalents without departing from the scope of the disclosure or the aspects and embodiments disclosed herein. Having now described the disclosure in detail, the same will be more clearly understood by reference to the following examples, which are intended merely to illustrate some aspects and embodiments of the disclosure, and should not be considered as limiting the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications mentioned herein are incorporated herein by reference in their entirety.

[0094] The present disclosure has multiple aspects, exemplified by the following non-limiting examples.

[0095] The following abbreviations are used in the examples: ACN is acetonitrile, DCM is dichloromethane, DIPEA is N,N-diisopropylethylamine, DMF is N,N-dimethylformamide, DMSO is dimethylsulfoxide, RT is room temperature, THF is tetrahydrofuran, and p-TsOH is p-toluenesulfonic acid.

[0096] Example 1 Sodium 3-((((2-methyl-2-nonyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (WZ-0547) [ka]

[0097] Step 1. (2-Methyl-2-nonyl-1,3-dioxolan-4-yl)methanol (WZ-0540) [ka]

[0098] To undecan-2-one (20.0 g, 117 mmol) in heptane and toluene (1:2, 300 mL) was added glycerol (13.0 g, 141 mmol) and p-toluenesulfonic acid (404 mg, 2.35 mmol). The mixture was heated to reflux using a Dean Stark trap condenser for 18 h. The reaction was neutralized with NaHCO3 (0.04 equiv.) in water (100 mL). The organic layer was washed with more water. The organic layer was dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to give the desired product (18.1 g, 63%) as a colorless oil. ESI MS m / z 245 [M+H]+.

[0099] Step 2. (2-Methyl-2-nonyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (WZ-0546) [ka]

[0100] To a solution of (2-methyl-2-nonyl-1,3-dioxolan-4-yl)methanol (5.0 g, 20.5 mmol) in THF (50 mL) at 0° C. was added 4-nitrophenyl carbonochloridate (6.2 g, 30.7 mmol) and pyridine (4.9 g, 61.4 mmol). The suspension was stirred at 0° C. for 2 h. The reaction was concentrated in vacuo, suspended in heptane, filtered, and the solid was washed with heptane. The filtrate was concentrated and purified by silica gel chromatography to give the desired product (7.8 g, 93%) as a colorless oil. MS m / z 410 [M+H]+.

[0101] Step 3. Sodium 3-((((2-methyl-2-nonyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (WZ-0547) [ka]

[0102] To a solution of (2-methyl-2-nonyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (0.60 g, 1.5 mmol) in THF (30 mL) was added tetrabutylammonium 3-aminopropane-1-sulfonate (0.67 g, 1.8 mmol). The mixture was stirred at room temperature for 30 min. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the tetrabutylammonium salt. The tetrabutylammonium product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin and lyophilized to give the desired product (0.094 g, 10%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.25 (t, J = 5.8 Hz, 1H), 4.21 - 4.11 (m, 1H), 4.07 - 3.96 (m, 2H), 3.90 (dd, J = 11.4, 6.2 Hz, 1H), 3.69 - 3.58 (m, 1H), 3.02 (q, J = 6.7 Hz, 2H), 2.45 - 2.32 (m, 2H), 1.75 - 1.63 (m, 2H), 1.57 - 1.47 (m, 2H), 1.35 - 1.18 (m, 14H), 0.91 - 0.79 (m, 3H);MS m / z 408 [M - H - Na]-.

[0103] Example 2 Sodium 3-((((2,2-dihexyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (TU-1179) [ka]

[0104] Step 1. (2,2-Dihexyl-1,3-dioxolan-4-yl)methanol (TU-1172) [ka]

[0105] To a solution of tridecan-7-one (11.5 g, 58.0 mmol) in heptane and toluene (1:2, 150 mL) was added glycerol (6.5 g, 70.6 mmol) and p-toluenesulfonic acid (230 mg, 1.2 mmol). The mixture was heated to reflux using a Dean Stark trap condenser for 18 h. The reaction was neutralized with NaHCO3 (0.04 equiv.) in water (100 mL) and extracted with ethyl acetate. The organic layer was dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to give the desired product (9.4 g, 59%) as a colorless oil. MS m / z 273 [M+H]+.

[0106] Step 2. (2,2-Dihexyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (TU-1175) [ka]

[0107] To a solution of (2,2-dihexyl-1,3-dioxolan-4-yl)methanol (6.1 g, 22.3 mmol) in THF (100 mL) at 0° C. was added 4-nitrophenyl carbonochloridate (6.8 g, 33.6 mmol) and pyridine (5.3 g, 67.0 mmol). The suspension was stirred for 4 h at 0° C. The reaction was concentrated in vacuo, dissolved in DCM, concentrated onto Celite, and purified by silica gel chromatography to give the desired product (9.5 g, 97%) as a pale yellow oil.

[0108] Step 3. Sodium 3-((((2,2-dihexyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (TU-1179) [ka]

[0109] To a solution of (2,2-dihexyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (1.47 g, 3.36 mmol) in THF (40 mL) was added tetrabutylammonium 3-aminopropane-1-sulfonate (3.08 g, 8.11 mmol). The mixture was stirred at room temperature for 3 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using DCM / MeOH to give the product as the tetrabutylammonium salt. The tetrabutylammonium product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin, and lyophilized to give the desired product (1.39 g, 90%) as a white solid. 1H NMR (400 MHz, heavy water) δ 4.37 - 4.16 (m, 1H), 4.12 - 3.91 (m, 3H), 3.75 - 3.58 (m, 1H), 3.23 - 3.09 (m, 2H), 2.91 - 2.74 (m, 2H), 1.93 - 1.80 (m, 2H), 1.67 - 1.42 (m, 4H), 1.42 - 1.15 (m, 16H), 0.82 (t, J = 6.2 Hz, 6H);MS m / z 436 [M - H - Na]-.

[0110] Example 3 Sodium 3-((((2-decyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (JRW-1564) [ka]

[0111] Step 1. (2-Decyl-1,3-dioxolan-4-yl)methanol (JRW-1537) [ka]

[0112] To a solution of undecanal (14.8 g, 86.9 mmol) in heptane and toluene (1:2, 300 mL) was added glycerol (9.6 g, 104 mmol) and p-toluenesulfonic acid (299 mg, 1.7 mmol). The mixture was heated to reflux using a Dean Stark trap condenser for 2 days. The reaction was neutralized with NaHCO3 (0.04 equiv.) in water (100 mL). The organic layer was washed with more water. The organic layer was dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to give the desired product (18.6 g, 87%) as a colorless oil.

[0113] Step 2. (2-Decyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (JRW-1539) [ka]

[0114] To a solution of (2-decyl-1,3-dioxolan-4-yl)methanol (18.6 g, 76.1 mmol) in THF (100 mL) at 0° C. was added 4-nitrophenyl carbonochloridate (23.0 g, 114 mmol) and pyridine (18.1 g, 228 mmol). The suspension was stirred at 0° C. and then warmed to room temperature for 18 h. The reaction was concentrated in vacuo, suspended in heptane, filtered, and the solid was washed with heptane. The filtrate was concentrated and purified by silica gel chromatography to give the desired product (21.3 g, 68%) as a white solid.

[0115] Step 3. Sodium 3-((((2-decyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (JRW-1564) [ka]

[0116] To a solution of (2-decyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (0.51 g, 1.25 mmol) in THF (20 mL) was added tetrabutylammonium 3-aminopropane-1-sulfonate (0.57 g, 1.5 mmol). The mixture was stirred at room temperature for 4 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the tetrabutylammonium salt. The tetrabutylammonium product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin and lyophilized to give the desired product (0.45 g, 83%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.30 - 7.21 (m, 1H), 4.90 (t, J = 4.8 Hz, 0.5H, diastereotopic), 4.82 (t, J = 4.8 Hz, 0.5H, diastereotopic), 4.24 - 4.12 (m, 1H), 4.11 - 3.92 (m, 2H), 3.92 - 3.78 (m, 1H), 3.68 - 3.61 (m, 0.5H, diastereotopic), 3.52-3.46 (m, 0.5H, diastereotopic), 3.08 - 2.92 (m, 2H), 2.44 - 2.36 (m, 2H), 1.75 - 1.63 (m, MS m / z 408 [M - H - Na]-.

