WO2004101597A2 - Methods for the reduction of disulfide bonds - Google Patents

Methods for the reduction of disulfide bonds Download PDF

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Publication number
WO2004101597A2
WO2004101597A2 PCT/IL2004/000392 IL2004000392W WO2004101597A2 WO 2004101597 A2 WO2004101597 A2 WO 2004101597A2 IL 2004000392 W IL2004000392 W IL 2004000392W WO 2004101597 A2 WO2004101597 A2 WO 2004101597A2
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unsubstituted
branched
dihydroxybutane
tris
cyclic
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PCT/IL2004/000392
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French (fr)
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WO2004101597A3 (en
Inventor
Michael Mizhiritskii
Yacov Shpernat
Boris Ashkenazi
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Frutarom Ltd.
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Publication of WO2004101597A3 publication Critical patent/WO2004101597A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains

Definitions

  • This invention provides methods for reducing disulfide bonds. In another embodiment, this invention provides methods for the reduction of disulfide bonds in peptides, proteins, cysteine derivatives and other organic compounds, containing S-S bond.
  • Oxidation of cysteine sulfhydryl groups during isolation, storage and use of proteins is often an important contributor to their deactivation.
  • the most effective and widely use reagents for protecting the ryst ine moietie° of enzyme? peptides and proteins against oxidation by adventitious oxygen are dithiothreitol (DTT, Cleland reagent) and dithioerythritol (DTE) - two optical isomers of 1,4- dimercapto-2,3 -dihydroxybutane.
  • DTT or DTE are used as reagents for reduction of disulfide bonds and for protection of thiol groups against oxidation.
  • DTT and DTE are considered to be equally effective as reducing agents, and no difference was observed between the two isomers [1].
  • DTT dithiothreitol
  • Phosphorous containing compounds such as trialkylphosphines can also reduce organic disulf ⁇ des to thiols smoothly and quantitatively:
  • Triphenylphosphines have no odor, but on the other hand, they are not soluble in water [11].
  • a commercially available water soluble tris (4- carboxyphenyl)phosphine is relatively expensive (Strem Chemicals).
  • Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) [10] a product of acidic hydrolysis of commercially available tris(2-cyanoethyl)phosphine, is an odorless, crystalline, air-stable solid, water soluble substance that reacts rapidly with disulfides. This reagent however, is even more expensive than DTT (BioVectra DCL. There is thus an industrial need for low cost, water soluble, low toxicity, odorless, easy to handle compounds that can be used as disulfide reducing agents.
  • this invention provides a method for reducing disulfide bonds in a chemical agent, the method may include, inter alia, the step of contacting the chemical agent with l,4-dimercapto-2,3-dihydroxybutane, wherein said 1,4- dimercapto-2,3-dihydroxybutane is prepared by a non-stereoselective reaction.
  • the chemical agent is a protein.
  • the chemical agent is a biopolymer.
  • the chemical agent is a peptide.
  • the chemical agent is a cysteine derivative.
  • this invention provides a method for protecting a protein against oxidation, the method may include, inter alia, the step of contacting the protein with l,4-dimercapto-2,3-dihydroxybutane, wherein the l,4-dimercapto-2,3- dihydroxybutane is prepared by a non-stereoselective reaction.
  • this invention provides a method for protecting cysteine sulfhydyl group of a protein against oxidation, the method may include, inter alia, the step of contacting the protein with l,4-dimercapto-2,3 -dihydroxybutane, wherein the l,4-dimercapto-2,3-dihydroxybutane is prepared by a non-stereoselective reaction.
  • this invention provides a method for reducing a disulfide bond in a chemical agent including, inter alia, the step of contacting the chemical agent with a compound represented by the structure of formula I
  • R l3 R 2 and R 3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
  • the chemical agent is a protein.
  • the chemical agent is a biopolymer.
  • the chemical agent is a peptide.
  • the chemical agent is a cysteine derivative.
  • this invention provides a method for protecting a protein against oxidation include, inter alia, the step of contacting the protein with a compound represented by the structure of formula I
  • R ⁇ , R and R 3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
  • this invention provides a method for protecting cysteine sulfhydyl group of a protein against oxidation including, inter alia, the step of contacting the protein with a compound represented by the structure of formula I
  • Ri, R 2 and R 3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
  • the compound is tris[hydroxyalkyl]phosphine.
  • the tris[hydroxyalkyl]phosphines is a tris[hydroxypropyl]phosphine.
  • the compound is [MeOCH 2 O(CH 2 )3]3P, [MeOCH 2 CH 2 O(CH2)3]3P,
  • this invention provides a-method for reducing disulfide bonds in a chemical agent
  • the method may include, inter alia, the step of contacting the chemical agent with l,4-dimercapto-2,3 -dihydroxybutane, wherein the 1,4- dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction.
  • the chemical agent is a protein.
  • the chemical agent is a biopolymer, which may comprise in one embodiment, a DNA or an RNA or in another embodiment may be a glycoprotein.
  • the chemical agent is a peptide.
  • the chemical agent is a cysteine derivative or any amino acid sequence which contains cysteine.
  • the chemical agent is an asymmetrical disulfide derivative.
  • the chemical agent is a symmetrical disulfide derivative.
  • a stereoselective reaction is a reaction wherein one stereoisomer is preferentially formed.
  • a non-stereoselective reaction is a reaction wherein no certain stereoisomer is preferentially formed.
  • the non- stereoselectivc leaction is a non-enantioselective reaction.
  • the non-enantioselective reaction is a reaction wherein no certain enentiomer is preferentially formed.
  • the non-stereoselective reaction is a non-diastereoselective reaction, i another embodiment, the non-diastereo selective reaction is a reaction wherein no certain diastereomer is preferentially formed.
  • l,4-dimercapto-2,3 -dihydroxybutane may be widely used in biotechnology, chemistry and pharmaceutical chemistry. Embodiments of this invention provide methods for using l,4-dimercapto-2,3 -dihydroxybutane manufactured in high yields as a reducing agent for disulfide bonds.
  • l,4-dimercapto-2,3 -dihydroxybutane can also be applied for protecting a peptide or a protein against oxidation.
  • 1 ,4-dimercapto-2,3 -dihydroxybutane is oxidized faster and more efficiently than certain moieties in a peptide or a protein (for example, amino acids such as methionine, tryptophan, histidine and cysteine). Therefore when l,4-dimercapto-2,3 -dihydroxybutane is contacted with a peptide or a protein it is easily be oxidized and prevents the oxidation of the peptide or the protein.
  • the presence of l,4-dimercapto-2,3 -dihydroxybutane in the reaction mixture during peptide synthesis or during protein isolation or purification allows working under ambient atmosphere without the need of an oxygen free environment.
  • this invention provides a method for protecting a protein against oxidation, the method may include, inter alia, the step of contacting the protein with l,4-dimercapto-2,3 -dihydroxybutane, wherein the l,4-dimercapto-2,3- dihydroxybutane is prepared by a non-stereoselective reaction.
  • this invention provides a method for protecting a peptide against oxidation, the method may include, inter alia, the step of contacting the peptide with 1, -dimercapto-2,3 -dihydroxybutane, wherein the l,4-dimercapto-2,3- dihydroxybutane is prepared by a non-stereoselective reaction.
  • this invention provides a use of l,4-dimercapto-2,3- dihydroxybutane prepared by a non-stereoselective reaction for protecting a peptide against oxidation.
  • this invention provides a method of l,4-dimercapto-2,3- dihydroxybutane for protecting a peptide against oxidation during peptide synthesis, the method may include, inter alia, the step of contacting the peptide with 1,4- dimercapto-2,3-dihydroxybutane, wherein the l,4-dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction.
