WO1999031124A1 - Echafaudages a base d'acide cholique pour la presentation moleculaire multidimensionnelle de peptides - Google Patents

Echafaudages a base d'acide cholique pour la presentation moleculaire multidimensionnelle de peptides Download PDF

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WO1999031124A1
WO1999031124A1 PCT/DK1998/000547 DK9800547W WO9931124A1 WO 1999031124 A1 WO1999031124 A1 WO 1999031124A1 DK 9800547 W DK9800547 W DK 9800547W WO 9931124 A1 WO9931124 A1 WO 9931124A1
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acid
peptide
amino
substituted
scaffold according
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PCT/DK1998/000547
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English (en)
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Thomas HØEG-JENSEN
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Novo Nordisk A/S
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Priority to AU15560/99A priority Critical patent/AU1556099A/en
Publication of WO1999031124A1 publication Critical patent/WO1999031124A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • 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
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • 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 present invention relates to the field of solid-phase chemistry and to the field of peptide mimicry.
  • the invention provides a method for solid-phase and combinatorial synthesis of organic compounds, namely scaffolds derived from cholic acid, substituted with peptide chains, peptoid chains and/or pseudo-peptide chains.
  • the invented molecules are potential mimetics of larger peptides.
  • Peptides constitute an important class of biologically active molecules. Peptides are present in all living organisms, where they are necessary for maintaining life functions. Several human disease states have been linked to lacking or decreased secretion or efficacy of peptide hormones in the patient. In some cases these states can be corrected by administration of peptides as drugs. Unfortunately, peptides are digested in the gut, and it is therefore necessary to give peptide drugs by injection. This situation has sparked an interest in peptide mimetics.
  • a peptide mimetic is an organic molecule, which can assume the role of a peptide, but which is non-peptidic and therefore potentially orally active. Mimetics of many peptides have been discovered, either by rational molecular design and by screening of large collections of random molecules.
  • Mimetics can be subdivided into two groups, true mimetics and functional mimetics.
  • a true peptide mimetic binds to the target in the same manner as the native peptide, thereby triggering the response. The effect of a true mimetic might thus be rationalised at the molecular level.
  • a functional mimetic triggers the signal without binding natively. Often the molecular mechanisms behind functional mimetics are not understood.
  • peptide mimetics are found in the group of smaller peptides, typically containing less then 10-20 amino acids (AA's).
  • AA's amino acids
  • a true mimetics may need to be of larger size to fill the native pocket of a given receptor. Since design and synthesis of artificial molecules generally gets more complicated as molecular size increases, this makes it generally more difficult to find mimetics of larger peptides.
  • typical compounds in libraries used for random screening are relatively small, molecular weight may typically be in the range of 200 to 800. In some cases, only smaller areas of a larger peptide are important for the biological function. In such cases it may be possible to mimic larger peptides using much smaller molecules. Experience show however that this is often not the case.
  • Insulin 51 AA's has so far no true mimetic, despite long-standing search.
  • hGH and EPO are other examples of large peptides with no small mimetic equivalent. Common for these long-chain peptides, they fold to form a compact, globular structure. Conclusively, there is a need for easy access to organic molecules, larger than those molecules that typically enter screening programs.
  • scaffold which is diversified by attaching various fragments, also called monomers.
  • the desired properties of a scaffold may be suitable rigidity and suitably spaced reactive sites, for coupling of monomers.
  • the synthetic sequence disclosed in this invention provides a scaffold for molecular presentation of up to 4 peptide chains, pseudo-peptide chains and/or peptoid chains in a compact, globular-like structure around the steroid nucleus of cholic acid.
  • the size of the resulting molecules are from around 1000 and up.
  • the synthetic technique is well-suited for solid-phase synthesis, thereby providing a route to large collection of compounds, either as mixtures or as discretes. Since the invented molecules may contain peptide chains, they may not always be orally active in themselves. If this is the case, they may however serve as leads for development of orally active mimetics.
  • Resin refers to any insoluble or partially insoluble material, to which compounds may be covalently attached.
  • Resins may be selected from the group consisting of any kind of organic or inorganic polymeric or oligomeric compound, e.g. polystyrene with different grades of crosslinking, polyethylene glycol (PEG), polyethylene glycol attached to polystyrene (e.g. TentaGel), polyacrylamides, poiyacrylates, polyurethanes, polycarbonates, polyamides, polysaccharides or silicates.