[0117] Example 4 Sodium 3-((((2-dodecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (JRW-1574) [ka]

[0118] Step 1. (2-Dodecyl-1,3-dioxolan-4-yl)methanol (JRW-1568) [ka]

[0119] To a solution of tridecanal (10.0 g, 50.4 mmol) in heptane and toluene (1:2, 300 mL) was added glycerol (5.6 g, 60.5 mmol) and p-toluenesulfonic acid (173 mg, 1.0 mmol). The mixture was heated to reflux using a Dean Stark trap condenser for 4 days. The reaction was neutralized with NaHCO3 (0.04 equiv.) in water (100 mL). The organic layer was washed with more water. The organic layer was dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to give the desired product (12.5 g, 91%) as a colorless oil.

[0120] Step 2. (2-Dodecyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (JRW-1571) [ka]

[0121] To a solution of (2-dodecyl-1,3-dioxolan-4-yl)methanol (12.5 g, 45.9 mmol) in THF (100 mL) at 0° C. was added 4-nitrophenyl carbonochloridate (11.1 g, 55.1 mmol) and pyridine (10.9 g, 137 mmol). The suspension was stirred for 3 h at 0° C. The reaction was concentrated in vacuo, suspended in heptane, filtered, and the solid was washed with heptane. The filtrate was concentrated and purified by silica gel chromatography to give the crude product (12.0 g) as a white solid.

[0122] Step 3. Sodium 3-((((2-dodecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (JRW-1574) [ka]

[0123] To a solution of (2-dodecyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (0.40 g, 0.91 mmol) in THF (20 mL) was added tetrabutylammonium 3-aminopropane-1-sulfonate (0.42 g, 1.1 mmol). The mixture was stirred at room temperature for 2.5 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the tetrabutylammonium salt. The tetrabutylammonium product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin and lyophilized to give the desired product (0.31 g, 73%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.29 - 7.21 (m, 1H), 4.90 (t, J = 4.8 Hz, 0.5H, diastereotopic) 4.82 (t, J = 4.8 Hz, 0.5H, diastereotopic), 4.24-4.12 (m, 1H), 4.11 - 3.93 (m, 2H), 3.92 - 3.78 (m, 1H), 3.67 - 3.61 (m, 0.5H, diastereotopic), 3.53 - 3.45 (m, 0.5H, diastereotopic), 3.08 - 2.96 (m, 2H), 2.42 - 2.36 (m, 2H), 1.74 - 1.63 (m, 2H), 1.62 - 1.47 (m, 2H), 1.24 (s, 20H), 0.92 - 0.80 (m, 3H).;MS m / z 436 [M - H - Na]-.

[0124] Example 5 Sodium 3-((((2-undecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (JRW-1565) [ka]

[0125] Step 1. (2-Undecyl-1,3-dioxolan-4-yl)methanol (JRW-1538) [ka]

[0126] To a solution of dodecanal (14.8 g, 80.3 mmol) in heptane and toluene (1:2, 200 mL) was added glycerol (8.8 g, 96.3 mmol) and p-toluenesulfonic acid (276 mg, 1.6 mmol). The mixture was heated to reflux using a Dean Stark trap condenser for 2 days. The reaction was neutralized with NaHCO3 (0.04 equiv.) in water (100 mL). The organic layer was washed with more water. The organic layer was dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to give the desired product (14.8 g, 71%) as a white solid.

[0127] Step 2. 4-Nitrophenyl((2-undecyl-1,3-dioxolan-4-yl)methyl)carbonate (JRW-1540) [ka]

[0128] To a solution of (2-undecyl-1,3-dioxolan-4-yl)methanol (14.8 g, 57.3 mmol) in THF (100 mL) at 0° C. was added 4-nitrophenyl carbonochloridate (17.3 g, 85.9 mmol) and pyridine (13.6 g, 172 mmol). The suspension was stirred for 2 h at 0° C. The reaction was concentrated in vacuo, suspended in heptane, filtered, and the solid was washed with heptane. The filtrate was concentrated and purified by silica gel chromatography to give the crude product (14.5 g) as a white solid.

[0129] Step 3. Sodium 3-((((2-undecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (JRW-1565) [ka]

[0130] To a solution of 4-nitrophenyl((2-undecyl-1,3-dioxolan-4-yl)methyl)carbonate (0.68 g, 1.61 mmol) in THF (20 mL) was added tetrabutylammonium 3-aminopropane-1-sulfonate (0.73 g, 1.9 mmol). The mixture was stirred at room temperature for 3 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the tetrabutylammonium salt. The tetrabutylammonium product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin and lyophilized to give the desired product (0.35 g, 49%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.35 - 7.18 (m, 1H), 4.90 (t, J = 4.8 Hz, 0.5H, diastereotopic), 4.82 (t, J = 4.8 Hz, 0.5H, diastereotopic), 4.24 - 4.13 (m, 1H), 4.09 - 3.92 (m, 2H), 3.92 - 3.78 (m, 1H), 3.67 - 3.61 (m, 0.5H, diastereotopic), 3.52 - 3.46 (m, 0.5H, diastereotopic), 3.07 - 2.97 (m, 2H), 2.46 - 2.34 (m, 2H), 1.74 - 1.64 (m, 2H), 1.60 - 1.49 (m, 2H), 1.37 - 1.18 (m, 18H), 0.91 - 0.81 (m, 3H).);MS m / z 422 [M - H - Na]-.

[0131] Example 6 Sodium 3,3'-((4-((((2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)phenyl)azanediyl)bis(propane-1-sulfonate) (SL-1735) [ka]

[0132] Step 1. (2-Methyl-2-undecyl-1,3-dioxolan-4-yl)methanol (AF-0046) [ka]

[0133] To a solution of tridecan-2-one (5.0 g, 25.2 mmol) in heptane and toluene (1:2, 200 mL) was added glycerol (2.8 g, 30.2 mmol) and p-toluenesulfonic acid (86 mg, 0.50 mmol). The mixture was heated to reflux using a Dean Stark trap condenser for 18 h. The reaction was neutralized with NaHCO3 (0.04 equiv.) in water (100 mL). The organic layer was washed with more water. The organic layer was dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to give the desired product (4.1 g, 66%) as a colorless oil.

[0134] Step 2. (2-Methyl-2-undecyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (AF-0047) [ka]

[0135] To a solution of (2-methyl-2-undecyl-1,3-dioxolan-4-yl)methanol (2.0 g, 7.3 mmol) in THF (50 mL) at 0° C. was added 4-nitrophenyl carbonochloridate (2.22 g, 11.0 mmol) and pyridine (1.7 g, 22 mmol). The suspension was stirred for 2 h at 0° C. The reaction was concentrated in vacuo, suspended in heptane, filtered, and the solid was washed with heptane. The filtrate was concentrated and purified by silica gel chromatography to give the crude product (3.0 g) as a white solid.

[0136] Step 3. (2-Methyl-2-undecyl-1,3-dioxolan-4-yl)methyl(4-aminophenyl)carbamate (SL-1733) [ka]

[0137] To a solution of (2-methyl-2-undecyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (1.0 g, 2.35 mmol) in DMF (6 mL) was added benzene-1,4-diamine (510 mg, 4.7 mmol) and diisopropylethylamine (0.61 g, 4.7 mmol). The reaction was stirred at room temperature for 4 h. The reaction was purified by silica gel chromatography to give the desired product (0.75 g, 78%) as a yellow solid. MS m / z 407 [M+H]+.

[0138] Step 4. Sodium 3,3'-((4-((((2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)phenyl)azanediyl)bis(propane-1-sulfonate) (SL-1735) [ka]

[0139] To a solution of (2-methyl-2-undecyl-1,3-dioxolan-4-yl)methyl(4-aminophenyl)carbamate (0.47 g, 1.16 mmol) in acetonitrile (9 mL) was added 1,2-oxathiolane 2,2-dioxide (423 mg, 3.47 mmol) and diisopropylethylamine (747 mg, 5.78 mmol). The mixture was heated to 80° C. for 2 days. The reaction was purified by silica gel chromatography to give the diisopropylethylamine salt as a yellow foam. The diisopropylethylamine product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin, and lyophilized to give the desired product (0.73 g, 91%) as a white foam. MS m / z 649 [MH-Na]-.

[0140] Example 7 Sodium 3-((((2-decyl-2-methyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (AF-0045) [ka]

[0141] To a solution of 4-nitrophenyl(2-decyl-2-methyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (1.0 g, 2.36 mmol) in THF (10 mL) was added tetrabutylammonium 3-aminopropane-1-sulfonate (1.1 g, 2.8 mmol). The mixture was stirred at room temperature for 30 min. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the tetrabutylammonium salt. The tetrabutylammonium product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin and lyophilized to give the desired product (0.77 g, 49%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.31 - 7.18 (m, 1H), 4.27 - 4.11 (m, 1H), 4.10 - 3.95 (m, 2H), 3.93 - 3.82 (m, 1H), 3.67 - 3.52 (m, 1H), MS m / z 422 [M - H - Na]-.

