WO2014033466A1 - Procédé et compositions pour l'élimination de groupes protecteurs labiles en milieu acide - Google Patents

Procédé et compositions pour l'élimination de groupes protecteurs labiles en milieu acide Download PDF

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WO2014033466A1
WO2014033466A1 PCT/GB2013/052280 GB2013052280W WO2014033466A1 WO 2014033466 A1 WO2014033466 A1 WO 2014033466A1 GB 2013052280 W GB2013052280 W GB 2013052280W WO 2014033466 A1 WO2014033466 A1 WO 2014033466A1
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acid
group
fmoc
hfip
composition
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PCT/GB2013/052280
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English (en)
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Dmitry Stetsenko
Pasquale Palladino
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Dmitry Stetsenko
Pasquale Palladino
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Publication of WO2014033466A1 publication Critical patent/WO2014033466A1/fr

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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
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C07K1/122—Hydrolysis with acids different from HF
    • 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
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the field of organic chemistry, in particular, to organic synthesis, where the use of protecting groups to protect organic compounds is necessary.
  • the invention relates to peptide chemistry, particularly to solid-phase peptide synthesis (SPPS) as well as to solution-phase peptide synthesis.
  • SPPS solid-phase peptide synthesis
  • Peptides are used extensively in medicine for drug design and drug discovery, and as probes for molecular imaging and disease diagnosis.
  • Peptides and their derivatives such as hormones, neurotransmitters and neuromodulators act as signal molecules in diverse biological and medicinal applications and thus have attracted considerable synthetic attention both for industrial manufacturing and small-scale lab production.
  • Boc SPPS remains a competitive method, especially for long peptides.
  • the Boc chemistry is based on a combination of two types of acid-labile protecting groups with vastly different sensitivity to acid: a temporary f-butoxycarbonyl group (Boc) for / ⁇ -protection and a semipermanent protecting group for nucleophilic amino acid side-chains, usually a (substituted) benzyl ester or ether (Bzl, 2-CI-Bzl, 2,6-di-Br-Bzl), benzyloxycarbonyl (Z) and its derivatives, such as 2- chlorobenzyloxycarbonyl (2-CIZ), and other groups with comparable acid stability such as p-toluenesulphonyl (tosyl, Tos) for Arg [Isidro-Llobet, A.; Alvarez, M.; Albericio, F.
  • Boc group is removed in each cycle of peptide elongation by a moderately strong acid, usually trifluoroacetic acid (TFA) at 10-100% concentration
  • TFA trifluoroacetic acid
  • Other protecting groups must be relatively TFA-resistant to prevent side-reactions resulting from premature unmasking of the amino acid side-chains.
  • Inorganic acids such as 6N hydrochloric acid HCI [Naharissoa, H.; Sarrade, V.; Follet, M.; Calas, B. Pept. Res. 1992, 5, 293] or concentrated sulphuric acid H 2 S0 4 in 1 ,4-dioxane (1 :9 v/v) [Trivedi, H. S.; Anson, M.; Steel, P. G.; Worley, J. SynLett 2001 , 1932] have also been tried for the Boc group deprotection during solid-phase peptide synthesis but they have not gained popularity.
  • 6N hydrochloric acid HCI Naharissoa, H.; Sarrade, V.; Follet, M.; Calas, B. Pept. Res. 1992, 5, 293
  • concentrated sulphuric acid H 2 S0 4 in 1 ,4-dioxane (1 :9 v/v) Trivedi, H. S.; Anson,
  • scavengers are usually employed such as anisole [Sakakibara, S.; Shimonishi, V. Bull.
  • TFA trifluoroacetic acid
  • the remaining 5-10% are usually water and various scavengers, in particular, sulphur-containing compounds such as thioanisole and 1 ,2- ethanedithiol [King, D. S.; Fields, C. G.; Fields, G. B. Int. J. Pept. Protein Res. 1989, 36, 255], or trialkylsilanes [Pearson, D. A.; Blanchette, M.; Baker, M. L; Guindon, C. A. Tetrahedron Lett. 1989, 30, 2739].
  • Scavengers are used to suppress side-reactions occurring with sensitive amino acid side-chains such as Trp under the action of reactive cations generated by TFA from protecting groups.
  • Trp [Fields, G. B. and Noble, R. L. Int. J. Peptide Prot. Res. 1990, 35, 161 ].
  • TFA is used in copious quantities (usually 50-100% v/v) to remove the /V-terminal Boc group in every cycle of peptide assembly.
  • TFA is considered a milder deprotecting agent than liquid HF, TFMSA in TFA or neat MSA.
  • TFA is an aggressive, extremely corrosive chemical capable of inflicting bodily harm through inhalation as well as skin contact, leaving hard to heal chemical burns. It readily attacks or infiltrates many common materials, and is relatively expensive, both for the initial purchase and for its ultimate disposal, especially when large-scale peptide synthesis requires large amounts of the chemical.