  • this invention provides a method for protecting cysteine sulfhydyl group of a protein against oxidation, the method may include, inter alia, the step of contacting the protein with l,4-dimercapto-2,3 -dihydroxybutane, wherein the l,4-dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction.
  • this invention provides a method for protecting cysteine sulfhydyl group of a peptide against oxidation, the method may include, inter alia, the step of contacting the peptide with l,4-dimercapto-2,3 -dihydroxybutane, wherein the l,4-dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction.
  • this invention provides a method for protecting an amino acid in a peptide or a protein against oxidation, the method may include, inter alia, the step of contacting the peptide or protein with l,4-dimercapto-2,3- dihydroxybutane, wherein the l,4-dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction.
  • the amino acid is methionine.
  • the amino acid is tryptophan.
  • the amino acid is histidine.
  • the amino acid is cysteine.
  • the non-stereoselective reaction may include, inter alia, the steps of reacting l,2:3,4-diepoxybutane with thioacetic acid to produce a reaction product comprising a 1,4-dith ⁇ oacetic -2,3-dihydroxybutane and hydrolysis of the 1,4-dithioacetic -2,3-dihydroxybutane to produce a reaction product comprising a distereomeric mixture of l,4-dimercapto-2,3 -dihydroxybutane.
  • the non-stereoselective reaction may include, inter alia, the steps of reacting l,2:3,4-diepoxybutane with thioacetic acid to produce a reaction product substantially comprising a 1,4-dithioacetic -2,3- dihydroxybutane and hydrolysis of the 1,4-dithioacetic -2,3-dihydroxybutane to produce a reaction product substantially comprising a distereomeric mixture of 1,4- dimercapto-2,3 -dihydroxybutane.
  • the hydrolysis is an acid hydrolysis. In another embodiment, the hydrolysis is a base hydrolysis.
  • l,4-dimercapto-2,3 -dihydroxybutane can be prepared from butadiene monoepoxide with yields of about 92%.
  • l,2:3,4-diepoxybutane is prepared from butadiene monoepoxide.
  • l,4-dimercapto-2,3 -dihydroxybutane can also be prepared from butadiene monoepoxide with yields of about 86%.
  • the l,2:3,4-diepoxybutane is prepared from butadiene.
  • the l,2:3,4-diepoxybutane is prepared from tetrahydroxybutane.
  • the l,4-dimercapto-2,3 -dihydroxybutane is prepared from 1,4- ditosyl-2,3-dihydroxybutane.
  • the reaction for the preparation of l,4-dimercapto-2,3 -dihydroxybutane from 1,4- ditosyl-2,3 -dihydroxybutane may include, inter alia, contacting the 1,4- ditosyl-2,3 -dihydroxybutane with thiourea and pyridine.
  • the reaction for the preparation of l,4-dimercapto-2,3 -dihydroxybutane from 1,4- ditosyl-2,3 -dihydroxybutane may include, inter alia, contacting the 1,4- ditosyl-2,3 -dihydroxybutane with potassium thioacetate.
  • the l,4-dimercapto-2,3 -dihydroxybutane is a distereomeric mixture.
  • the mixture may include 1-99% dithiothreitol and 99-1% dithioerythritol. In another embodiment, the mixture may include 1-50% dithiothreitol and 50-1% dithioerythritol. In another embodiment, the mixture may include 30-80% dithiothreitol and 70-20% dithioerythritol. In another embodiment, the mixture may include 20-70% dithiothreitol and 80-30% dithioerythritol.
  • this invention provides a method for reducing a disulfide bond in a chemical agent, the method may include, inter alia, the step of contacting the chemical agent with a compound represented by the structure of formula I
  • Rr, R 2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
  • the chemical agent is a protein.
  • the chemical agent is a biopolymer, which may comprise in one embodiment, a DNA or an RNA or in another embodiment may be a glycoprotein.
  • the chemical agent is a peptide.
  • the chemical agent is a cysteine derivative or any amino acid sequence which contains cysteine.
  • the chemical agent is an asymmetrical disulfide derivative.
  • the chemical agent is a symmetrical disulfide derivative.
  • one or more compounds represented by the structures of formula I are effective in protecting a peptide or a protein, against oxidation. Since these compounds ' are oxidized faster and more efficiently than certain moieties in a peptide or a protein (for example, amino acids such as methionine, tryptophan, histidine and cysteine), they will prevent the oxidation of the peptide or the protein with which they are in contact. As described above, this allows working under ambient atmosphere without the need of an oxygen free environment during peptide synthesis or during protein isolation or purification.
  • this invention provides a method for protecting a protein against oxidation, the method may include, inter alia, the step of contacting the protein with a compound represented by the structure of formula I
  • R l3 R 2 and R 3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
  • this invention provides a method for protecting a peptide against oxidation, the method may include, inter alia, the step of contacting the peptide with a compound represented by the structure of formula I
  • R l3 R 2 and R 3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
  • this invention provides a method for protecting a peptide against oxidation during peptide synthesis, the method may include, inter alia, the step of contacting the peptide with a compound represented by the structure of formula I
  • R ls R 2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
  • this invention provides a method for protecting cysteine sulfhydyl group of a protein against oxidation, the method may include, inter alia, the step of contacting the protein with a compound represented by the structure of formula I
  • Ri, R 2 and R 3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
  • this invention provides a method for protecting cysteine sulfhydyl group of a peptide against oxidation, the method may include, inter alia, the step of contacting the peptide with a compound represented by the structure of formula I
  • R l5 R 2 and R 3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or -cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
  • this invention provides a method for protecting an amino acid in a peptide or a protein against oxidation, the method may include, inter alia, the step of contacting the peptide or protein with a compound represented by the structure of formula I
  • Ri, R 2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
  • the amino acid is methionine.
  • the amino acid is tryptophan.
  • the amino acid is histidine.
  • the amino acid is cysteine.
  • This invention provides a method for the removal of a protecting group from cysteine-containing peptide, the method may include, inter alia, the step of contacting the protein with a compound represented by the structure of formula I
  • Ri, R 2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
  • Tris[hydroxyalkyl(aryl)]phosphines are water soluble, low toxicity, odorless, easy to handle compounds that are commercially available and can be used in reducing disulfide bonds (and in protecting a peptide or a protein against oxidation) at a wide range of pH, short time, mild reaction condition and in lower amounts.
  • the compound is tris[hydroxyalkyl]phosphine.
  • the tris[hydroxyalkyl]phosphine is a tris[hydroxypropyl]phosphine.
  • the tris[hydroxypropyl]phosphine is a tris[3-hydroxypropyl]phosphine.
  • the tris[hydroxypropyl]phosphine is a tris[2- hydroxypropyl]phosphine.
  • the tris[hydroxypropyl]phosphine is atris[l-hydroxypropyl]phosphine.
  • the compound is [MeOCH 2 O(CH 2 ) 3 ] 3 P, [MeOCH 2 CH 2 O(CH 2 ) 3 ] 3 P, [CCl 3 CH 2 O(CH 2 )3] 3 P,
  • R 4 R 5 NCH 2 O(CH 2 ) 3 ] 3 P, [CH 3 -S-CH 2 O(CH 2 )3] 3 P, [tetrahydropyranyloxy(CH 2 ) 3 ] 3 P or any combination thereof, wherein R 4 and R 5 are the same or different, independently of each other, H or an unsubstituted or substituted alkyl.
  • an "alkyl” group refers to a saturated aliphatic hydrocarbon, may include straight-chain, branched-chain and cyclic alkyl groups.
  • the alkyl group has 1-12 carbons.
  • the alkyl group has 1-7 carbons.
  • the alkyl group has 1-6 carbons.
  • the alkyl group has 1-4 carbons.
  • the alkyl group is a methyl group.
  • the alkyl group is an ethyl group.
  • the alkyl group is a propyl group.