[0142] Example 8 Sodium 3-((((2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (JRW-1573) [ka]

[0143] To a solution of (2-methyl-2-undecyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (0.30 g, 0.68 mmol) in THF (20 mL) was added tetrabutylammonium 3-aminopropane-1-sulfonate (0.31 g, 0.82 mmol). The mixture was stirred at room temperature for 18 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the tetrabutylammonium salt. The tetrabutylammonium product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin and lyophilized to give the desired product (0.21 g, 67%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.29 - 7.19 (m, 1H), 4.28 - 4.11 (m, 1H), 4.07 - 3.96 (m, 2H), 3.94 - 3.81 (m, 1H), 3.69 - 3.52 (m, 1H), 3.02 (q, J = 6.7 Hz, 2H), 2.44 - 2.34 (m, 2H), 1.75 - 1.63 (m, 2H), 1.60 - 1.47 (m, 2H), 1.35 - 1.18 (m, 21H), 0.91 - 0.81 (m, 3H);MS m / z 436 [M - H - Na]-.

[0144] Example 9 Sodium 2-hydroxy-3-((((2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (HW-0797) [ka]

[0145] To a solution of (2-methyl-2-undecyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (0.17 g, 0.39 mmol) in acetonitrile (20 mL) was added a solution of sodium 3-amino-2-hydroxypropane-1-sulfonate (0.066 g, 0.43 mmol) and sodium bicarbonate (49 mg, 0.58 mmol) in water (10 ml). The mixture was stirred at room temperature for 3 h. The reaction was concentrated under vacuum and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the sodium salt (86 mg, 49%). 1H NMR (400 MHz, DMSO-d6) δ 7.19 (t, J = 5.8 Hz, 1H), 5.03 (s, 1H), 4.21 - 4.11 (m, 1H), 4.06 - 3.97 (m, 2H), 3.94 - 3.79 (m, 2H), 3.96 - 3.87 (m, 1H), 3.86 - 3.76 (m, 1H), 3.65 - 3.61 (m, 1H), 3.10 - 2.94 (m, 2H), 2.6 - 2.59 (m, 1H), 2.43 - 2.32 (m, 1H), 1.59 - 1.47 (m, 2H), 1.35 - 1.18 (m, 21H), 0.91 - 0.79 (m, 3H);MS m / z 453 [M - H - Na]-.

[0146] Example 10 Sodium 3-((((2-decyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)-2-hydroxypropane-1-sulfonate (HW-0801) [ka]

[0147] To a solution of (2-decyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (0.23 g, 0.56 mmol) in acetonitrile (20 mL) was added a solution of sodium 3-amino-2-hydroxypropane-1-sulfonate (0.11 g, 0.67 mmol) and sodium bicarbonate (72 mg, 0.56 mmol) in water (10 ml). The mixture was stirred at room temperature for 3 h. The reaction was concentrated under vacuum and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the sodium salt (0.16 mg, 67%). 1H NMR (400 MHz, DMSO-d6) δ 7.19 (t, J = 5.8 Hz, 1H), 5.03 (s, 1H), 4.91 - 4.80 (m, 1H), 4.23 - 4.14 (m, 1H), 4.06 - 3.95 (m, 2H), 3.90 - 3.78 (m, 2H), 3.65 - 3.48 (m, 1H), 3.10 - 2.93 (m, 2H), 2.69 - 2.58 (m, 1H), 2.43 - 2.30 (m, 1H), 1.62 - 1.47 (m, 2H), 1.35 - 1.18 (m, 16H), 0.91 - 0.79 (m, 3H);MS m / z 425 [M - H - Na]-.

[0148] Example 11 Sodium 3-((((2-dodecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)-2-hydroxypropane-1-sulfonate (HW-0807) [ka]

[0149] To a solution of (2-dodecyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (0.38 g, 0.87 mmol) in acetonitrile (20 mL) was added a solution of sodium 3-amino-2-hydroxypropane-1-sulfonate (0.15 g, 0.96 mmol) and sodium bicarbonate (146 mg, 1.74 mmol) in water (10 ml). The mixture was stirred at room temperature for 3 h. The reaction was concentrated under vacuum and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the sodium salt (160 mg, 41%). 1H NMR (400 MHz, DMSO-d6) δ 7.19 (t, J = 5.8 Hz, 1H), 5.03 (s, 1H), 4.91 - 4.80 (m, 1H), 4.23 - 4.14 (m, 1H), 4.06 - 3.95 (m, 2H), 3.90 - 3.78 (m, 2H), 3.65 - 3.48 (m, 1H), 3.10 - 2.94 (m, 2H), 2.71 - 2.59 (m, 1H), 2.43 - 2.31 (m, 1H), 1.61 - 1.47 (m, 2H), 1.35 - 1.18 (m, 20H), 0.91 - 0.79 (m, 3H);MS m / z 453 [M - H - Na]-.

[0150] Example 12 Sodium 2-hydroxy-3-((((2-undecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (HW-0805) [ka]

[0151] To a solution of 4-nitrophenyl((2-undecyl-1,3-dioxolan-4-yl)methyl)carbonate (0.38 g, 0.90 mmol) in acetonitrile (20 mL) was added a solution of sodium 3-amino-2-hydroxypropane-1-sulfonate (0.15 g, 0.99 mmol) and sodium bicarbonate (151 mg, 1.79 mmol) in water (10 ml). The mixture was stirred at room temperature for 3 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the sodium salt (150 mg, 38%). 1H NMR (400 MHz, DMSO-d6) δ 7.19 (t, J = 5.8 Hz, 1H), 5.03 (s, 1H), 4.91 - 4.80 (m, 1H), 4.23 - 4.14 (m, 1H), 4.06 - 3.95 (m, 2H), 3.90 - 3.78 (m, 2H), 3.65 - 3.48 (m, 1H), 3.10 - 2.93 (m, 2H), 2.68 - 2.59 (m, 1H), 2.43 - 2.32 (m, 1H), 1.60 - 1.47 (m, 2H), 1.35 - 1.18 (m, 18H), 0.91 - 0.79 (m, 3H);MS m / z 439 [M - H - Na]-.

[0152] Example 13 Sodium 3-((((2,2-dipentyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)-2-hydroxypropane-1-sulfonate (JZ-0164) [ka]

[0153] Step 1. (2,2-dipentyl-1,3-dioxolan-4-yl)methanol (JZ-0144) [ka]

[0154] To a round bottom flask with undecan-6-one (5.11 g, 30 mmol), glycerol (3.32 g, 1.2 eq, 36 mmol), and 90 mL of mixed solvent (80 mL toluene and 10 mL heptane) was added p-TsOH (103 mg, 2 mol%). The mixture was refluxed under N2 with a Dean Stark for 18 h. The mixture was cooled to room temperature and diluted with 300 mL EtOAc. The organic layer was washed twice with 2% NaHCO3 (80 mL) and then dried over Na2SO4. The crude was then purified by flash column to give a colorless oil product (6.73 g, 92%).1 HNMR (400 MHz, chloroform-d): δ 4.24 (m, 1H), 4.05 (dd, J = 8.0, 6.6 Hz, 1H), 3.85 - 3.74 (m, 2H), 3.61 (dd, J = 11.7, 5.1 Hz, 1H), 1.79 (s, 1H), 1.70 - 1.58 (m, 4H), 1.47 - 1.25 (m, 12H), 0.91 (t, J = 6.9 Hz, 6H).

[0155] Step 2. (2,2-dipentyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (JZ-0151) [ka]

[0156] To a round bottom flask containing (2,2-dipentyl-1,3-dioxolan-4-yl)methanol (3.07, 12.56 mmol), p-nitrophenyl chloroformate (3.80 g, 1.5 eq, 18.84 mmol), and 30 mL of THF was added pyridine (3.04 mL, 3 eq) at 0° C. The reaction was then warmed to room temperature and stirred for 2 hours. The mixture was concentrated in vacuo, washed with heptane, and the solid was filtered off. The filtrate was collected and then purified by flash column. A pale yellow oil product was obtained (3.84 g, 75%). 1 HNMR (400 MHz, chloroform-d): δ 8.38 - 8.22 (m, 2H), 7.49 - 7.36 (m, 2H), 4.51 - 4.29 (m, 3H), 4.21 - 4.12 (m, 1H), 3.81 (dd, J = 8.4, 6.5 Hz, 1H), 1.74 - 1.58 (m, 4H), 1.46 - 1.21 (m, 12H), 0.91 (q, J = 6.8 Hz, 6H).