  • Linker A molecule with at least two reactive sites, which permit its covalent attachment to other molecules or to a resin.
  • Either the bond of the linker to the resin or the bond of the linker to other molecules attached to it or the linker itself must be cleavable upon selective exposure to an activator such as a selected chemical activator or other specific conditions, e.g. by treatment with a strong acid or by exposure to electromagnetic radiation or by metal catalysis.
  • an activator such as a selected chemical activator or other specific conditions, e.g. by treatment with a strong acid or by exposure to electromagnetic radiation or by metal catalysis.
  • Array A collection of N single compounds or N mixtures of compounds with a common structural element, synthesised simultaneously in a parallel fashion using the same synthetic reaction sequence.
  • the precise structure of a single compound within an array of compounds or the components of a mixture within an array of mixtures is determined by the sequence of reactants which gave rise to this compound or mixture and can be deduced from the recorded reaction-protocol. The spatial arrangement of the array is irrelevant.
  • Scaffold A central molecule, to which variant, diversifying monomers are attached.
  • Monomer A molecular fragment, which is used in combination with other monomers for diversifying a scaffold.
  • a protecting group is a material, which is chemically bound to a molecule or a substrate and which may be removed upon exposure to an activator such as a selected chemical activator or other specific conditions, e.g. by treatment with a strong acid or by exposure to electromagnetic radiation or by metal catalysis. Orthogonal protecting groups can be removed selectively in any order.
  • Combinatorial synthesis An ordered strategy for parallel synthesis of arrays of single compounds or of mixtures, by sequential addition of reagents.
  • Mix-and-split combinatorial synthesis provides bead libraries where each bead contain only one structure. When peptides are involved, identification of a single compound on a single bead can be performed by use of amino acid sequence analysis.
  • Mix-and-split A combinatorial synthetic technique where the resin is divided into separate portions before each step of monomer coupling. After each coupling step, the resin portions are pooled and mixed (Furka et al., Int. J. Peptide Protein Res. 1991 , 37, 1991). This procedure yields mixtures of compounds, where each resin bead in principle contain only one structure (Lam et al., Nature 1991 , 354, 82).
  • Receptor A material that has an affinity for a given ligand.
  • Receptors may be naturally- occurring or synthetic molecules or aggregates of molecules. Also, they can be employed in their unaltered state or as aggregates with other species.
  • Receptors may be attached, covalentiy or non-covalently, to a binding material or a substrate, either directly or via a linking substance.
  • receptors which can be employed by this invention include, but are not restricted to, antibodies, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as viruses, cells or other materials), cell membrane receptors, drugs, oligonucleotides, polynucleotides, nucleic acids, peptides, cofactors, small organic molecules, lectins, sugars, oligosaccharides, cells, cellular membranes, organelles, microorganism receptors, enzymes, catalytic polypeptides, hormone receptors, primary metabolite receptors such as carbohydrate receptors, nucleotide receptors or lipid receptors and secondary metabolite receptors such as opiate receptors, prostaglandine receptors, etc.
  • specific antigenic determinants such as viruses, cells or other materials
  • cell membrane receptors drugs, oligonucleotides, polynucleotides, nucleic acids, peptides, cofactors, small organic molecules, lectins, sugar
  • Mimetic An organic molecule that simulates the structure or function of a biologically active compound, such as a peptide.
  • a true mimetic binds to the target in the same manner as the native compound, thereby triggering the response. The effect of a true mimetic can thus be rationalised at the molecular level.
  • a functional mimetic triggers the signal without binding natively. Often the molecular mechanisms behind functional mimetics are not understood.
  • Peptide structure A molecular structure, where amino acid monomers are assembled in chains by amide bonds.
  • the monomers may be of both natural and unnatural origin, e.g. L-amino acids and D-amino acids, beta-amino acids or other compounds containing both an amine and a carboxylic acid.
  • Pseudo-peptide structure A structure is pseudo-peptide when one or more amide bonds in a peptide chain are substituted with other bonds.
  • amide bond substitutes are thioamides, N-methyl amides and methylene amines.
  • Peptoid structure In peptoid structures, amino acid side chains are placed on the amino group, rather then the ⁇ -carbon. Peptoids can be build-up by alternating couplings of bromoacetic acid and primary amines (Zuckermann et al., Proc. Natl. Acad. Sci. USA 1992, 89, 9367).