  • the alkyl group is a butyl group.
  • the alkyl group may be unsubstituted or substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.
  • substituted may refer to substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.
  • an "aryl” group refers to an aromatic group having at least one carbocyclic aromatic group or heterocyclic aromatic group, which may be unsubstituted or substituted by one or more groups selected from halogen, haloalkyl, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxy or thio or thioalkyl.
  • Nonlimiting examples of aryl rings are phenyi, naphthyl, pyranyl, pyrroiyl, pyrazinyl, pyrimidinyl, pyrazolyl, pyridinyl, furanyl, thiophenyl, thiazolyl, imidazolyl, isoxazolyl, and the like.
  • a "hydroxyl” group refers to an OH group.
  • an "alkoxy” group refers to an O-alkyl group, wherein alkyl has the same definition as described above.
  • a "phenoxy” group refers to an O-phenyl group.
  • a "thio" group refers to a SH group.
  • an "alkylthio” group refers to an S-alkyl group wherein alkyl has the same definition as described above.
  • an "alkylthio” group refers to an S-aryl group wherein aryl has the same definition as described above.
  • a halogen or halo group refers to F, Cl, Br or I.
  • Fmoc amino acids were obtained from ArtChem Division of Frutarom.
  • the phosphines were obtained from Cytec. Other chemicals from Sigma- Aldrich.
  • butadiene can be used instead of butadiene monoepoxide, providing 86% yield of diepoxide.
  • Other peroxides can be used instead of m-chloroperbenzoic acid (for example magnesium monoperphthalate, 5-hydroperoxycarbonylphthalimide and trifluoroperacetic acid).
  • the l,2:3,4-diepoxibutane can be prepared as an optical isomer by reaction of trans- 1,4-butenediol with bromine, followed by cyclization of 2,3-dibromo-l,4- dihydroxybutane with potassium hydroxide in two-phase system (or with quaternary ammonium hydroxide in organic solvent).
  • the overall yield of this process is not higher than 45-50%.
  • Table 1 Rates of disulfides reduction by l,4-dimercapto-2,3 -dihydroxybutane (D), DTT and DTE (at pH 7.0 and 298 K).
  • k m is the approximate rate constant of the disulfide reduction.
  • M ' V 1 (Molar "1 second "1 ) are the units of A aPP .
  • DDT was prepared from 4,5-dihydroxy-l,2-dithiane according to example 3 i.
  • the yield was 88% (after crystallization from MTBE), m.p. 48-50°C.
  • the ethyl acetate solution was separated from water, dried over sodium sulfate, evaporated in vacuum to give 3.4 g (100 %) yield of Fmoc-cysteine, which was 94.7% pure, according to HPLC.
  • the compound was purified by precipitation from ethyl acetate solution with hexane (3.4 g of compound, 40 ml of ethyl acetate, 50 ml of hexane). The yield was 87%, 99.1 % purity according to HPLC.
  • Fragment of thioredoxin was synthesized as N-acetyl and C-amide octapeptide, according to [12], using t-butylsulphenyl group for cysteine protection.
  • Fmoc-cystine amide a compound which is difficult to reduce in clean and high yield reaction, using ordinary methods such as Zn/AcOH, sodium borohydride or lithium aluminum hydride, Mg/MeOH, Hg(OAc)2 and other substances [14].

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Abstract

This invention provides a method for reducing disulfide bonds in a chemical agent, the method may include, inter alia, the step of contacting the chemical agent with 1,4-dimercapto-2,3-dihydroxybutane, wherein the 1,4-dimercapto-2,3­dihydroxybutane is prepared by a non-stereoselective reaction. This invention further provides a method for protecting a protein against oxidation, the method may include, inter alia, the step of contacting the protein with 1,4-dimercapto-2,3­dihydroxybutane, wherein the 1,4-dimercapto-2,3-dihydroxybutane is prepared by a non-stereoselective reaction. This invention further provides a method for reducing a disulfide bond in a. chemical agent, the method may include, inter alia, the step of contacting the chemical agent with a compound represented by the structure of formula (I) wherein R1, R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether. This invention further provides a method for protecting a protein against oxidation, the method may include, inter alia, the step of contacting the protein with a compound represented by the structure of formula (I) wherein R1, R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted hydroxyaryl, an unsubstituted or subsituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.

Description

METHODS FOR THE REDUCTION OF DISULFIDE BONDS
FIELD OF THE INVENTION
[001] This invention provides methods for reducing disulfide bonds. In another embodiment, this invention provides methods for the reduction of disulfide bonds in peptides, proteins, cysteine derivatives and other organic compounds, containing S-S bond.
BACKGROUND OF THE INVENTION
[002] Oxidation of cysteine sulfhydryl groups during isolation, storage and use of proteins is often an important contributor to their deactivation. The most effective and widely use reagents for protecting the ryst ine moietie° of enzyme? peptides and proteins against oxidation by adventitious oxygen are dithiothreitol (DTT, Cleland reagent) and dithioerythritol (DTE) - two optical isomers of 1,4- dimercapto-2,3 -dihydroxybutane.
Figure imgf000002_0001
DTT DTE
[003] DTT or DTE are used as reagents for reduction of disulfide bonds and for protection of thiol groups against oxidation. DTT and DTE are considered to be equally effective as reducing agents, and no difference was observed between the two isomers [1].
[004] The synthesis of optically active form of dithiothreitol (DTT) (-) -1,4- dithiothreitol was also described [2]. DTT reduces protein disulfide groups rapidly and completely, maintains the monothiols in a completely reduced state and is convenient to handle. The DTT reagent, on the other hand, is exorbitantly expensive. [005] Since DTT and DTE have similar activity, and since their enantiomeric and distereomeric purity is almost certainly irrelevant in most applications, a synthesis for the preparation of a mixture of diastereomeric l,4-dimercapto-2,3-butanediols (i.e., a mixture of DTT and DTE) by reaction of l,2:3,4-diepoxybutane with thioacetic acid followed by acid-catalyzed deacylation (transesterification) in methanol [3] was proposed. However, the preparation of the l,2:3,4-diepoxybutane starting material, gave only 22% yield [4]. Analogues preparation of 1,2:3,4- diepoxybutane from butadiene gave only 8% yield of desired diepoxide [5]. The overall yield of the process for l,4-dimercapto-2,3-butanediol preparation is ~ 15- 18% which make this process less attractive from economical point of view than other methods [1,2].
[006] Organic selenols and diselenides were proposed as potential catalysts for DTT-disulfide interchange reactions [6]. However, the organic derivatives of selenium are highly toxic and have an unpleasant odor.
[007] Whitesides described structure reactivity relations and rate and equilibrium constants for thiol disulfide interchange reactions [7]. On the basis of these investigations meso-2,5-dimercapto-N,N,N',N'-tetramethyladipamide (DTA) was synthesized [8]. This compound was reported to be 8 time kinetically faster than DDT at pH 7.0 [9]. DTA was prepared in five steps (39% overall yield) from adipic acid. However, DTA is -10 time more expensive than DDT, ~3 time less soluble in water and less active as a reducing agent than DDT because of steric interactions (DTA is a secondary thiol, and DTT is a primary thiol). There is thus a need for low cost chemical agents for reducing disulfide bonds.
[008] Phosphorous containing compounds such as trialkylphosphines can also reduce organic disulfϊdes to thiols smoothly and quantitatively:
R'3P + RSSR + H2O - R'3P=O + 2RSH
[009] The strength of the P=O bond renders the reduction irreversible in contrast to thiol-disulfide interchange. Since trialkylphosphines are stable in aqueous solution, selective for the reduction of the disulfide bond and unreactive toward many other functional groups, they are attractive as reducing agents [10]. However, their use has been limited by low solubility of most simple trialkylphosphines in water and their acceptability as reagents was limited by the odor and ease of autoxidation. In addition, some of these compounds, for example, thibutylphosphine and thiethylphosphine, are toxic.