  • Sulphonic acids such as methanesulphonic acid (MSA) [Yajima, H.; Ogawa, H.; Fujii, N.; Funakoshi, S. Chem. Pharm. Bull. 1977, 25, 740; Kiso, Y.; Fujiwara, Y.; Kimura, T.; Nishitani, A.; Akaji, K. Int. J. Peptide Protein Res. 1992, 40, 308] and, in particular, p-toluenesulphonic acid (p-TSA) [Goodacre, J.; Ponsford, R. J.; Stirling, I.
  • MSA methanesulphonic acid
  • p-TSA p-toluenesulphonic acid
  • the sulphonic acids have insufficient solubility in dichloromethane (DCM), which is a preferred solvent for low-crosslinked (1 - 2%) polystyrene resins usually employed as polymeric supports in the Boc SPPS. That requires addition of suboptimal solvents such as 1 ,4-dioxane to reach the necessarily high concentration of the acid.
  • DCM dichloromethane
  • the sulphonic acids are stronger acids than TFA and have been used at higher concentration, such as 100% MSA, for the full deprotection of peptides [Yajima, H.; Kiso, Y.; Ogawa, H.; Fujii, N.; Irie, H. Chem. Pharm. Bull. 1975, 23, 1 164].
  • One aspect of the invention is the use of the method and compositions for full or partial removal of acid- labile protecting groups from a compound such as a protected peptide.
  • Another aspect of the invention is the use of the method and compositions to cleave an acid-labile linker to a solid support used in solid-phase synthesis, such as solid-phase peptide synthesis (SPPS).
  • SPPS solid-phase peptide synthesis
  • the method of the present invention uses less hazardous and more environmentally friendly compositions than the currently used trifluoroacetic acid (TFA) or liquid hydrofluoric acid HF based cocktails.
  • a method for removing acid-labile protecting groups from a protected compound that has one or more acid-labile protecting groups comprising: a. dissolving or dispersing the protected compound, or immersing a solid support to which the protected compound is attached, in a mixture comprising a fluoro alcohol, an acid and, optionally, an organic solvent and/or a scavenger; and b. maintaining the resulting solution or dispersion, or keeping the solid support immersed, for a period of time sufficient to ensure the removal of one or more acid-labile protecting groups from said protected compound, thereby producing a deprotected or partially protected compound.
  • the deprotected or partially protected compound is recovered at the end of the method.
  • the method is preferably performed at ambient temperature.
  • a composition for use in a method for removing acid-labile protecting groups from a protected compound that has one or more acid-labile protecting groups comprising i) a fluoro alcohol, ii) an acid and, optionally, iii) an organic solvent and/or a scavenger.
  • the composition contains at least one fluoro alcohol and at least one acid and, optionally, at least one organic solvent and/or at least one scavenger; but if desired may contain combinations of two or more different fluoro alcohols, acids, solvents and/or scavengers.
  • the method involves the use of a mixture or composition that contains a combination of at least two key components.
  • a fluoro alcohol such as 2,2,2-trifluoroethanol (TFE), 1 , 1 , 1 ,3,3,3- hexafluoroisopropanol (HFIP), 2,2,3,3,3-pentafluoro-1 -propanol, 2,2,3,3,4,4,4-heptafluoro-1 -butanol, nonafluoro-tert-butyl alcohol and the like, or a combination thereof, in a concentration of 0.01 -99.99% (v/v).
  • the fluoro alcohol is preferably present in a concentration within the range of 5- 99% (v/v), and more preferably within the range of 10-30% (v/v).
  • key component two is a protic (Br0nsted) acid such as hydrochloric acid HCI, hydrobromic acid HBr, sulphuric acid, benzenesulphonic acid, p-toluenesulphonic acid (p-TSA), methanesulphonic acid, ethanesulphonic acid, trifluoromethanesulphonic acid (TFMSA), trifluoroacetic acid (TFA), trichloroacetic acid (TCA), dichloroacetic acid (DCA), chloroacetic acid, formic acid, acetic acid and the like, or a combination thereof.
  • protic (Br0nsted) acid such as hydrochloric acid HCI, hydrobromic acid HBr, sulphuric acid, benzenesulphonic acid, p-toluenesulphonic acid (p-TSA), methanesulphonic acid, ethanesulphonic acid, trifluoromethanesulphonic acid (TFMSA), tri
  • key component two is a Lewis acid or a silicon compound such as chlorotrimethylsilane Me 3 SiCI, bromotrimethylsilane Me 3 SiBr, boron trifluoride BF 3 , boron trichloride BCI 3 , silicon tetrachloride SiCI 4 , trimethylsilyl trifluoromethane sulphonate (TMSOTf) and the like, or a combination thereof.