[0157] Step 3. Sodium 3-((((2,2-dipentyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)-2-hydroxypropane-1-sulfonate (JZ-0164) [ka]

[0158] To a round bottom flask containing (2,2-dipentyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (327 mg, 0.8 mmol), 3-amino-2-hydroxypropane-1-sulfonic acid (186 mg, 1.5 equiv, 1.2 mmol) and an 8 mL can was added aqueous NaOH (1.6 mL) at room temperature. The reaction was stirred at 50° C. for 4 hours. The crude mixture was dried in vacuum and then purified by flash column. A white solid product (200 mg, 56% yield) was obtained. 1 HNMR (400 MHz, DMSO-d6): δ 7.17 (t, J = 6.0 Hz, 1H), 5.04 (d, J = 1.8 Hz, 1H), 4.19 (m, 1H), 4.11 - 3.97 (m, 2H), 3.94 - 3.76 (m, 2H), 3.57 (t, J = 7.6 Hz, 1H), 3.12 - 2.91 (m, 2H), 2.63 (dd, J = 13.5, 3.1 Hz, 1H), 2.37 (dd, J = 13.5, 9.1 Hz, 1H), 1.52 (m, 4H), 1.36 - 1.14 (m, 12H), 0.86 (t, J = 6.8 Hz, 6H);MS m / z 424 [M - Na - H]-.

[0159] Example 14 Sodium 3-((((2,2-dipentyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)propane-1-sulfonate (JZ-0177) [ka]

[0160] To a round bottom flask containing (2,2-dipentyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (205 mg, 0.5 mmol), 3-aminopropane-1-sulfonic acid (104 mg, 0.75 mmol) and a 5 mL can was added 1M aqueous NaOH solution (1 mL) at room temperature. The reaction was stirred at 50° C. for 3 hours. The crude mixture was then concentrated in vacuum and purified by flash column. A white solid product was obtained (210 mg, 97%). 1 H NMR (400 MHz, DMSO-d6): δ 7.24 (t, J = 5.8 Hz, 1H), 4.19 (qd, J = 6.7, 4.2 Hz, 1H), 4.09 - 3.97 (m, 2H), 3.87 (dd, J = 11.4, 6.5 Hz, 1H), 3.57 (dd, J = 8.2, 6.9 Hz, 1H), 3.01 (q, J = 6.7 Hz, 2H), 2.43 - 2.35 (m, 2H), 1.68 (m, 2H), 1.52 (m, 4H), 1.36 - 1.15 (m, 12H), 0.90 - 0.80 (m, 6H). MS m / z 408 [M - Na - H]-.

[0161] Example 15 Sodium 3-((((2,2-dihexyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)-2-hydroxypropane-1-sulfonate (JZ-0179) [ka]

[0162] To a round bottom flask containing (2,2-dihexyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (88 mg, 0.2 mmol), 3-amino-2-hydroxypropane-1-sulfonic acid (47 mg, 0.3 mmol) and a 2 mL can was added 1M aqueous NaOH solution (0.4 mL) at room temperature. The reaction was stirred at room temperature for 4 hours. The mixture was concentrated in vacuum and then purified by flash column. A white solid product was obtained (41 mg, 43%). 1 H NMR (400 MHz, DMSO-d6) δ 7.17 (t, J = 6.0 Hz, 1H), 5.04 (d, J = 1.8 Hz, 1H), 4.25 - 4.15 (m, 1H), 4.09 - 3.97 (m, 2H), 3.93 - 3.79 (m, 2H), 3.62 - 3.54 (m, 1H), 3.01 (m, 2H), 2.64 (dd, J = 13.5, 3.1 Hz, 1H), 2.37 (dd, J = 13.5, 9.1 Hz, 1H), 1.52 (m, 4H), 1.26 (q, J = 5.0, 2.8 Hz, 16H), 0.91 - 0.82 (m, 6H). MS m / z 452 [M - Na - H]-.

[0163] Example 16 Sodium 3-((2,2-bis(pentyloxy)propoxy)carbonyl)propane-1-sulfonate (RBK-0168) [ka]

[0164] Step 1. Pentyl 2,2-bis(pentyloxy)propanoate (RBK-0155-1) [ka]

[0165] To a solution of methyl pyruvate (10.0 g, 97.95 mmol) and 1-proponal (40.1 g, 454.91 mmol) in toluene (100 mL) was added p-toluenesulfonic acid (190 mg, 1.1 mmol). The mixture was heated to reflux using a Dean Stark trap condenser for 2 days. The reaction was neutralized with NaHCO3 (0.04 equiv.) in water (150 mL). The organic layer was washed with more water. The organic layer was dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography (0-10% EtOAc / heptane) to give the desired product (22.5 g, 17%) as a colorless oil.

[0166] Step 2. 2,2-Bis(pentyloxy)propan-1-ol (RBK-0156-1) [ka]

[0167] To a solution of pentyl 2,2-bis(pentyloxy)propanoate (9.2 g, 29.1 mmol) in THF (100 mL) at 0° C. was added lithium aluminum hydride (6.4 mL of a 2.4 M solution in THF, 15.5 mmol). The mixture was then stirred at room temperature for 1 h. The reaction was again cooled to 0° C. and diethyl ether (300 mL) was added, followed by dropwise addition of a 9:1 mixture of EtOAc / H2O (100 mL). The white precipitate was filtered. The filtrate was concentrated and purified by silica gel chromatography (0-10% EtOAc / heptane) to give the product (5.7 g, 75%) as a colorless oil.

[0168] Step 3. 2,2-Bis(pentyloxy)propyl 4-nitrophenyl carbonate (RBK-0158-2) [ka]

[0169] To a solution of 2,2-bis(hexyloxy)propan-1-ol (5.4 g, 23.2 mmol) in THF (60 mL) at 0° C. was added 4-nitrophenyl carbonochloridate (7.6 g, 37.9 mmol) and pyridine (4.2 mL, 69.7 mmol). The suspension was stirred for 2 h at 0° C. The reaction was diluted with heptane (100 mL) and the white solid was filtered. The filtrate was concentrated and purified by silica gel chromatography to give the crude product (5.6 g) as a yellow oil.

[0170] Step 4. Sodium 3-((2,2-bis(pentyloxy)propoxy)carbonyl)propane-1-sulfonate (RBK-0168) [ka]

[0171] To a solution of 2,2-bis(pentyloxy)propyl 4-nitrophenyl carbonate (0.45 g, 1.0 mmol) in THF (50 mL) was added tetrabutylammonium 3-aminopropane-1-sulfonate (0.43 g, 1.1 mmol). The mixture was stirred at room temperature for 3 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the tetrabutylammonium salt. The tetrabutylammonium product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin and lyophilized to give the desired product (0.48 g, 50%) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.27 - 7.24 (m, 1H), 3.90 (s, 2H), 3.42 - 3.35 (m, 4H), 3.02 (q, J = 6.7 Hz, 2H), 2.45 - 2.37 (m, 2H), 1.76 - 1.63 (m, 2H), 1.51 - 1.41 (m, 4H), 1.36 - 1.22 (m, 11H), 0.93 - 0.82 (m, 6H);MS m / z 395 [M - H - Na]-.

[0172] Example 17 Sodium 3-((2,2-bis(hexyloxy)propoxy)carbonyl)propane-1-sulfonate (RBK-169) [ka]

[0173] Step 1. Hexyl 2,2-bis(hexyloxy)propanoate (RBK-0161-1) [ka]

[0174] To a solution of methyl pyruvate (10.0 g, 97.95 mmol) and 1-hexanol (40.1 g, 392.5 mmol) in toluene (100 mL) was added p-toluenesulfonic acid (190 mg, 1.1 mmol). The mixture was heated to reflux using a Dean Stark trap condenser for 2 days. The reaction was neutralized with NaHCO3 (0.04 equiv.) in water (150 mL). The organic layer was washed with more water. The organic layer was dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography (0-10% EtOAc / heptane) to give the desired product (25.3 g, 18%) as a colorless oil.