[0010] Triphenylphosphines have no odor, but on the other hand, they are not soluble in water [11]. A commercially available water soluble tris (4- carboxyphenyl)phosphine is relatively expensive (Strem Chemicals).
[0011] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) [10], a product of acidic hydrolysis of commercially available tris(2-cyanoethyl)phosphine, is an odorless, crystalline, air-stable solid, water soluble substance that reacts rapidly with disulfides. This reagent however, is even more expensive than DTT (BioVectra DCL. There is thus an industrial need for low cost, water soluble, low toxicity, odorless, easy to handle compounds that can be used as disulfide reducing agents.
SUMMARY OF THE INVENTION
[0012] In one embodiment, this invention provides a method for reducing disulfide bonds in a chemical agent, the method may include, inter alia, the step of contacting the chemical agent with l,4-dimercapto-2,3-dihydroxybutane, wherein said 1,4- dimercapto-2,3-dihydroxybutane is prepared by a non-stereoselective reaction. In one embodiment, the chemical agent is a protein. In another embodiment, the chemical agent is a biopolymer. In another embodiment, the chemical agent is a peptide. In another embodiment, the chemical agent is a cysteine derivative.
[0013] In one embodiment, this invention provides a method for protecting a protein against oxidation, the method may include, inter alia, the step of contacting the protein with l,4-dimercapto-2,3-dihydroxybutane, wherein the l,4-dimercapto-2,3- dihydroxybutane is prepared by a non-stereoselective reaction.
[0014] In one embodiment, this invention provides a method for protecting cysteine sulfhydyl group of a protein against oxidation, the method may include, inter alia, the step of contacting the protein with l,4-dimercapto-2,3 -dihydroxybutane, wherein the l,4-dimercapto-2,3-dihydroxybutane is prepared by a non-stereoselective reaction.
[0015] In one embodiment, this invention provides a method for reducing a disulfide bond in a chemical agent including, inter alia, the step of contacting the chemical agent with a compound represented by the structure of formula I
Figure imgf000005_0001
I
wherein Rl3 R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether. In one embodiment, the chemical agent is a protein. In another embodiment, the chemical agent is a biopolymer. In another embodiment, the chemical agent is a peptide. In another embodiment, the chemical agent is a cysteine derivative.
[0016] In one embodiment, this invention provides a method for protecting a protein against oxidation include, inter alia, the step of contacting the protein with a compound represented by the structure of formula I
Figure imgf000005_0002
I
wherein R\, R and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
[0017] In one embodiment, this invention provides a method for protecting cysteine sulfhydyl group of a protein against oxidation including, inter alia, the step of contacting the protein with a compound represented by the structure of formula I
Ri-P 2
R3
I
wherein Ri, R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether. In another embodiment, the compound is tris[hydroxyalkyl]phosphine. In another embodiment, the tris[hydroxyalkyl]phosphines is a tris[hydroxypropyl]phosphine. In another embodiment, the compound is [MeOCH2O(CH2)3]3P, [MeOCH2CH2O(CH2)3]3P,
[CCl3CH2O(CH2)3]3P, [R4R5NCH2O(CH2)3]3P, [CH3-S-CH2O(CH2)3]3P, [tetrahydropyranyloxy(CH2)3]3P or any combination thereof, wherein Rt and R5 are the same or different, independently of each other, H or an unsubstituted or substituted alkyl.
DETAILED DESCRIPTION OF THIS INVENTION
[0018] In one embodiment, this invention provides a-method for reducing disulfide bonds in a chemical agent, the method may include, inter alia, the step of contacting the chemical agent with l,4-dimercapto-2,3 -dihydroxybutane, wherein the 1,4- dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction. In one embodiment, the chemical agent is a protein. In another embodiment, the chemical agent is a biopolymer, which may comprise in one embodiment, a DNA or an RNA or in another embodiment may be a glycoprotein. In another embodiment, the chemical agent is a peptide. In another embodiment, the chemical agent is a cysteine derivative or any amino acid sequence which contains cysteine. In another embodiment, the chemical agent is an asymmetrical disulfide derivative. In another embodiment, the chemical agent is a symmetrical disulfide derivative.
[0019] A stereoselective reaction is a reaction wherein one stereoisomer is preferentially formed. A non-stereoselective reaction is a reaction wherein no certain stereoisomer is preferentially formed. In one embodiment of this invention, the non- stereoselectivc leaction is a non-enantioselective reaction. In another embodiment, the non-enantioselective reaction is a reaction wherein no certain enentiomer is preferentially formed. In another embodiment, the non-stereoselective reaction is a non-diastereoselective reaction, i another embodiment, the non-diastereo selective reaction is a reaction wherein no certain diastereomer is preferentially formed.
[0020] It has now been surprisingly found that there is no difference in reactivity of reduction of disulfide bonds between l,4-dimercapto-2,3 -dihydroxybutane, prepared by a non-stereoselective reaction, DTT and DTE. Therefore, l,4-dimercapto-2,3- dihydroxybutane can be used for the same applications as its optical isomers. The 1 ,4-dimercapto-2,3 -dihydroxybutane which may be a mixture of DTT and DTE is an efficient reducing agent, is easily prepared and is less costly than preparing each of the isomers alone, and therefore can be used as a reagent for reducing disulfide bonds. l,4-dimercapto-2,3 -dihydroxybutane may be widely used in biotechnology, chemistry and pharmaceutical chemistry. Embodiments of this invention provide methods for using l,4-dimercapto-2,3 -dihydroxybutane manufactured in high yields as a reducing agent for disulfide bonds.
[0021] l,4-dimercapto-2,3 -dihydroxybutane can also be applied for protecting a peptide or a protein against oxidation. 1 ,4-dimercapto-2,3 -dihydroxybutane is oxidized faster and more efficiently than certain moieties in a peptide or a protein (for example, amino acids such as methionine, tryptophan, histidine and cysteine). Therefore when l,4-dimercapto-2,3 -dihydroxybutane is contacted with a peptide or a protein it is easily be oxidized and prevents the oxidation of the peptide or the protein. The presence of l,4-dimercapto-2,3 -dihydroxybutane in the reaction mixture during peptide synthesis or during protein isolation or purification, allows working under ambient atmosphere without the need of an oxygen free environment.
[0022] In one embodiment, this invention provides a method for protecting a protein against oxidation, the method may include, inter alia, the step of contacting the protein with l,4-dimercapto-2,3 -dihydroxybutane, wherein the l,4-dimercapto-2,3- dihydroxybutane is prepared by a non-stereoselective reaction.
[0023] In one embodiment, this invention provides a method for protecting a peptide against oxidation, the method may include, inter alia, the step of contacting the peptide with 1, -dimercapto-2,3 -dihydroxybutane, wherein the l,4-dimercapto-2,3- dihydroxybutane is prepared by a non-stereoselective reaction.
[0024] In one embodiment, this invention provides a use of l,4-dimercapto-2,3- dihydroxybutane prepared by a non-stereoselective reaction for protecting a peptide against oxidation.
[0025] In one embodiment, this invention provides a method of l,4-dimercapto-2,3- dihydroxybutane for protecting a peptide against oxidation during peptide synthesis, the method may include, inter alia, the step of contacting the peptide with 1,4- dimercapto-2,3-dihydroxybutane, wherein the l,4-dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction.
[0026] In one embodiment, this invention provides a method for protecting cysteine sulfhydyl group of a protein against oxidation, the method may include, inter alia, the step of contacting the protein with l,4-dimercapto-2,3 -dihydroxybutane, wherein the l,4-dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction.