  • a Lewis acid or a silicon compound such as chlorotrimethylsilane Me 3 SiCI, bromotrimethylsilane Me 3 SiBr, boron trifluoride BF 3 , boron trichloride BCI 3 , silicon tetrachloride SiCI 4 , trimethylsilyl trifluoromethane sulphonate (TMSOTf) and the like, or a combination thereof.
  • key component two is a polymeric protic or Lewis acid or a silylated polymeric acid such as the sulphonated polystyrene resins, for example Dowex® or Amberlyst® resins, Nafion® resin or trimethylsilyl-Nafion® resin [Murata, S.; Noyori, R. Tetrahedron Lett. 1980, 21, 767] and the like.
  • sulphonated polystyrene resins for example Dowex® or Amberlyst® resins, Nafion® resin or trimethylsilyl-Nafion® resin [Murata, S.; Noyori, R. Tetrahedron Lett. 1980, 21, 767] and the like.
  • said key component two is selected from the group of polymeric protic acids in the H form.
  • said key component two is an Amberlyst® resin in the H form.
  • an organic solvent may be added such as dichloromethane (DCM), chloroform CHCI 3 , 1 ,2-dichloroethane (DCE), carbon tetrachloride CCI 4 , benzene C6H 6 , toluene CH 3 C6H 5 , pentane C5H12, hexane ⁇ and the like, or a combination thereof.
  • DCM dichloromethane
  • DCE 1,2-dichloroethane
  • CCI 4 carbon tetrachloride
  • benzene C6H 6 toluene CH 3 C6H 5
  • pentane C5H12 hexane ⁇ and the like
  • a scavenger may be added.
  • scavengers examples include, but are not limited to, thioanisole (TA), dimethylsulphide (DMS), ethylmethylsulphide, tetrahydrothiophene, 1 ,2-ethanedithiol, 1 ,3- propanedithiol, benzylmercaptan, thiophenol, anisole, phenol, m-cresol, water, pentamethylbenzene, triethylsilane and triisopropylsilane, or a combination thereof.
  • TA thioanisole
  • DMS dimethylsulphide
  • ethylmethylsulphide etrahydrothiophene
  • 1 ,2-ethanedithiol 1 ,2-ethanedithiol
  • 1 ,3- propanedithiol propanedithiol
  • benzylmercaptan thiophenol
  • anisole phenol
  • phenol m-cresol
  • water penta
  • the concentration of a scavenger or scavengers is preferably within the range of 0.1 -95% (v/v), and more preferably within the range of 1 -50% (v/v).
  • said protected compound is attached to a solid support for solid-phase synthesis.
  • a protected compound attached to a solid support by a chemical bond that is non-cleavable using the method of this invention in the current embodiment will be deprotected or partially deprotected of acid-labile protecting groups by the method of this invention; but will remain attached to the solid support.
  • the protected compound is attached to a solid support by an acid-labile linker (i.e. a cleavable bond using the method of this invention in the current embodiment) referred to herein as an acid- labile solid support; and the method then results in the production of a deprotected or partially protected compound that is detached from the solid support.
  • an acid-labile linker i.e. a cleavable bond using the method of this invention in the current embodiment
  • a composition which comprises hydrochloric acid, 1 , 1 , 1 , 3,3, 3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises hydrochloric acid, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • the concentration of hydrochloric acid is preferably within the range of 0.001 - 6N, and more preferably within the range of 0.05 - 1 N.
  • a composition which comprises p- toluenesulphonic acid (p-TSA), 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • p-TSA p- toluenesulphonic acid
  • 2,2,2-trifluoroethanol 2,2,2-trifluoroethanol
  • solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises p- toluenesulphonic acid (p-TSA), 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • p-TSA p- toluenesulphonic acid
  • 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • the concentration of p-toluenesulphonic acid is preferably within the range of 0.001 - 2M, and more preferably within the range of 0.05 - 1 M.
  • a composition which comprises benzenesulphonic acid, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises benzenesulphonic acid, 1 , 1 , 1 , 3,3, 3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • the concentration of benzenesulphonic acid is preferably within the range of 0.001 - 2M, and more preferably within the range of 0.05 - 1 M.
  • a composition which comprises hydrobromic acid, 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises hydrobromic acid, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • concentration of hydrobromic acid is preferably within the range of 0.001 - 6M, and more preferably within the range of 0.05 - 1 M.
  • a composition which comprises methanesulphonic acid (MSA), 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • MSA methanesulphonic acid
  • 2,2,2-trifluoroethanol 2,2,2-trifluoroethanol
  • solvent and/or a scavenger or a combination of solvents and/or scavengers optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises methanesulphonic acid (MSA), 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • MSA methanesulphonic acid
  • 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • the concentration of methanesulphonic acid is preferably within the range of 0.001 - 2M, and more preferably within the range of 0.05 - 1 M.