[0175] Step 2. 2,2-Bis(hexyloxy)propan-1-ol (RBK-0162-1) [ka]

[0176] To a solution of hexyl 2,2-bis(hexyloxy)propanoate (13.2 g, 36.8 mmol) in THF (100 mL) at 0° C. was added lithium aluminum hydride (6.4 mL of a 2.4 M solution in THF, 15.5 mmol). The mixture was then stirred at room temperature for 1 h. The reaction was again cooled to 0° C. and diethyl ether (300 mL) was added, followed by dropwise addition of a 9:1 mixture of EtOAc / H2O (100 mL). The white precipitate was filtered. The filtrate was concentrated and purified by silica gel chromatography (0-10% EtOAc / heptane) to give the product (5.9 g, 59%) as a colorless oil.

[0177] Step 3. 2,2-Bis(hexyloxy)propyl 4-nitrophenyl carbonate (RBK-0165-1) [ka]

[0178] To a solution of 2,2-bis(hexyloxy)propan-1-ol (5.4 g, 23.2 mmol) in THF (60 mL) at 0° C. was added 4-nitrophenyl carbonochloridate (7.6 g, 37.9 mmol) and pyridine (4.2 mL, 69.7 mmol). The suspension was stirred for 2 h at 0° C. The reaction was diluted with heptane (100 mL) and the white solid was filtered. The filtrate was concentrated and purified by silica gel chromatography to give the crude product (3.6 g) as a yellow oil.

[0179] Step 4. Sodium 3-((2,2-bis(hexyloxy)propoxy)carbonyl)propane-1-sulfonate (RBK-0169-1) [ka]

[0180] To a solution of 2,2-bis(hexyloxy)propyl 4-nitrophenyl carbonate (0.40 g, 0.94 mmol) in THF (50 mL) was added tetrabutylammonium 3-aminopropane-1-sulfonate (0.43 g, 1.1 mmol). The mixture was stirred at room temperature for 3 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the tetrabutylammonium salt. The tetrabutylammonium product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin and lyophilized to give the desired product (0.24 g, 56%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.25 (t, J = 5.8 Hz, 1H), 3.90 (s, 2H), 3.41-3.34 (m, 4H), 3.02 (q, J = 6.7 Hz, 3H), 2.45 - 2.36 (m, 2H), 1.75 - 1.64 (m, 2H), 1.51 - 1.40 (m, 4H), 1.36 - 1.22 (m, 15H), 0.93 - 0.82 (m, 6H);MS m / z 423 [M - H - Na]-.

[0181] Example 18 Sodium 3-(((2,2-diheptyl-1,3-dioxolan-4-yl)methoxy)carbonyl)propane-1-sulfonate (RBK-170) [ka]

[0182] Step 1. (2,2-Diheptyl-1,3-dioxolan-4-yl)methanol (JZ-0142) [ka]

[0183] To a round bottom flask with pentadecan-8-one (2.26 g, 10 mmol), glycerol (1.11 g, 1.2 eq, 12 mmol), and 36 mL of mixed solvent (24 mL toluene and 12 mL heptane) was added p-TsOH (34.5 mg, 2 mol%). The mixture was refluxed under N2 with a Dean Stark for 18 h. The mixture was cooled to room temperature and diluted with 100 mL EtOAc. The organic layer was washed twice with 2% NaHCO3 (20 mL) and then dried over Na2SO4. The crude was then purified by flash column to give a colorless oil product (2.65 g, 88%). 1 H NMR (400 MHz, chloroform-d) δ 4.28 - 4.20 (m, 1H), 4.09 - 4.01 (m, 1H), 3.82 - 3.72 (m, 2H), 3.65 - 3.58 (m, 1H), 1.70 - 1.56 (m, 4H), 1.46 - 1.21 (m, 20H), 0.95 - 0.83 (m, 6H).

[0184] Step 2. (2,2-Diheptyl-1,3-dioxolan-4-yl)methyl(4-nitrophenyl)carbonate (JZ-0143) [ka]

[0185] To a round bottom flask containing (2,2-diheptyl-1,3-dioxolan-4-yl)methanol (2.65 g, 8.82 mmol), p-nitrophenyl chloroformate (2.67 g, 1.5 eq, 13.2 mmol), and 25 mL of THF was added pyridine (2.14 mL, 3 eq) at 0° C. The reaction was then warmed to room temperature and stirred for 2 hours. The mixture was concentrated in vacuo, washed with heptane, and the solid was filtered off. The filtrate was collected and then purified by flash column. A pale yellow oil product was obtained (3.93 g, 96%). 1H NMR (400 MHz, chloroform-d) δ 8.38 - 8.25 (m, 2H), 7.47 - 7.35 (m, 2H), 4.51 - 4.28 (m, 3H), 4.21 - 4.10 (m, 1H), 3.80 (dd, J = 8.4, 6.4 Hz, 1H), 1.73 - 1.59 (m, 4H), 1.48 - 1.20 (m, 20H), 0.95 - 0.83 (m, 6H).

[0186] Step 3. Sodium 3-(((2,2-diheptyl-1,3-dioxolan-4-yl)methoxy)carbonyl)propane-1-sulfonate (RBK-170) [ka]

[0187] To a solution of (2,2-diheptyl-1,3-dioxolan-4-yl)methyl 4-nitrophenyl carbonate (0.40 g, 0.86 mmol) in THF (50 mL) was added tetrabutylammonium 3-aminopropane-1-sulfonate (0.39 g, 1.0 mmol). The mixture was stirred at room temperature for 3 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the tetrabutylammonium salt. The tetrabutylammonium product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin and lyophilized to give the desired product (90 mg, 22%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.25 (t, J = 5.7 Hz, 1H), 4.23 - 4.14 (m, 1H), 4.07 - 3.96 (m, 2H), 3.91 - 3.82 (m, 1H), 3.61 - 3.53 (m, 1H), 3.02 (q, J = 6.7 Hz, 2H), 2.44 - 2.36 (m, 2H), 1.75 - 1.62 (m, 2H), 1.56 - 1.46 (m, 4H), 1.33 - 1.17 (m, 20H), 0.98 - 0.82 (m, 6H). MS m / z 463 [M - H - Na]-.

[0188] Example 19 Sodium 3-((2,2-bis(pentyloxy)propoxy)carbonyl)-2-hydroxypropane-1-sulfonate (RBK-0171) [ka]

[0189] To a solution of 2,2-bis(pentyloxy)propyl 4-nitrophenyl carbonate (0.50 g, 1.26 mmol) in THF (10 mL) and HO (10 mL) was added sodium 3-amino-2-hydroxypropane-1-sulfonate (0.46 g, 2.5 mmol) and NaHCO3 (211 mg, 2.5 mmol). The mixture was stirred at room temperature for 3 h and celite was added. The reaction mixture was concentrated and purified by silica gel chromatography using heptane / ethyl acetate (0-100%) followed by DCM / MeOH. Fractions were collected via TLC stained with potassium permanganate solution. Fractions were concentrated and lyophilized to give the desired product (0.13 g, 23%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.19 (t, J = 5.9 Hz, 1H), 5.05 (s, 1H), 3.97 - 3.80 (m, 3H), 3.43 - 3.33 (m, 4H), 3.30 (s, 2H), 3.09 - 2.95 MS m / z 411 [M - H - Na]-.

[0190] Example 20 Sodium 3-((2,2-bis(hexyloxy)propoxy)carbonyl)-2-hydroxypropane-1-sulfonate (RBK-0172) [ka]

[0191] To a solution of 2,2-bis(hexyloxy)propyl 4-nitrophenyl carbonate (0.50 g, 1.2 mmol) in THF (10 mL) and HO (10 mL) was added 3-amino-2-hydroxypropane-1-sulfonate sodium (0.42 g, 2.4 mmol) and NaHCO3 (197 mg, 2.4 mmol). The mixture was stirred at room temperature for 3 h and celite was added. The reaction mixture was concentrated and purified by silica gel chromatography using heptane / ethyl acetate (0-100%) followed by DCM / MeOH. Fractions were collected via TLC stained with potassium permanganate solution. Fractions were concentrated and lyophilized to give the desired product (0.18 g, 34%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.24 - 7.15 (m, 1H), 5.03 (s, 1H), 3.96 - 3.78 (m, 3H), 3.42 - 3.30 (m, 4H), 3.22 - 3.14 (m, 1H), MS m / z 439 [M - H - Na]-.