[0027] In one embodiment, this invention provides a method for protecting cysteine sulfhydyl group of a peptide against oxidation, the method may include, inter alia, the step of contacting the peptide with l,4-dimercapto-2,3 -dihydroxybutane, wherein the l,4-dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction. [0028] In one embodiment, this invention provides a method for protecting an amino acid in a peptide or a protein against oxidation, the method may include, inter alia, the step of contacting the peptide or protein with l,4-dimercapto-2,3- dihydroxybutane, wherein the l,4-dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction. In one embodiment of this invention the amino acid is methionine. In another embodiment, the amino acid is tryptophan. In another embodiment, the amino acid is histidine. In another embodiment, the amino acid is cysteine.
[0029] In one embodiment of this invention, the non-stereoselective reaction may include, inter alia, the steps of reacting l,2:3,4-diepoxybutane with thioacetic acid to produce a reaction product comprising a 1,4-dithιoacetic -2,3-dihydroxybutane and hydrolysis of the 1,4-dithioacetic -2,3-dihydroxybutane to produce a reaction product comprising a distereomeric mixture of l,4-dimercapto-2,3 -dihydroxybutane.
[0030] In one embodiment of this invention, the non-stereoselective reaction may include, inter alia, the steps of reacting l,2:3,4-diepoxybutane with thioacetic acid to produce a reaction product substantially comprising a 1,4-dithioacetic -2,3- dihydroxybutane and hydrolysis of the 1,4-dithioacetic -2,3-dihydroxybutane to produce a reaction product substantially comprising a distereomeric mixture of 1,4- dimercapto-2,3 -dihydroxybutane.
[0031] In one embodiment of this invention, the hydrolysis is an acid hydrolysis. In another embodiment, the hydrolysis is a base hydrolysis.
[0032] l,4-dimercapto-2,3 -dihydroxybutane can be prepared from butadiene monoepoxide with yields of about 92%. Thus, in one embodiment of this invention, l,2:3,4-diepoxybutane is prepared from butadiene monoepoxide.
[0033] l,4-dimercapto-2,3 -dihydroxybutane can also be prepared from butadiene monoepoxide with yields of about 86%. Thus, in one embodiment of this invention the l,2:3,4-diepoxybutane is prepared from butadiene.
[0034] In one embodiment of this invention, the l,2:3,4-diepoxybutane is prepared from tetrahydroxybutane. [0035] In one embodiment of this invention, the l,4-dimercapto-2,3 -dihydroxybutane is prepared from 1,4- ditosyl-2,3-dihydroxybutane. In one embodiment of this invention, the reaction for the preparation of l,4-dimercapto-2,3 -dihydroxybutane from 1,4- ditosyl-2,3 -dihydroxybutane may include, inter alia, contacting the 1,4- ditosyl-2,3 -dihydroxybutane with thiourea and pyridine. In one embodiment of this invention, the reaction for the preparation of l,4-dimercapto-2,3 -dihydroxybutane from 1,4- ditosyl-2,3 -dihydroxybutane may include, inter alia, contacting the 1,4- ditosyl-2,3 -dihydroxybutane with potassium thioacetate.
[0036] In one embodiment of this invention, the l,4-dimercapto-2,3 -dihydroxybutane is a distereomeric mixture.
[0037] In one embodiment of this invention, the mixture may include 1-99% dithiothreitol and 99-1% dithioerythritol. In another embodiment, the mixture may include 1-50% dithiothreitol and 50-1% dithioerythritol. In another embodiment, the mixture may include 30-80% dithiothreitol and 70-20% dithioerythritol. In another embodiment, the mixture may include 20-70% dithiothreitol and 80-30% dithioerythritol.
[0038] As is shown in the Examples section, it has been surprisingly found that one or more compounds represented by the structures of formula I below are effective in reducing disulfide bonds.
[0039] In one embodiment, this invention provides a method for reducing a disulfide bond in a chemical agent, the method may include, inter alia, the step of contacting the chemical agent with a compound represented by the structure of formula I
Rι-P 2
R3
I
wherein Rr, R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
[0040] In one embodiment, the chemical agent is a protein. In another embodiment, the chemical agent is a biopolymer, which may comprise in one embodiment, a DNA or an RNA or in another embodiment may be a glycoprotein. In another embodiment, the chemical agent is a peptide. In another embodiment, the chemical agent is a cysteine derivative or any amino acid sequence which contains cysteine. In another embodiment, the chemical agent is an asymmetrical disulfide derivative. In another embodiment, the chemical agent is a symmetrical disulfide derivative.
[0041] It has now further been surprisingly found that one or more compounds represented by the structures of formula I are effective in protecting a peptide or a protein, against oxidation. Since these compounds ' are oxidized faster and more efficiently than certain moieties in a peptide or a protein (for example, amino acids such as methionine, tryptophan, histidine and cysteine), they will prevent the oxidation of the peptide or the protein with which they are in contact. As described above, this allows working under ambient atmosphere without the need of an oxygen free environment during peptide synthesis or during protein isolation or purification.
[0042] In one embodiment, this invention provides a method for protecting a protein against oxidation, the method may include, inter alia, the step of contacting the protein with a compound represented by the structure of formula I
Figure imgf000011_0001
I
wherein Rl3 R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
[0043] In one embodiment, this invention provides a method for protecting a peptide against oxidation, the method may include, inter alia, the step of contacting the peptide with a compound represented by the structure of formula I
Rι-P( 2
R3
I
wherein Rl3 R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
[0044] In one embodiment, this invention provides a method for protecting a peptide against oxidation during peptide synthesis, the method may include, inter alia, the step of contacting the peptide with a compound represented by the structure of formula I
Ri-P 2
R3
I
wherein Rls R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
[0045] In one embodiment, this invention provides a method for protecting cysteine sulfhydyl group of a protein against oxidation, the method may include, inter alia, the step of contacting the protein with a compound represented by the structure of formula I
Figure imgf000013_0001
T
wherein Ri, R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
[0046] In one embodiment, this invention provides a method for protecting cysteine sulfhydyl group of a peptide against oxidation, the method may include, inter alia, the step of contacting the peptide with a compound represented by the structure of formula I
Figure imgf000013_0002
I
wherein Rl5 R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or -cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
[0047] In one embodiment, this invention provides a method for protecting an amino acid in a peptide or a protein against oxidation, the method may include, inter alia, the step of contacting the peptide or protein with a compound represented by the structure of formula I
Rι-P( λ2
R3
I
wherein Ri, R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether. In one embodiment of this invention the amino acid is methionine. In another embodiment, the amino acid is tryptophan. In another embodiment, the amino acid is histidine. In another embodiment, the amino acid is cysteine.
[0048] In one embodiment, This invention provides a method for the removal of a protecting group from cysteine-containing peptide, the method may include, inter alia, the step of contacting the protein with a compound represented by the structure of formula I
Figure imgf000014_0001
wherein Ri, R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
[0049] Tris[hydroxyalkyl(aryl)]phosphines are water soluble, low toxicity, odorless, easy to handle compounds that are commercially available and can be used in reducing disulfide bonds (and in protecting a peptide or a protein against oxidation) at a wide range of pH, short time, mild reaction condition and in lower amounts.
[O0501 Ip one embodiment of the present invention, the compound is tris[hydroxyalkyl]phosphine. In another embodiment, the tris[hydroxyalkyl]phosphine is a tris[hydroxypropyl]phosphine. In another embodiment, the tris[hydroxypropyl]phosphine is a tris[3-hydroxypropyl]phosphine. In another embodiment, the tris[hydroxypropyl]phosphine is a tris[2- hydroxypropyl]phosphine. In another embodiment, the tris[hydroxypropyl]phosphine is atris[l-hydroxypropyl]phosphine.