  • a composition which comprises trifluoromethanesulphonic acid (TFMSA), 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • TFMSA trifluoromethanesulphonic acid
  • a composition which comprises trifluoromethanesulphonic acid, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • the concentration of trifluoromethanesulphonic acid is preferably within the range of 0.001 - 2M, and more preferably within the range of 0.05 - 1 M.
  • a composition which comprises sulphuric acid, 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises sulphuric acid, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • the concentration of sulphuric acid is preferably within the range of 0.001 - 2M, and more preferably within the range of 0.05 - 1 M.
  • a composition which comprises trifluoroacetic acid (TFA), 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • TFA trifluoroacetic acid
  • 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises trifluoroacetic acid, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • concentration of trifluoroacetic acid is preferably within the range of 0.05 - 95 % (v/v), and more preferably within the range of 0.5 - 50 % (v/v).
  • a composition is used, which comprises formic acid, 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises formic acid, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • the concentration of formic acid is preferably within the range of 0.05 - 95 % (v/v), and more preferably within the range of 0.5 - 50 % (v/v).
  • a composition which comprises acetic acid, 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition is used, which comprises acetic acid, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • the concentration of acetic acid is preferably within the range of 0.05 - 95 % (v/v), and more preferably within the range of 0.5 - 50 % (v/v).
  • a composition which comprises chlorotrimethylsilane Me 3 SiCI, 1 , 1 , 1 , 3,3, 3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition is used, which comprises chlorotrimethylsilane, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • the concentration of chlorotrimethylsilane is preferably within the range of 0.001 - 3M, and more preferably within the range of 0.05 - 1 M.
  • a composition which comprises bromotnmethylsilane Me 3 SiBr, 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises bromotnmethylsilane, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • the concentration of bromotrimethylsilane is preferably within the range of 0.001 - 3M, and more preferably within the range of 0.05 - 1 M.
  • a composition which comprises trimethylsilyl trifluoromethanesulphonate (TMSOTf), 1 , 1 , 1 ,3,3,3- hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • TMSOTf trimethylsilyl trifluoromethanesulphonate
  • a composition which comprises trimethylsilyl trifluoromethanesulphonate, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • concentration of trimethylsilyl trifluoromethanesulphonate is preferably within the range of 0.001 - 3M, and more preferably within the range of 0.05 - 1 M.
  • a composition which comprises an Amberlyst® resin in the H form, 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises an Amberlyst® resin in the H form, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises a Dowex® resin in the H form, 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises a Dowex® resin in the H form, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises a Nafion® resin, 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises a Nafion® resin, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition is used, which comprises a trimethylsilyl-Nafion® resin, 1 , 1 , 1 ,3,3,3-hexafluoroisopropanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • a composition which comprises a trimethylsilyl-Nafion® resin, 2,2,2-trifluoroethanol and, optionally, a solvent and/or a scavenger or a combination of solvents and/or scavengers.
  • mixture used in the method of this invention may be added as a pre-formed composition or, alternatively, one or more of the components may be added separately to the other component(s) during the performance of the method.
  • the method and compositions can be used in organic synthesis to deprotect organic compounds protected by acid-labile protecting groups or cleave acid-labile linkers to solid supports in solid-phase synthesis.
  • the method and compositions may be used in solid-phase peptide synthesis as well as in liquid-phase peptide synthesis to remove acid-labile protecting groups from protected peptides, and in solid-phase peptide synthesis to remove a fully deprotected or partially protected peptide from a solid support.
  • the method and compositions of the invention replace currently used highly hazardous deprotection reagents trifluoroacetic acid (TFA) or liquid hydrofluoric acid HF.
  • Fluoro alcohols such as 2,2,2-trifluoroethanol (TFE) or 1 , 1 , 1 ,3,3,3- hexafluoroisopropanol (HFIP) have been widely used as solvents and reagents in organic synthesis in general and in peptide chemistry in particular.
  • Typical fluoro alcohols are acidic: pK A of TFE is 12.4 [Ballinger, P.; Long, F. A. J. Am. Chem. Soc. 1959, 81, 1050], pK A of HFIP is 9.3, and that of perfluoro-fe/f-butanol (CF 3 ) 3 COH is 5.2 [Filler, R.; Schure, R. M. J. Org. Chem.
  • fluoro alcohols are good solubilising agents for peptides, especially those prone to form secondary structures [Yanagi, K.; Ashizaki, M.; Yagi, H.; Sakurai, K.; Lee,
  • Cleavage of fully protected peptides from trityl ester resin may be effected by dilute HCI in aqueous DMF with optional TFE (up to 10% v/v)
  • TFE up to 10% v/v
  • the p-benzyloxybenzyl ester of the Fmoc-Leu-Wang resin was cleaved by 0.1 N HCI in HFIP - DCM (1 :4 v/v) mixture more slowly (91 .6% release after 3 h). It should be noted that the ratio of HCI to the polymeric ester was nearly equimolar: ca. 0.1 mmol (10 ⁇ of concentrated ca. 37% aqueous HCI per 1 cm 3 of the mixture) per ca. 0.095 mmol of the resin-bound amino acid (100 mg of the resin with the Fmoc group loading of 0.95 mmol g "1 ).