[0192] Example 21 Sodium 3-(((2,2-diheptyl-1,3-dioxolan-4-yl)methoxy)carbonyl)-2-hydroxypropane-1-sulfonate (RBK-0173) [ka]

[0193] To a solution of (2,2-diheptyl-1,3-dioxolan-4-yl)methyl 4-nitrophenyl carbonate (0.50 g, 1.1 mmol) in THF (10 mL) and H2O (10 mL) was added sodium 3-amino-2-hydroxypropane-1-sulfonate (0.46 g, 2.5 mmol) and NaHCO3 (211 mg, 2.5 mmol). The mixture was stirred at room temperature for 3 h and celite was added. The reaction mixture was concentrated and purified by silica gel chromatography using heptane / ethyl acetate (0-100%) followed by DCM / MeOH. Fractions were collected via TLC stained with potassium permanganate solution. Fractions were concentrated and lyophilized to give the desired product (0.21 g, 38%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.18 (t, J = 6.0 Hz, 1H), 5.02 (s, 1H), 4.23 - 4.14 (m, 1H), 4.08 - 3.96 (m, 2H), 3.93 - 3.77 (m, 2H), 3.62 - 3.52 (m, 1H), 3.10 - 2.93 (m, 2H), 2.69 - 2.60 (m, 1H), 2.39 (dd, J = 13.5, 8.9 Hz, 1H), 1.57 - 1.44 (m, 4H), 1.33 - 1.16 (m, 18H), 0.90 - 0.82 (m, 6H);MS m / z 479 [M-H-Na]-.

[0194] Example 22 Sodium 3,3'-((4-((((2-undecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)phenyl)-azanediyl)bis(propane-1-sulfonate) (CS-0843) [ka]

[0195] Step 1. (2-Undecyl-1,3-dioxolan-4-yl)methyl(4-aminophenyl)carbamate (CS-0838) [ka]

[0196] To a solution of 4-nitrophenyl((2-undecyl-1,3-dioxolan-4-yl)methyl)carbonate (423 mg, 1.0 mmol) in DMF (10 mL) at room temperature was added p-phenylenediamine (216.3 mg, 2.0 mmol) and DIPEA (870 μL, 5.0 mmol). The reaction was stirred at room temperature for 3 h. LC-MS showed complete conversion. The reaction was poured onto crushed ice and extracted with EtOAc (3×100 mL). The combined organic layers were washed with saturated aqueous NaHCO3 (25 mL), H2O (25 mL), brine (25 mL), dried (Na2SO4) and concentrated in vacuo to give the crude product. The product was isolated using silica gel purification (Hept / EtOAc, 3 / 2). 1 H NMR (400 MHz, CDCl3) δ 7.22 - 7.08 (m, 2H), 6.65 (m, 2H), 6.48 (br s, 1H), 5.08 - 4.84 (m, 1H), 4.39 - 4.07 (m, 3H), 4.03 - 3.75 (m, 2H), 3.71 - 3.35 (br s, 2H), 1.81 - 1.11 (m, 20H), 0.97 - 0.76 (m, 3H). LC-MS [M + H] + 393.

[0197] Step 2. Sodium 3,3'-((4-((((2-undecyl-1,3-dioxolan-4-yl)methoxy)carbonyl)amino)phenyl)-azanediyl)bis(propane-1-sulfonate) (CS-0843) [ka]

[0198] To a solution of (2-undecyl-1,3-dioxolan-4-yl)methyl(4-aminophenyl)carbamate (300 mg, 0.76 mmol) in CH3CN (20 mL) was added 1,3-propane sultone (373 mg, 3.1 mmol) and DIPEA (1.0 mL, 6.2 mmol). The solution was set to reflux for 48 h. The reaction was cooled, concentrated, and purified by silica gel chromatography (CH2Cl2 / MeOH 7 / 3). The desired product was purified as the DIPEA salt and converted to the Na salt using an ion exchange column. 1 H NMR (400 MHz, D2O) δ 7.40 - 7.19 (m, 2H), 7.10 - 7.10 (m, 2H), 5.03 (br s, 1H), 4.59 - 3.95 (m, 4H), 3.84 - 3.64 (m, 1H), 3.55 - LC-MS [M - Na]+ 659.

[0199] Example 23 Sodium 3,3'-((4-(((2,2-bis(pentyloxy)propoxy)carbonyl)amino)phenyl)azanediyl)bis(propane-1-sulfonate) (CS-0853) [ka]

[0200] Step 1. 2,2-Bis(pentyloxy)propyl(4-aminophenyl)carbamate (CS-0840) [ka]

[0201] To a solution of 4-nitrophenyl 2,2-bis(pentyloxy)propyl(4-nitrophenyl)carbonate (278 mg, 0.7 mmol) in DMF (10 mL) at room temperature was added p-phenylenediamine (113 mg, 1.1 mmol) and DIPEA (488 μL, 2.8 mmol). The reaction was stirred at room temperature for 3 h. LC-MS showed complete conversion. The reaction was poured onto crushed ice and extracted with EtOAc (3×100 mL). The combined organic layers were washed with saturated aqueous NaHCO3 (25 mL), H2O (25 mL), brine (25 mL), dried (Na2SO4) and concentrated in vacuo to give the crude product. The product was isolated using silica gel purification (Hept / EtOAc, 3 / 2). 1 H NMR (400 MHz, CDCl3) δ 7.18 (d, J = 8.2 Hz, 2H), 6.86 - 6.65 (m, 2H), 6.54 (s, 1H), 4.64 (d, J = 54.4 Hz, 2H), 4.13 (s, 2H), 3.59 - 3.29 (m, 4H), 1.70 - 1.46 (m, 4H), 1.43 - 1.19 (m, 11H), 0.98 - 0.76 (m, 6H). LC-MS [M + H]+ 367.

[0202] Step 2. Sodium 3,3'-((4-(((2,2-bis(pentyloxy)propoxy)carbonyl)amino)phenyl)azanediyl)bis(propane-1-sulfonate) (CS-0853) [ka]

[0203] To a solution of 2,2-bis(pentyloxy)propyl(4-aminophenyl)carbamate (240 mg, 0.65 mmol) in CH3CN (20 mL) was added 1,3-propane sultone (172 μL, 2.0 mmol) and DIPEA (560 μL, 3.0 mmol). The solution was set to reflux for 48 h. The reaction was cooled, concentrated, and purified by silica gel chromatography (CH2Cl2 / MeOH 7 / 3). The desired product was purified as the DIPEA salt and converted to the Na salt using an ion exchange column. 1 H NMR (400 MHz, CD3OD) δ 7.21 (d, J = 8.5 Hz, 2H), 6.76 (d, J = 8.5 Hz, 2H), 4.06 (s, 2H), 3.54 - 3.37 (m, 8H), 2.89 - 2.75 (m, 4H), 2.12 - 1.93 (m, 4H), 1.53 (qd, J = 7.2, 2.9 Hz, 4H), 1.41 - 1.26 (m, 11H), 0.97 - 0.82 (m, 6H). LC-MS [M - Na]+ 633.

[0204] Example 24 Sodium 3-(((2,2-bis((3,3,4,4,5,5,6,6,6-nonafluorohexyl)oxy)propoxy)carbonyl)amino)propane-1-sulfonate (ZZ-0842) [ka]

[0205] Step 1. 3,3,4,4,5,5,6,6,6-Nonafluorohexyl 2,2-bis((3,3,4,4,5,5,6,6,6-nonafluorohexyl)oxy)propanoate (ZZ-0835) [ka]

[0206] To a solution of methyl pyruvate (5.0 g, 49 mmol) and 3,3,4,4,5,5,6,6,6-nonafluorohexan-1-ol (51.7 g, 196 mmol) in toluene (50 mL) was added p-toluenesulfonic acid (843 mg, 4.9 mmol). The mixture was heated to reflux using a Dean Stark trap condenser for 2 days. The reaction was neutralized with NaHCO3 (sat) (20 mL). The mixture was extracted with ethyl acetate and the organic layer was dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography (0-10% EtOAc / heptane) to give the desired product (27.0 g, 65%) as an oil.

[0207] Step 2. 2,2-Bis((3,3,4,4,5,5,6,6,6-nonafluorohexyl)oxy)propan-1-ol (ZZ-0838) [ka]

[0208] To a solution of 3,3,4,4,5,5,6,6,6-nonafluorohexyl 2,2-bis((3,3,4,4,5,5,6,6,6-nonafluorohexyl)oxy)propanoate (5.0 g, 5.9 mmol) in THF (40 mL) at 0° C. was added lithium aluminum hydride (2.7 mL of a 2.4 M solution in THF, 6.5 mmol). The mixture was then stirred at room temperature for 1 h. The reaction was quenched with water:ethyl acetate (4:4 mL) and potassium sodium tartrate (50 mL). The mixture was washed with ethyl acetate, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography (0-20% EtOAc / heptane) to give the desired product (1.0 g, 29%) as an oil.