[0051] In one embodiment of the present invention, the compound is [MeOCH2O(CH2)3]3P, [MeOCH2CH2O(CH2)3]3P, [CCl3CH2O(CH2)3]3P,
[R4R5NCH2O(CH2)3]3P, [CH3-S-CH2O(CH2)3]3P, [tetrahydropyranyloxy(CH2)3]3P or any combination thereof, wherein R4 and R5 are the same or different, independently of each other, H or an unsubstituted or substituted alkyl.
[0052] In one embodiment of the present invention,, an "alkyl" group refers to a saturated aliphatic hydrocarbon, may include straight-chain, branched-chain and cyclic alkyl groups. In one embodiment, the alkyl group has 1-12 carbons. In another embodiment, the alkyl group has 1-7 carbons. In another embodiment, the alkyl group has 1-6 carbons. In another embodiment, the alkyl group has 1-4 carbons. In another embodiment, the alkyl group is a methyl group. In another embodiment, the alkyl group is an ethyl group. In another embodiment, the alkyl group is a propyl group.
[0053] In another embodiment, the alkyl group is a butyl group. In another embodiment, the alkyl group may be unsubstituted or substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl. In another embodiment, the term "substituted" may refer to substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.
[0054] In one embodiment of the present invention, an "aryl" group refers to an aromatic group having at least one carbocyclic aromatic group or heterocyclic aromatic group, which may be unsubstituted or substituted by one or more groups selected from halogen, haloalkyl, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxy or thio or thioalkyl. Nonlimiting examples of aryl rings are phenyi, naphthyl, pyranyl, pyrroiyl, pyrazinyl, pyrimidinyl, pyrazolyl, pyridinyl, furanyl, thiophenyl, thiazolyl, imidazolyl, isoxazolyl, and the like.
[0055] In one embodiment of the present invention, a "hydroxyl" group refers to an OH group. In one embodiment of the present invention, an "alkoxy" group refers to an O-alkyl group, wherein alkyl has the same definition as described above. In one embodiment of the present invention, a "phenoxy" group refers to an O-phenyl group. In one embodiment of the present invention, a "thio" group refers to a SH group. In one embodiment of the present invention, an "alkylthio" group refers to an S-alkyl group wherein alkyl has the same definition as described above. In one embodiment of the present invention, an "alkylthio" group refers to an S-aryl group wherein aryl has the same definition as described above. A halogen or halo group refers to F, Cl, Br or I.
[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, may include definitions, will control. [0057] This invention is further illustrated in the Experimental Details section, which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims that follow thereafter.
EXPERIMENTAL DETAILS
MATERIALS AND METHODS
[0058] Fmoc amino acids were obtained from ArtChem Division of Frutarom. The phosphines were obtained from Cytec. Other chemicals from Sigma- Aldrich.
[0059] Peptide synthesis was performed according to known methods. Thin-layer chromatography was carried out on Merck silica coated aluminum plates, HPLC was performed on HP 1090 model. NMR spectra were recorded on a 400 MHz Bruker apparatus.
Example 1: Preparation of l,4-dimercapto-2,3-dihydroxybutane
i) Preparation of 1,2:3.4-diepoxibutane from butadiene or butadiene monoepoxide
[0060] 70 g (1 mol) of butadiene monoepoxide (Eastman Co.) was dissolved in 100 ml of methylene chloride. 172.6 g (1 mol) of m-chloroperbenzoic acid in 200 ml of methylene chloride was added to this solution at 0-5 °C upon stirring. After complete consumption of monoepoxide (GC control), m-chlorobenzoic acid was filtered off, the solvent was distilled off, and the residue was distilled, yielding 79.2g (92%) of 1,2:3,4- diepoxibutane, b.p. 52-58°C (25 mm). It should be noted that butadiene can be used instead of butadiene monoepoxide, providing 86% yield of diepoxide. Other peroxides can be used instead of m-chloroperbenzoic acid (for example magnesium monoperphthalate, 5-hydroperoxycarbonylphthalimide and trifluoroperacetic acid).
[0061] Because of the disactivating effect of epoxy groups which are adjacent to double bonds, methods based of hydrogen peroxide or alkyl peroxide activations differed from carboxylic acid catalysis (for example, hydrogen peroxide/ maleic, phthalic or trifluroacetic anhydrides, hydrogen peroxide/ urea complex, tetrabutylammonium peroxydisulfate, hydrogen peroxide/DCC and Sharpless epoxidation) are ineffective. To overcome disactivating effect of epoxy group, indirect methods for preparation of l,2:3,4-diepoxibutane are useful. For example, Sharpless epoxidation was performed, using l-toxyloxy-2-hydroxy-3-butene, followed by treatment with base. But these methods are not practical for scale-up and can be suitable for preparation of milligram quantities of desired material.
ii). Preparation of l,2:3,4-diepoxibutane from 1,2.3.4 - tetrahydroxybutane.
[0062] A solution of 1,2,3,4 — tetrahydroxybutane (200 g, 1.62 mol) in pyridine (6 L) was cooled to 0-5°C upon stirring. Tosyl chloride (592 g, 3.12 mol) was added dropwise or in small portions maintaining the temperature at 5-10°C. A clear solution resulted which became opaque 30 min after the last portion of tosyl chloride was added. The mixture was stirred for 12 h at 3-5°C then was allowed to warm to room temperature. The resulting thick suspension was evaporated under reduced pressure at 45-50°C to give a thick orange syrup (-1 L). This was dissolved in ethyl acetate (4 L) and 5M HC1 was added dropwise with vigorous stirring until the aqueous solution became acidic. The mixture was poured into a separating funnel, the organic layer was removed and washed with water (400-500 ml). The organic solution was dried over sodium sulfate, filtered and evaporated under reduced pressure to give the crude 1,4- ditosyl-2,3 -dihydroxybutane (-445-450 g). This compound was suspended in methylene chloride (3 L) and powdered potassium hydroxide (112 g, 1 mol) was added portionwise over 30-40 min with vigorous mechanical stirring. The mixture was stirred for 2-3h until TLC (silica gel, ethyl acetate :hexane = 1:1) showed that no bis-tosylate remained. The suspension was filtered and solvent was evaporated to give a liquid, which was distilled under reduced pressure to give 35% yield of l,2:3,4-diepoxibutane.
[0063] Mesyl chloride (or other sulphochlorides) can be used instead of tosyl chloride, but the yield are lower (22-25% vs. 35-40%).
[0064] The l,2:3,4-diepoxibutane can be prepared as an optical isomer by reaction of trans- 1,4-butenediol with bromine, followed by cyclization of 2,3-dibromo-l,4- dihydroxybutane with potassium hydroxide in two-phase system (or with quaternary ammonium hydroxide in organic solvent). The overall yield of this process is not higher than 45-50%.
iii) Preparation of 1.4-dimercapto-2.3- dihydroxybutane from l,2:3,4-diepoxibutane.
[0065] 152,4 g (2 mol) of thioacetic acid was mixed with 172 g (2 mol) of 1,2:3,4- diepoxibutane at 0-5°C. The mixture was allowed to stir at room temperature. Within 48 h a crystalline mass of the epimeric l,4-dimercaptoacetyl-2,3-butanediols has developed (the process of crystallization is highly exotermic and must be performed under temperature control). l,4-dimercapto-2,3 -dihydroxybutane can be prepared from l,2:3,4-diepoxibutane by acid or base hydrolysis:
[0066] Acid hydrolysis. To this mixture 50 ml of methyl acetate was added, followed by 300 ml of methanolic HC1. The mixture was refluxed under inert atmosphere for 6-8 h until TLC showed that no dithioacetate remained. If dithioacetate still remained, 50 ml of methanolic HC1 should be added and the reflux should be continued. After completion of reaction the solvents were evaporated and the residue was distilled under reduced pressure (115-125°C at 0.1-0.5 mm) giving 84 g (90%) yield of 1,4- dimercapto-2,3 -butandiol.