  • the p-hydroxymethylphenoxyacetyl ester (Fmoc-Gly-HMPA-PEG-PS® resin) has slightly lower lability to 0.1 N HCI in HFIP (84.2% release after 3 h).
  • the addition of 1 % v/v dimethylsulphide had no noticeable accelerating effect on the cleavage.
  • the two common resins for peptide amides were cleaved even faster.
  • Rink-NovaPEG® resin released 82% Fmoc-glycinamide after 2 h, and PAL-PEG-PS® lost 91 % of the same after 1 h of reaction.
  • the type of the resin had no clear-cut influence on the rate of deprotection.
  • a peptide Fmoc-Lys- Thr-Thr-Lys-Ser-OH was prepared by the Fmoc SPPS as described [Amblard, M.; Fehrentz, J-A.; Martinez, J.; Subra, G. Mol. Biotechnol. 2006, 3, 239] on TentaGel® S carboxytrityl ester-linked resin [Grubler, G.; Zimmermann, H.; Echner, H.; Stoeva, S.; Bernardi, E.; Pourrias, B.; Voelter, W.
  • fluoro alcohols are poor swelling solvents for polystyrene resins.
  • the latter can be improved by adding good swelling solvent such as DCM.
  • DCM swelling solvent
  • a mixture of just 10% (v/v) TFE in DCM is an excellent swelling medium for low crosslinked polystyrene supports such as MBHA or Merrifield resin used in the Boc SPPS.
  • p-TSA non-volatile crystalline p-toluenesulphonic acid monohydrate
  • the deprotected species in the case of ethanol was the corresponding ethyl ester H-Lys(Fmoc)-OEt detected as a dominant peak (intensity 100%) after 72 h of reaction in the HR-MS spectrum (m/z 397.2042) (Example 14).
  • the corresponding trifluoroethyl ester was also detectable in the reaction mixture after 72 h of reaction as a low intensity peak in the HR-MS spectrum (m/z 452.1839) (Example 14).
  • No trifluoroethyl ester side-product was seen on RP-HPLC within the time necessary for complete Boc deprotection (5 min). It should be noted that no such side-reaction of esterification of the free carboxyl group was ever observed with HFIP in our experiments.
  • fluoro alcohol refers to compounds of the formula R 1 R 2 R 3 C-OH, where R 1 is a fluorinated lower alkyl, and R 2 and R 3 are each independently H or a fluorinated lower alkyl radical.
  • exemplary fluoro alcohols include, without limitation, 2,2,2-trifluoroethanol CF 3 CH 2 OH ("TFE"), 1 , 1 , 1 ,3,3,3- hexafluoroisopropanol (CF 3 ) 2 CHOH (“HFIP”), 2,2,3,3-tetrafluoro-1 -propanol CHF 2 CF 2 CH 2 OH, 2,2,3,3,3-pentafluoro-1 -propanol CF 3 CF 2 CH 2 OH, 2,2,3,3,4,4,4-heptafluoro-1 -butanol CF3CF2CF2CH2OH, perfluoro-fe/f- butanol (CF 3 ) 3 COH and the like.
  • lower alkyl refers to monovalent hydrocarbon radicals composed of carbon and hydrogen, and having no unsaturation. Lower alkyl radicals may be straight or branched, and contain from 1 to 6 carbon atoms, inclusive.
  • fluorinated lower alkyl refers to a lower alkyl radical in which one or more hydrogen atoms have been replaced by fluorine. Exemplary fluorinated lower alkyl radicals include, without limitation, CF 3 - CHF 2 - CF 3 CF 2 - CF 3 CF 2 CF 2 - CHF 2 CF 2 - (CF 3 ) 3 C- and the like.
  • non-hydrogen-bonding solvent refers to an organic solvent thatdoes not form strong hydrogen bonds.
  • the term "acid” as used herein refers to a protic (Bransted) acid including, without limitation, hydrofluoric acid HF, hydrochloric acid HCI, hydrobromic acid HBr, sulphuric acid H 2 S0 4 , p-toluenesulphonic acid (p-TSA), methanesulphonic acid (MSA), trifluoromethanesulfonic acid (TFMSA), tetrafluoroboric acid HBF 4 , trifluoroacetic acid (TFA), formic acid HC0 2 H, acetic acid CH 3 C0 2 H and the like.