[0209] Step 3. 2,2-Bis((3,3,4,4,5,5,6,6,6-nonafluorohexyl)oxy)propyl(4-nitrophenyl)carbonate (ZZ-0839) [ka]

[0210] To a solution of 2,2-bis((3,3,4,4,5,5,6,6,6-nonafluorohexyl)oxy)propan-1-ol (1.0 g, 1.7 mmol) in THF (20 mL) at 0° C. was added 4-nitrophenyl carbonochloridate (0.52 g, 2.6 mmol) and pyridine (0.41 g, 5.1 mmol). The suspension was stirred for 2 h at 0° C. The reaction was diluted with heptane (100 mL) and the white solid was filtered. The filtrate was concentrated and purified by silica gel chromatography to give the crude product.

[0211] Step 4. Sodium 3-(((2,2-bis((3,3,4,4,5,5,6,6,6-nonafluorohexyl)oxy)propoxy)carbonyl)amino)propane-1-sulfonate (ZZ-0842) [ka]

[0212] To a solution of 2,2-bis((3,3,4,4,5,5,6,6,6-nonafluorohexyl)oxy)propyl(4-nitrophenyl)carbonate (0.50 g, 0.67 mmol) in THF (20 mL) was added tetrabutylammonium 3-aminopropane-1-sulfonate (0.38 g, 1.0 mmol). The mixture was stirred at room temperature for 2 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the product as the tetrabutylammonium salt (0.6 g). The tetrabutylammonium product was dissolved in water and methanol (10:1, 10 mL), passed through disodium ion exchange resin and lyophilized to give the desired product as a white solid. 1H NMR (400 MHz, methanol-d4) δ 4.09 (s, 2H), 3.90 - 3.78 (m, 4H), 3.26 (t, J = 6.8 Hz, 2H), 2.89 - 2.78 (m, 2H), 2.57 - 2.38 (m, 4H), 2.06 - 1.90 (m, 2H), 1.41 (s, 3H);MS m / z 748 [M - H - Na]-.

[0213] Example 25 Sodium 3-(((2,2-bis((3,3,4,4,5,5,6,6,6-nonafluorohexyl)oxy)propoxy)carbonyl)amino)-2-hydroxypropane-1-sulfonate (ZZ-0850) [ka]

[0214] To a solution of 2,2-bis((3,3,4,4,5,5,6,6,6-nonafluorohexyl)oxy)propyl(4-nitrophenyl)carbonate (0.50 g, 0.67 mmol) in THF / water (20 / 5 mL) was added 3-amino-2-hydroxypropane-1-sulfonate (0.18 g, 1.0 mmol) and sodium carbonate (0.056 g, 0.67 mmol). The mixture was stirred at room temperature for 4 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the desired product (0.15 g, 29%) as a white solid. 1 H NMR (400 MHz, methanol-d4) δ 4.20 - 4.03 (m, 3H), 3.90 - 3.78 (m, 4H), 3.35 - 3.24 (m, 2H), 3.01 (dd, J = 14.0, 4.0 Hz, 1H), 2.89 (dd, J = 14.0, 7.9 Hz, 1H), 2.58 - 2.39 (m, 4H), 1.42 (s, 3H);MS m / z 764 [M - H - Na]-.

[0215] Example 26 Sodium 3-(((2,2-bis((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)propoxy)carbonyl)amino)-2-hydroxypropane-1-sulfonate (ZZ-0846) [ka]

[0216] Step 1. 3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluorooctyl 2,2-bis((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)propanoate (ZZ-0836) [ka]

[0217] To a solution of methyl pyruvate (5.0 g, 49 mmol) and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctan-1-ol (71.3 g, 196 mmol) in toluene (50 mL) was added p-toluenesulfonic acid (843 mg, 4.9 mmol). The mixture was heated to reflux using a Dean Stark trap condenser for 2 days. The reaction was neutralized with NaHCO3 (sat) (20 mL). The mixture was extracted with ethyl acetate and the organic layer was dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography (0-10% EtOAc / heptane) to give the desired product as an oil.

[0218] Step 2. 2,2-Bis((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)propan-1-ol (ZZ-0837) [ka]

[0219] To a solution of 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl 2,2-bis((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)propanoate (8.0 g, 7.0 mmol) in THF (40 mL) at 0° C. was added lithium aluminum hydride (3.2 mL of a 2.4 M solution in THF, 7.7 mmol). The mixture was then stirred at room temperature for 1 h. The reaction was quenched with water:ethyl acetate (4:4 mL) and potassium sodium tartrate (50 mL). The mixture was washed with ethyl acetate, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography (0-20% EtOAc / heptane) to give the desired product (3.9 g, 71%) as an oil.

[0220] Step 3. 2,2-Bis((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)propyl(4-nitrophenyl)carbonate (ZZ-0841) [ka]

[0221] To a solution of 2,2-bis((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)propan-1-ol (3.0 g, 3.6 mmol) in THF (20 mL) at 0° C. was added 4-nitrophenyl carbonochloridate (1.1 g, 5.4 mmol) and pyridine (0.85 g, 10.8 mmol). The suspension was stirred for 2 h at 0° C. The reaction was diluted with heptane (100 mL) and the white solid was filtered. The filtrate was concentrated and purified by silica gel chromatography to give the crude product.

[0222] Step 4. Sodium 3-(((2,2-bis((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)propoxy)carbonyl)amino)-2-hydroxypropane-1-sulfonate (ZZ-0846) [ka]

[0223] To a solution of 2,2-bis((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)propyl(4-nitrophenyl)carbonate (0.60 g, 0.63 mmol) in THF / water (20 / 5 mL) was added 3-amino-2-hydroxypropane-1-sulfonate (0.17 g, 0.95 mmol) and sodium carbonate (0.053 g, 0.63 mmol). The mixture was stirred at room temperature for 4 h. The reaction was concentrated under vacuum and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the desired product (0.15 g, 25%) as a white solid. 1 H NMR (400 MHz, D2O) δ 4.73 - 4.62 (m, 3H), 4.49 - 4.33 (m, 4H), 3.96 - 3.87 (m, 1H), 3.83 - 3.70 (m, 1H), 3.62 - 3.43 (m, 2H), 3.17 - 2.97 (m, 4H), 1.42 (s, 3H);MS m / z 964 [M - H - Na]-.

[0224] Example 27 Sodium 3-(((2,2-bis((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)propoxy)carbonyl)amino)propane-1-sulfonate (ZZ-0849) [ka]

[0225] To a solution of 2,2-bis((3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)oxy)propyl(4-nitrophenyl)carbonate (0.60 g, 0.63 mmol) in ACN / water (5 / 5 mL) was added 3-aminopropane-1-sulfonate (0.153 g, 0.95 mmol) and sodium carbonate (0.053 g, 0.63 mmol). The mixture was stirred at room temperature for 4 h. The reaction was concentrated in vacuo and purified by silica gel chromatography using heptane / ethyl acetate followed by DCM / MeOH to give the desired product (0.15 g, 25%) as a white solid. 1 H NMR (400 MHz, D2O) δ 3.95 (s, 2H), 3.78 - 3.57 (m, 4H), 3.14 (t, J = 6.9 Hz, 2H), 2.83 - 2.71 (m, 2H), 2.38 - 2.17 (m, 4H), 1.91 - 1.78 (n, 2H), 1.27 (s, 3H);MS m / z 948 [M - H - Na]-.

[0226] Example 28 Stability Data To test thermal stability (% remaining at 95° C. for 1 hour), 20 mM stock solutions of exemplary compounds in DMSO were prepared. The stock solutions were diluted 100-fold in Tris-based PCR buffer and heated to 95° C. for 1 hour in a thermocycler. The solutions were then analyzed by LCMS.

[0227] To test acid stability, 3% stock solutions (9 mg / 300 μL) of each exemplary compound were prepared in NanoPure water. A secondary 3 mM stock solution was prepared in NanoPure water. The secondary stock was diluted 6-fold in NanoPure water as a control or 6-fold with 2.4% TFA-NanoPure water to 2% TFA-NanoPure water for acid stability experiments. The solutions were heated to 40° C. for 1 hour and analyzed by LCMS.