[0067] Base hydrolysis. The mixture of l,4-dimercaptoacetyl-2,3-butanediols was dissolved in 200 ml of dioxane under inert atmosphere, 2.2 equivalents (eq.) of triethylamine and water was added. The mixture was stirred overnight. The reaction end was controlled by TLC. The solvents were evaporated, and the residue was distilled in vacuum giving 85% yield of l,4-dimercapto-2,3-butandiol.
iv) Preparation of l,4-dimercapto-2,3-dihydroxybutane from 1,4- ditosyl-2,3- dihvdroxybutane.
[0068] 1,4- ditosyl-2,3 -dihydroxybutane (exp. ii) (1 eq.) , 2,1 eq. of thiourea and 5 eq. of pyridine in 300 ml of methanol were refluxed for 3 h until full consumption of tosylate was reached (TCL control). The mixture was filtered and filtrate was refluxed with a solution of 12 g of sodium hydroxide in 150 ml of methanol. The mixture was cooled, acidified to pH 6 with methanolic HC1, filtered, and the solvents were distilled in vacuum. The dark brown residue was purified by distillation, giving 42% of 1,4- dimercapto-2,3 -dihydroxybutane.
[0069] 1,4- ditosyl-2,3-dihydroxybutane (exp. ii) (1 eq.) , 2,1 eq. of potassium thioacetate in 300 ml of dimethylformamide (DMF) were heated at 100°C for 4 h until full consumption of tosylate was reached (TCL control). The mixture was filtered and filtrate was hydrolyzed according to examples (iii a) or (iii b) giving 35-46% of 1,4- dimercapto-2,3 -dihydroxybutane. Example 2; Reactivity of 1.4-dimercapto-2,3-butanedio, DTE and DTT.
[0070] The reactivity of l,4-dimercapto-2,3 -dihydroxybutane, DTT and DTE in disulfide reduction in glutathione disulfide and mercaptoethanol was compared by using IH NMR spectroscopy as described in [8]. The results are demonstrated in Table 1.
Table 1: Rates of disulfides reduction by l,4-dimercapto-2,3 -dihydroxybutane (D), DTT and DTE (at pH 7.0 and 298 K).
Figure imgf000021_0001
[0071] km is the approximate rate constant of the disulfide reduction. M'V1 (Molar"1 second"1) are the units of AaPP.
[0072] The results clearly indicate that there is no difference in reactivity of the disulfide reduction between l,4-dimercapto-2,3 -dihydroxybutane (D), DTT and DTE and therefore, compound (D) can be used in the same applications that its optical isomers.
Example 3: Reduction of disulfide bonds with tris(hydroxypropyl)phosphine (THPP) i) Reduction of glutathione disulfide
[0073] 0.016 g (0.1 mmol) of tris(hydroxypropyl)phosphine (THPP) in 5 ml of methanol was added to 0.06 g (0.1 mmol) of glutathione disulfide in 5 ml of 50% methanol. The mixture was stirred for 5-10 min. at room temperature. HPLC analysis indicated the complete conversion of disulfide to glutathione-SH. Then the mixture was evaporated to dryness. Crystalization from 50% ethanol gave glutathione, m.p. 192- 195°C, which was identical to the standard material (Sigma-Aldrich). ii) Reduction of 4,5-dihvdroxy-1.2-dithiane (oxidized form of DTT or DDE)
[0074] DDT was prepared from 4,5-dihydroxy-l,2-dithiane according to example 3 i.
The yield was 88% (after crystallization from MTBE), m.p. 48-50°C.
iii) Reduction of Fmoc-Cystine [0075] 3.4 g (0.5 mol) of Fmoc-cystine was mixed with 50 ml of ethanol. To this mixture 5 ml of water was added, followed by 1.1 g (0.5 mol) of THPP and the mixture was stirred for 5-10 min at room temperature (TLC control showed disappearance of starting material and presence of Fmoc-cysteine as a single product). The solvent was evaporated under reduced pressure, residue was dissolved in 100 of water and 50 ml of ethyl acetate, and the organic solution was washed twice with 50 ml of water. The ethyl acetate solution was separated from water, dried over sodium sulfate, evaporated in vacuum to give 3.4 g (100 %) yield of Fmoc-cysteine, which was 94.7% pure, according to HPLC. The compound was purified by precipitation from ethyl acetate solution with hexane (3.4 g of compound, 40 ml of ethyl acetate, 50 ml of hexane). The yield was 87%, 99.1 % purity according to HPLC.
iv) Removal of protecting groups from cysteine-containing peptides
[0076] Fragment of thioredoxin was synthesized as N-acetyl and C-amide octapeptide, according to [12], using t-butylsulphenyl group for cysteine protection.
[0077] Ac-Ala-Cys(S-t-Bu)-Ala-Thr-Cys(S-t-Bu)-Asp-Gly-PheNH2 was dissolved in 95% trifluoroethanol (TFE) and was treated with 2.2 eq. of THPP for 0.5 h. On addition of MTBE hexane, the fully deprotected octapeptide was obtained as a homogeneous compound according to TLC (solvent: CHC13/MeOH/AcOH/water = 60:25:2:4, RF = 0.45) and HPLC. This compound was oxidized, affording the same compound as described in [13].
Example 4: Reactivity of different reducing reagents towards cystine derivative
[0078] As a model, we selected Fmoc-cystine amide, a compound which is difficult to reduce in clean and high yield reaction, using ordinary methods such as Zn/AcOH, sodium borohydride or lithium aluminum hydride, Mg/MeOH, Hg(OAc)2 and other substances [14].
Figure imgf000023_0001
*) Reaction in DMF at temperature higher than 60°C led to formation of Fmoc- cysteine dimethylamide as a main by-product, which is very difficult to purified off. References:
1. Cleland, W.W. , (1964) Biochemistry, Vol. 3, 481.
2. Carmack, M. and CJ.Kelley (1968) Journal of Organic Chemistry, Vol. 33, 171.
3. Whitesides, G.M. et al (1977) J. Org. Chem, Vol. 42, 332.
4. US Patent No. 3,275,662.
5. Zhurenbaev A.N., Gabdzhaev Z.G. (1976) Zhurnal Obschei Khimii, Vol. 47, 1420. 6. Singh, R. e.a. et al (1991) J. Org.Chem., Vol. 56, 6931.
7. Whitesides G. M. et al (1993) J. Org. Chem., Vol. 58, 633.
8. Whitesides G. M. e.a. J. Org. Chem., 1991, v.56, p.7328
9. US Patent No. 5,378,813
10. Burns J.A. et al (1991) J.Org.Chem., Vol. 56, 2648. 11. Valentine D.H., Jr., Hillhouse J.H. (2003) Synthesis, 317.
12. Sweetman B.J. and MacLaren J.A. (1996) Aust. J. Chem., Vol. 19, 2347.
13. Musiol H-J., et al (1994) Biopolymers, Vol. 34, 1553.
14. Wolman Y. (1974) The chemistry of the Thiol Group, Patai S Ed., London.
[0081] It will be appreciated that this invention is not limited by what has been described hereinabove and that numerous modifications, all of which fall within the scope of the present invention, exist. Rather the scope of the invention is defined by the claims that follow:

Claims

What we claim is:
1. A method for reducing disulfide bonds in a chemical agent comprising the step of contacting said chemical agent with l,4-dimercapto-2,3 -dihydroxybutane, wherein said l,4-dimercapto-2,3 -dihydroxybutane is prepared by a non- stereoselective reaction.
2. The method according to claim 1, wherein said chemical agent is a biopolymer, a protein, a peptide or a cysteine derivative.