  • p-TSA p-toluenesulphonic acid
  • MSA methanesulphonic acid
  • TFMSA trifluoromethanesulfonic acid
  • TFA tetrafluoroboric acid
  • an acid may comprise a Lewis acid including, without limitation, boron trifluoride BF 3 , boron trichloride BCI3, boron tribromide BBr 3 , aluminium chloride AICI3, tin chloride SnCI 4 , titanium chloride TiCI 4 and the like.
  • an acid may comprise a polymer-supported protic or Lewis acid or a silylated polymeric acid including, without limitation, sulphonated polystyrene resins such as Dowex® or Amberlyst® resins, Nafion® resin or trimethylsilyl-Nafion® resin and the like.
  • the term "acid” as used here may refer to a silicon compound including, without limitation, silicon tetrachloride SiCI 4 , chlorotrimethylsilane Me 3 SiCI, bromotrimethylsilane Me 3 SiBr, iodotrimethylsilane Me 3 Sil, trimethylsilyl trifluoromethanesulphonate (TMSOTf) and the like.
  • an acid may comprise a combination thereof.
  • acid-labile protecting group refers to a protecting group that is removable by acid.
  • exemplary acid-labile protecting groups include, without limitation, trityl (Trt), tetrahydropyranyl (Thp), f-butoxycarbonyl (Boc), f-butyl ester and ether, benzyloxycarbonyl (Z), benzyl ester and ether, 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulphonyl (Pbf), p-toluenesulphonyl (Tos) and the like.
  • acid-labile solid supports are regarded for present purposes essentially as polymeric acid-labile protecting groups.
  • labile refers to the relative ease of removing an acid-labile protecting group.
  • protecting group refers to a chemical group that is used to block temporarily a reactive site in a compound. A protecting group must be removable under specific conditions. Exemplary protecting groups include, without limitation, trityl (Trt), f-butoxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl (Fmoc), 2,4-dinitrophenyl (Dnp), p- toluenesulphonyl (Tos) and the like. Examples of the protecting groups that are not removed by the method of the invention include, without limitation, 9-fluorenylmethoxycarbonyl group (Fmoc), ethyl ester and the like.
  • acid-labile solid support refers to solid supports that incorporate acid-labile linkers.
  • acid-labile solid supports include, without limitation, Wang resin, SASRIN® resin, o-chlorotrityl (Barlos) resin, Rink amide resin, Sieber resin and the like.
  • acid-labile linker refers to a chemical group that links a compound to a solid support and is cleavable by acid to detach said compound from said solid support.
  • acid-labile linkers include, without limitation, p-benzyloxybenzyl ester (Wang linker), p- hydroxymethylphenoxyacetyl ester (HMPA linker), 2,4-dialkoxybenzyl ester (SASRIN® linker), trialkoxybenzhydrylamide (Rink or Knorr linker), trialkoxybenzylamide (PAL® linker), alkoxy-xanthenyl amide (Sieber linker) and the like.
  • solid support refers to a polymeric solid support used in solid- phase synthesis such as solid-phase peptide synthesis.
  • Exemplary solid supports include, without limitation, polystyrene resins such as Wang resin, polystyrene-polyethylene glycol grafted resins such as PEG-PS® or TentaGel® resins, polyacrylamide resins such as PEGA® resin, or polyethylene glycol resins such as NovaPEG® resin.
  • solid support as used herein also refers to non-resin types of solid supports used, for example, in combinatorial chemistry including, without limitation, multipin systems: SynPhaseTM crowns, SynPhaseTM lanterns [Parsons, J. G.; Sheehan, C. S.; Wu, Z.; James, I. W.; Bray, A. M. Methods Enzymol. 2003, 369, 39 and the references therein] and the like.
  • protected compound refers to an organic compound, which incorporates one or more acid-labile protecting groups. It is possible for a protected compound to have several acid-labile protecting groups of different type simultaneously.
  • deprotected compound refers to a compound from which one or more acid-labile protecting groups have been removed. Note that a deprotected compound within the scope of this invention may still retain other protecting groups, which are generally undisturbed by the method of this invention.
  • partially protected compound refers to a compound that retains some protecting groups.
  • scavenger refers to a compound that is used to avoid or minimise side-reactions.
  • exemplary scavengers include, without limitation, water, thioanisole (TA), dimethylsulphide (DMS), ethylmethylsulphide, tetrahydrothiophene, 1 ,2-ethanedithiol, 1 ,3- propanedithiol, benzylmercaptan, thiophenol, anisole, phenol, m-cresol, indole, pentamethylbenzene, triethylsilane, triisopropylsilane and the like.
  • ambient temperature refers to temperatures in the range of 16- 28°C, more preferably in the range of 20-25°C inclusive.
  • Example 2 The experiments were carried out essentially as described in Example 1 . Two milligrams of the amino acid derivative were weighed into a screw-cap glass vial (1 cm 3 ), and 1 cm 3 of either 0.1 N HCI in HFIP or 1 N HCI in HFIP or 0.1 N HCI in HFIP in the presence of extra solvent was added. The nature and percentage of a solvent is indicated below. The solution was left standing for the period of time indicated below and then analysed by RP- HPLC.