[0228] Compound purity and percent remaining were determined by integrating peaks in total ion chromatograms using LCMS. Compound peaks were identified in negative mode using molecular weights without sodium. A mobile phase of acetonitrile and water contained 10 mM ammonium acetate using a Synergi Max-RP column, 50×4.6 mm, 2.5 micron. [Table 1-1] [Table 1-2]

[0229] Example 29 Protein solubilization and denaturation efficiency To test protein solubilization ability, 4% stock solutions (12 mg / 300 μL) of exemplary compounds were prepared in NanoPure water. Stock solutions were diluted 8-fold in 50 mM ammonium bicarbonate, pH 7.8 (0.5% final) and added to acetone-precipitated protein pellets from lysed E. coli. Samples were incubated at 50° C. with shaking at 1200 rpm overnight and then analyzed by SDS PAGE and BCA. As a control, acetone-precipitated protein pellets were solubilized under the same conditions with 0.5% SDS in 50 mM ammonium bicarbonate, pH 7.8. A representative SDS-PAGE gel of solubilized E. coli protein pellets is shown in FIG. 1. Representative BCA assay results of solubilized E. coli protein pellets are shown in FIG. 2. SDS is one of the best performing detergents for protein solubilization and extraction, but is not compatible with mass spectrometry. These results demonstrate that many of the exemplary compounds are able to match or exceed the performance of sodium dodecyl sulfate (SDS) in solubilizing or extracting proteins.

[0230] To test protein denaturing ability, 4% stock solutions (12 mg / 300 μL) of each exemplary compound were prepared in NanoPure water. Stock solutions were diluted 40-fold in 50 mM ammonium bicarbonate, pH 7.8 (0.1% final) with 20 μg myoglobin (Sigma) and 0.5 μg Trypsin Gold (Promega). Samples were incubated at 37° C. with shaking at 1200 rpm for 40 minutes and then analyzed by SDS PAGE. The assay measures protein denaturation and trypsin compatibility. Myoglobin is only digested if it is sufficiently denatured by the detergent. A representative SDS-PAGE gel of myoglobin denaturation and trypsin digestion products is shown in FIG. 3. The results of this assay show that the exemplary compounds can sufficiently denature myoglobin to be successfully digested by trypsin protease. The exemplary compounds offer the advantage of denaturing proteins to allow improved digestion by trypsin.

[0231] Example 30 Trypsin compatibility The compatibility of different compounds with trypsin was tested using the trypsin-specific substrate, benzoyl-L-arginine ethyl ester hydrochloride (BAEE). 4% stock solutions (12 mg / 300 μL) of exemplary compounds were prepared in NanoPure water. Stock solutions were diluted 40-fold in 50 mM ammonium bicarbonate, pH 7.8 (0.1% final) with 200 μM BAEE. The entire sample was transferred to individual wells in a 96-well UV-compatible microplate. The absorbance of the samples was measured at 253 nm using a TECAN M1000 plate reader. After 2 minutes, 0.2 μg of Trypsin Gold (Promega) was added to the reaction and the absorbance at 253 nm was monitored every minute over a 10-minute period. The resulting activity measurements were compared to a control of trypsin without the presence of surfactant. Results from a representative trypsin assay are shown in FIG. 4 and the data are summarized in Table 2. Trypsin is the most commonly used protease for digestion of protein mass spectrometry samples. The results from this experiment show that the compounds have minimal effect on trypsin activity and can be used during the sample digestion step. [Table 2]

[0232] Example 31 Proteomics Mass Spectrometry To determine the effect of detergents on protein sample preparation for mass spectrometry analysis, 4% stock solutions (12 mg / 300 μL) of exemplary compounds were prepared in NanoPure water. Stock solutions were diluted 8-fold in 50 mM ammonium bicarbonate, pH 7.8 (0.5% final) and added to acetone-precipitated protein pellets from lysed E. coli. Samples were incubated at 50° C. with shaking at 1200 rpm overnight to solubilize the protein pellets. The resulting samples were reduced with 5 mM dithiothreitol (DTT) at 37° C. for 1 hour, followed by alkylation with 15 mM iodoacetamide (IAM) at room temperature for 1 hour. Each sample was digested with Trypsin Gold (Promega) at a ratio of 1:40 μg of trypsin per μg protein at 50° C. with shaking at 1200 rpm for 3 hours. Detergent was removed by centrifugation at 16,000 xg for 5 minutes at room temperature, followed by washing with ZipTips (OMIX) according to the manufacturer's instructions.

[0233] All samples were analyzed by LC-MS / MS using a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. Data was analyzed with MASCOT and Scaffold software to determine the number of protein and peptide identifications. As a control, protein pellets from lysed E. coli were solubilized and digested under the same conditions in the presence of 0.1% RapiGest (a detergent provided by Waters). Results are summarized in Table 3. Mass spectrometry data show that the exemplary compounds increased overall protein and peptide identifications when compared to RapiGest (Waters), a currently available mass spectrometry compatible detergent. [Table 3]

Claims

1. Compounds of formula (I): 【Chemistry 1】 or a salt thereof, wherein A, 【Chemistry 2】 and R 1 is hydrogen, C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, and C 1 -C 12 haloalkyl; R 2 But, C 4 -C 20 Alkyl, C 4 -C 20 Alkenyl, and C 4 -C 20 haloalkyl; m is 1, 2, or 3; R a and R b is, at each occurrence, independently selected from hydrogen and C 1 -C 3 alkyl, Q is C 1 -C 6 -alkylene or the group -Q-SO 3 M is of the formula: 【Chemistry 3】 having Each M is independently hydrogen, an alkali metal cation, or NR 4 + wherein each R is independently selected from hydrogen and C 1 -C 12 The compound or a salt thereof, wherein the compound is selected from the group consisting of alkyl, aryl ... and aryl.

2. R2 is C 6 -C 12 is alkyl, R is hydrogen, methyl, or C 4 -C 8 2. The compound of claim 1, or a salt thereof, wherein:

3. A, 【Chemistry 4】 3. The compound according to claim 1 or 2, or a salt thereof, selected from:

4. m is 1, R a and R b The compound according to any one of claims 1 to 3, or a salt thereof, wherein each of is hydrogen.

5. Q is C 1 -C 6 5. The compound according to claim 1, wherein -alkylene, or a salt thereof.

6. Q is -CH 2 CH 2 CH 2 The compound according to claim 5, or a salt thereof, wherein

7. The compound according to any one of claims 1 to 6, or a salt thereof, wherein each M is independently an alkali metal.

8. 8. The compound according to claim 7, or a salt thereof, wherein the alkali metal is sodium. 【Request 9】 【Chemical 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 【Chemistry 5-4】 A compound selected from:

10. 1. A method for digesting a protein, comprising: A sample containing at least one protein is treated with a protein digestion reagent. and a compound according to any one of claims 1 to 9, or a salt thereof, The method thereby comprises providing a sample comprising at least one digested protein.

11. The method of claim 10, wherein the sample is a gel, a solid support, or a solution.

12. The method of claim 11 , wherein the sample is a gel.

13. The method of claim 11 , wherein the sample is an aqueous solution.

14. 14. The method of claim 13, wherein the aqueous solution is a solution from in-solution protein digestion with a protease, protein pellet solubilization, protein extract from cells, protein extract from tissues, protein deglycosylation with a deglycosidase, or removal of non-specifically bound proteins from a protein immobilization support.

15. The method of any one of claims 10 to 14, wherein the protein digestion reagent comprises a protease, cyanogen bromide, hydroxylamine, or any combination thereof.

16. The method of claim 15 , wherein the protein digestion reagent comprises a protease.

17. 17. The method of claim 16, wherein the protease is a serine protease.

18. 18. The method of claim 17, wherein the serine protease is trypsin, chymotrypsin, or Lys-C.

19. The method of any one of claims 10 to 18, further comprising decomposing the surfactant after the contacting step.

20. 20. The method of claim 19, wherein the decomposing step comprises contacting the surfactant with an acid.

21. 20. The method of claim 19, wherein the surfactant autolyzes after the contacting step.

22. The method of any one of claims 10 to 21, further comprising isolating at least one digested protein fragment.

23. The method of any one of claims 10 to 22, further comprising analysing at least one digested protein fragment.

24. 24. The method of claim 23, wherein the analyzing step comprises mass spectrometry, liquid chromatography, gel electrophoresis, or a combination thereof.

25. A composition comprising a gel and a compound according to any one of claims 1 to 9.

26. 1. A method for extracting peptides from a gel, comprising: contacting the gel with a compound according to any one of claims 1 to 9 and an aqueous solution; Separating the aqueous liquid from the gel; thereby providing a solution comprising the peptide.

Images