3. The method according to claim 1, wherein said non-stereoselective reaction comprising the steps of reacting 1,2: 3, 4-diepoxy butane with thioacetic acid to produce a reaction product comprising a 1,4-dimercaptoacetyl -2,3- dihydroxybutane; and hydrolysis of said 1,4-dithioacetic -2,3-dihydroxybutane to produce a reaction product comprising 1 ,4-dimercapto-2,3 -dihydroxybutane.
4. The method according to claim 3, wherein said l,2:3,4-diepoxybutane is prepared from butadiene.
5. The method according to claim 3, wherein said l,2:3,4-diepoxybutane is prepared from butadiene monoepoxide.
6. The method according to claim 1, wherein said l,4-dimercapto-2,3- dihydroxybutane is a distereomeric mixture.
7. The method according to claim 6, wherein said mixture comprising 1-99% dithiothreitol and 99-1% dithioerythritol.
8. The method according to claim 6, wherein said mixture comprising
1-50% dithiothreitol and 50-99% dithioerythritol.
9. A method for protecting a protein against oxidation comprising the step of contacting said protein with 1 ,4-dimercapto-2,3 -dihydroxybutane, wherein said l,4-dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction.
10. The method according to claim 9, wherein said non-stereoselective reaction comprising the steps of reacting l,2:3,4-diepoxybutane with thioacetic acid to produce a reaction product comprising a 1,4-dimercaptoacetyl -2,3- dihydroxybutane; and hydrolysis of said 1,4-dithioacetic -2,3-dihydroxybutane to produce a reaction product comprising 1 ,4-dimercapto-2,3-dihydroxybutane.
11. The method according to claim 10, wherein said l,2:3,4-diepoxybutane is prepared from butadiene.
12. The method according to claim 10, wherein said l,2:3,4-diepoxybutane is prepared from butadiene monoepoxide.
13. Tbft method according to claim 10, wherein said l,4-dimercapto-2,3- dihydroxybutane is a distereomeric mixture.
14. The method according to claim 13, wherein said mixture comprising 1-99% dithiothreitol and 99-1% dithioerythritol.
15. The method according to claim 13 , wherein said mixture comprising
1-50% dithiothreitol and 50-99% dithioerythritol.
16. A method for protecting cysteine sulfhydyl group of a protein against oxidation comprising the step of contacting said protein with l,4-dimercapto-2,3- dihydroxybutane, wherein said l,4-dimercapto-2,3 -dihydroxybutane is prepared by a non-stereoselective reaction.
17. The method according to claim 16, wherein said non-stereoselective reaction comprising the steps of reacting l,2:3,4-diepoxybutane with thioacetic acid to produce a reaction product comprising a 1,4-dimercaptoacetyl -2,3- dihydroxybutane; and hydrolysis of said 1,4-dithioacetic -2,3-dihydroxybutane to produce a reaction product comprising l,4-dimercapto-2,3 -dihydroxybutane.
18. The method according to claim 17, wherein said l,2:3,4-diepoxybutane is prepared from butadiene.
19. The method according to claim 17, wherein said l,2:3,4-diepoxybutane is prepared from butadiene monoepoxide.
20. The method according to claim 17, wherein said l,4-dimercapto-2,3- dihydroxybutane is a distereomeric mixture.
21. The method according to claim 20, wherein said mixture comprising 1-99% dithiothreitol and 99-1% dithioerythritol.
22. The method according to claim 20, wherein said mixture comprising
1-50% dithiothreitol and 50-99% dithioerythritol.
23. A method for reducing a disulfide bond in a chemical agent comprising the step of contacting said chemical agent with a compound represented by the structure of formula I
Figure imgf000027_0001
I
wherein Rls R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
24. The method according to claim 23, wherein said chemical agent is a biopolymer, protein, a peptide or a cysteine derivative.
25. The method according to claim 23, wherein said compound is tris[hydroxyalkyl]phosphine.
26. The method according to claim 25, wherein said tris[hydroxyalkyl]phosphines is a tris[hydroxypropyl]phosphine.
27 The method according to claim 26, wherein said tris[hydroxypropyl]phosphine is a tris[3-hydroxypropyl]phosphine.
28. The method according to claim 26, wherein said tris[hydroxypropyl]phosphine is a tris[2-hydroxypropyl]phosphine.
29. The method according to claim 26, wherein said tris[hydroxypropyl]phosphine is atris[l-hydroxypropyl]phosphine.
30. The method according to claim 23, wherein said compound is
[MeOCH2O(CH2)3]3P, [MeOCH2CH2O(CH2)3]3P, [CCl3CH2O(CH2)3]3P, [R4R5NCH2O(CH2)3]3P, [CH3-S-CH2O(CH2)3]3P, [tetrahydropyranyloxy(CH2)3]3P or any combination thereof, wherein R4 and R5 are the same or different, independently of each other, H or an'unsubstituted or substituted alkyl.
31. A method for protecting a protein against oxidation comprising the step of contacting said protein with a compound represented by the structure of formula I
Ri-P λ2
R3
I
wherein Rj, R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
32. The method according to claim 31, wherein said compound is tris [hydroxyalkyl]pho sphine .
33. The method according to claim 32, wherein said tris[hydroxyalkyl]phosphines is a tris[hydroxypropyl]phosphine.
34. The method according to claim 33, wherein said tris[hydroxypropyl]phosphine is a tris[3-hydroxypropyl]phosphine.
35. The method according to claim 33, wherein said tris[hydroxypropyl]phosphine is a tris[2-hydroxypropyl]phosphine.
36. The method according to claim 33, wherein said tris[hydroxypropyl]phosphine is a tris[l-hydroxypropyl]phosphine.
37. The method according to claim 31, wherein said compound is [MeOCH2O(CH2)3]3P, [MeOCH2CH2O(CH2)3]3P, [CCl3CH2O(CH2)3]3P, [R4R5NCH2O(CH2)3]3P, [CH3-S-CH2O(CH2)3]3P, [tetrahydropyranyloxy(CH2)3]3P or any combination thereof, wherein IU and R5 are the same or different, independently of each other, H or an unsubstituted or substituted alkyl.
38. A method for protecting cysteine sulfhydyl group of a protein against oxidation comprising the step of contacting said protein with a compound represented by the structure of formula I
Rι-P λ2
R3
I
wherein Rl3 R2 and R3 are the same or different, independently of each other represent H, an unsubstituted or substituted linear, branched or cyclic hydroxyalkyl, an unsubstituted or substituted linear, branched or cyclic hydroxyalkenyl or an unsubstituted or substituted hydroxyaryl, an unsubstituted or substituted linear, branched or cyclic alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkoxy alkoxyalkyl ether, an unsubstituted or substituted linear, branched or cyclic alkylthioalkyl ether.
39. The method according to claim 38, wherein said compound is tris[hydroxyalkyl]phosphine.
40. The method according to claim 39, wherein said tris[hydroxyalkyl]phosphines is a tris[hydroxypropyl]phosphine.
41. The method according to claim 40, wherein said tris[hydroxypropyl]phosphine is a tris[3-hydroxypropyl]phosphine.
42. The method according to claim 40, wherein said tris[hydroxypropyl]phosphine is a tris[2-hydroxypropyl]phosphine.
43. The method according to claim 40, wherein said tris[hydroxypropyl]phosphine is a tris[l-hydroxypropyl]phosphine.
44. The Method according to claim 38, wherein said compound is [MeOCH2O(CH2)3]3P, [MeOCH2CH2O(CH2)3]3P, [CCl3CH2O(CH2)3]3P, [R4R5NCH2O(CH2)3]3P, [CH3-S-CH2O(CH2)3]3P, [tetrahydropyranyloxy(CH2)3]3P or any combination thereof, wherein R4 and R5 are the same or different, independently of each other, H or an unsubstituted or substituted alkyl.
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