  • hydrogen-bonding solvents such as dimethylformamide (DMF) or /V-methylpyrrolidone-2 (NMP) had a markedly negative effect on the rate of Pbf deprotection.
  • An important feature of the method of the present invention is a markedly suppressive effect of even small quantities of hydrogen-bonding solvents on the rate of deprotection of the acid-labile protecting groups.
  • a presence of just 5% (v/v) of isopropanol /-PrOH has led to a noticeable slowdown of Pbf removal by 0.1 N HCI in HFIP (Fig. 3.4, grey) while the increase in the percentage of the solvent to 10% (v/v) and higher has resulted in a complete suppression of the deprotection (Fig. 3.4, black).
  • Example 2 The experiments were carried out essentially as described in Example 1. Two milligrams of the amino acid derivative were weighed into a screw-cap glass vial (1 cm 3 ), and 1 cm 3 of either neat HFIP or 0.1 N HCI in HFIP or 0.1 N HCI in HFIP with 1 % (v/v) triisopropylsilane was added. The solution was left standing for the period of time indicated below and then analysed by RP-HPLC. The cysteine derivative was not completely soluble in HFIP at 2 mg cm The yellow colour of the trityl cation appeared instantly when HFIP was added to solid Fmoc-Cys(Trt)-OH.
  • Example 1 1 .3 Removal of the i-butyl ether group from Fmoc-Tyr(tBu)-OH by Amberlyst 15/HFIP The experiment was carried out as described in Example 8.3. Fmoc- Tyr(tBu)-OH was deprotected rapidly and quantitatively by Amberlyst® 15 resin H form in HFIP within 30 min at ambient temperature (Fig. 1 1 .3). Notably, the resin-mediated deprotection in HFIP has shown high sensitivity to the nature and electronic environment of the protected group. The order of lability is the same as in the case of a thermal deprotection of the f-butyl ether by HFIP alone: Tyr > Thr > Ser.
  • Example 2 The experiments were carried out essentially as described in Example 1. Two milligrams of the amino acid derivative were weighed into a screw-cap glass vial (1 cm 3 ), and 1 cm 3 of either 0.1 N HCI in HFIP or 1 N HCI in HFIP or 0.1 M p-toluenesulphonic acid (p-TSA) in TFE was added. The solution was left standing for the period of time indicated below and then analysed by RP-HPLC. Deprotection of the benzyloxycarbonyl group (Z) from the ⁇ - nitrogen of Lys was monitored by disappearance of the peak of the starting amino acid H-Lys(Z)-OH at either 0.1 N or 1 N HCI in HFIP.
  • p-TSA p-toluenesulphonic acid
  • the benzyl ester group of Fmoc-Glu(OBzl)-OH was relatively resistant to 1 N HCI in HFIP in the absence of any scavenger, the conditions that induce fast removal of the benzyloxycarbonyl group (Z) from Lys (Example 13). Less than 10% Bzl ester deprotection was detected after 4 h 10 min (Fig. 15.1.1 ).
  • p-TSA p-toluenesulphonic acid
  • benzyl esters and ethers are not affected by the conditions of rapid Boc deprotection: 0.1 M concentration of the acid, 10-15% (v/v) of TFE in DCM, 5-10 min of reaction.
  • benzyl ester group of Fmoc-Glu(OBzl)-OH is stable to 0.1 M p-TSA in TFE for at least 2 h at ambient temperature (Fig. 15.2, black).
  • the same ester could be removed at a higher concentration of the acid (1 M) in HFIP (Fig. 15.2, grey).
  • Example 8.3 The experiment was carried out essentially as described in Example 8.3 except that 1 % (v/v) of dimethylsulphide was added. Likewise, the benzyl ester of Fmoc-Glu(OBzl)-OH was resistant to Amberlyst® 15 resin H form in HFIP in the presence of 1 % (v/v) of dimethylsulphide for at least 2 h at ambient temperature (Fig. 15.3).
  • Example 14 The experiment was carried out essentially as described in Example 15.2 except that only 0.1 M p-TSA in TFE - DCM (1 :9 v/v) was used. Similarly, the benzyl ether of Fmoc-Thr(Bzl)-OH was resistant to 0.1 M p-TSA in TFE - DCM (1 :9 v/v) in the absence of a scavenger for at least 4 h 30 min at ambient temperature (Fig. 16.2). Under the same conditions the Boc group is removed within 5 min (Example 14).
  • the tosyl (p-toluenesulphonyl) group on the guanidino function of Arg was not significantly affected by either 1 N HCI/HFIP + 1 % (v/v) thioanisole (TA) for 2.5 h (Fig. 17.1 , grey) or 1 M chlorotrimethylsilane (TMSCI) in HFIP for 12 h (Fig. 17.2).
  • TMSCI chlorotrimethylsilane
  • the latter reagent at 1 M concentration in a mixture with 3M phenol and DCM has been proposed for the removal of the Boc group in SPPS [Kaiser Sr, E.; Tarn, J. P.; Kubiak, T. M.; Merrifield, R. B. Tetrahedron Lett. 1988, 29, 303; Kaiser Sr, E.; Picart, F.; Kubiak, T.; Tarn, J. P.; Merrifield, R. B. J. Org. Chem. 1993, 58, 5167].
  • Example 18 Cleavage of common resin linkers by 0.1 N HCI in the presence or absence of HFIP and a scavenger dimethylsulphide (DMS)
  • a sample of an appropriate resin 100 mg was weighed into a 1.5 cm 3 Eppendorf polypropylene tube.
  • a 1 cm 3 of a deprotection mixture consisting of 0.1 N HCI in an appropriate solvent or a mixture of solvents with or without a scavenger dimethylsulphide was added, and the reaction vessel was left standing with occasional agitation for 3 h at ambient temperature. Aliquots of the resin (ca.
  • Fmoc-Leu-Wang resin (Novabiochem®) with the initial loading of 0.958 mmol g "1 was treated with a mixture of 10 ⁇ concentrated (ca. 37% w/v) aqueous HCI, 200 ⁇ HFIP and 790 ⁇ DCM. After 3 h of reaction, the extent of cleavage reached over 91 %.
  • a peptide Fmoc-Lys-Thr-Thr-Lys-Ser-OH 19.1 was prepared by the Fmoc SPPS as described [Amblard, M.; Fehrentz, J-A.; Martinez, J.; Subra, G. Mol. Biotechnol.
  • HATU 2-(7-Aza-1 /-/-benzotriazol-1 -yl)-1 , 1 ,3,3- tetramethyluronium hexafluorophosphate
  • DIEA N,N- diisopropylethylamine
  • NMP 1 -methylpyrrolidin-2-one
  • the resin was agitated by a gentle stream of nitrogen for 1 h, then washed with NMP (25 cm 3 ) and DMF (50 cm 3 ), and the deprotection-coupling cycle was repeated until the last amino acid (Lys) was incorporated. After that the resin was washed with methanol (50 cm 3 ), dichloromethane (50 cm 3 ) and diethyl ether (25 cm 3 ) and dried in vacuo. A sample of pre-dried resin (0.6 g) was swollen in 0.1 N HCI/HFIP (5 cm 3 ) and left to cleave for 15 min at ambient temperature, washed twice by 0.1 N HCI/HFIP (5 cm 3 ), and the washings (ca.
  • Palmitoyl-KTTKS ESI HR-MS calc. [M+H] + for C 39 H 76 N 7 Oi 0 802.5654 Da, obs. 802.5673 Da.
  • Linoleyl-KTTKS MALDI-TOF calc. [M+H] + for C 4 i H 76 N 7 Oi 0 826.56 Da, obs. 826.16 Da.

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Abstract

La présente invention concerne un procédé pour éliminer des groupes protecteurs labiles en milieu acide d'un composé protégé qui comporte un ou plusieurs groupes protecteurs labiles en milieu acide, ledit procédé comprenant : a. la dissolution ou la dispersion du composé protégé, ou l'immersion d'un support solide auquel est fixé le composé protégé, dans un mélange comprenant i) un alcool fluoré, ii) un acide et, éventuellement, iii) un solvant organique et/ou un capteur ; et b. le maintien de la solution ou de la dispersion résultante, ou la conservation du support solide immergé, pendant une durée suffisante pour assurer l'élimination d'un ou plusieurs groupes protecteurs labiles en milieu acide dudit composé protégé, produisant ainsi un composé déprotégé ou partiellement déprotégé. Le composé protégé peut être, par exemple, un peptide. L'invention concerne également une composition destinée à être utilisée avec un tel procédé, ladite composition comprenant i) un alcool fluoré ; ii) un acide et, éventuellement, iii) un solvant organique et/ou un capteur.
PCT/GB2013/052280 2012-08-31 2013-08-30 Procédé et compositions pour l'élimination de groupes protecteurs labiles en milieu acide WO2014033466A1 (fr)

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US11492369B2 (en) 2017-12-15 2022-11-08 Chugai Seiyaku Kabushiki Kaisha Method for producing peptide, and method for processing bases
US11542299B2 (en) 2017-06-09 2023-01-03 Chugai Seiyaku Kabushiki Kaisha Method for synthesizing peptide containing N-substituted amino acid
US11891457B2 (en) 2011-12-28 2024-02-06 Chugai Seiyaku Kabushiki Kaisha Peptide-compound cyclization method

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