  • EPO erythropoietin
  • Fmoc fluorenylmethyloxycarbonyl hGH: human growth hormone
  • HOBt 1 -hydroxybenzotriazole Ms: methylsulfonyl
  • NBS N-bromsuccinimide
  • Phg phenylglycine R: organic radical
  • Fig. 1 SDS page gel, monitoring insulin receptor phosphorylation levels.
  • Entry 1 is compound A at 4x10-5 M with insulin 2.5 nM.
  • Entry 2 is compound B at 4x10-5 M with insulin 2.5 nM.
  • Entry 3 is compound A at 2x10-4 M with insulin 2.5 nM.
  • Entry 4 is compound B at 2x10-4 M with insulin 2.5 nM.
  • Entry 5 is blank.
  • Entry 6 is insulin 2.5 nM.
  • Track 7 is insulin 100 nM. Description of the invention
  • the present invention relates to substituted cholic acid-based scaffolds of formula 1
  • R1 , R2, R3 and R4 independently represent amino acids or chains of peptide, pseudo-peptide or peptoid structure containing from 2 to 10 monomers.
  • R1 , R2, R3 and R4 are either identical or discrete and may each consist homogeneously or heterogeneously of peptide, pseudo-peptide and peptoid monomers.
  • the orientation of substituent in positions 3, 7 and 12 are either ⁇ or ⁇ .
  • the position of the hydrogen in position 5 of the scaffold is either ⁇ or ⁇ .
  • the orientation of the substituent in position 3 is preferably ⁇ .
  • the orientation of the substituent in position 7 is preferably ⁇ .
  • the orientation of the substituent in position 12 is preferably ⁇ .
  • the orientation of the hydrogen in position 5 is preferably ⁇ .
  • R1 represents preferably an amino acid or a chain of peptide, pseudo-peptide or peptoid structure containing from 2 to 6 monomers, more preferably 3 or 4 monomers.
  • R2 represents preferably an amino acid or a chain of peptide, pseudo-peptide or peptoid structure containing from 2 to 6 monomers, more preferably 3 or 4 monomers.
  • R3 represents preferably an amino acid or a chain of peptide, pseudo-peptide or peptoid structure containing from 2 to 6 monomers, more preferably 3 or 4 monomers.
  • R4 represents preferably an amino acid or a chain of peptide, pseudo-peptide or peptoid structure containing from 2 to 6 monomers, more preferably 3 or 4 monomers.
  • R1 , R2, R3 and R4 each independently represent an amino acid or a peptide chain.
  • Monomers in the peptide chains are preferably, in either L- or D-form where relevant: glycine, alanine, valine, leucine, isoleucine, methionine, threonine, serine, cysteine, histidine, phenylalanine, tyrosine, tryptophane, lysine, arginine, aspartic acid, asparagine, glutamic acid, glutamine, proline, 4-hydroxyproline, aminobutyric acid, norvaline, norleucine, 3- pyridinealanine, cyclohexylalanine, phenylglycin, tetrahydroisoquinoline-3-carboxylic acid, biphenylalanine, 1-naphthylalanine, 2-naphthylalanine, anthranilic acid, 3-aminobenzoic acid, 4-aminobenzoic, 4-aminobutyric acid, beta-alanine, 3-amin
  • Monomers in chains of pseudo-peptide structure are amide bond substituted derivatives of the monomers described for peptide chains above.
  • the amide bond substitutions are preferably thioamide, N-methyl amide or methylene amine.
  • Monomers in the chains of peptoids structure are preferably: phenethylamine, tryptamine, benzylamine, 2,3-dimetoxybenzylamine, 3,5-dimethoxybenzylamine, 4-
  • the substituted scaffolds according to the present invention preferably have a molecular weight in the range of 800 to 5000, more preferably 1000 to 2500.
  • the invention is furthermore concerned with a collection of N discrete, non-identical substituted scaffolds according to the invention, where N is in the range of 5 to 1000, preferably 10 to 500.
  • the invention is also concerned with a mixture containing N non-identical substituted scaffolds according to the invention, where N is in the range of 10 2 to 10 9 , preferably 10 3 to 10 5 .
  • Mixtures of substituted scaffolds can be screened towards a receptor either while still attached to resin beads or in solution.
  • Collections and mixtures of a large number of the compounds of the invention may be used to identify compounds which act as peptide mimetics. Since the invented molecules may contain peptide chains, they may not always be orally active in themselves. If this is not the case, they may however serve as leads for development of orally active mimetics.
  • the present invention furthermore relates to a method for identifying lead compounds for the development of mimetics of a biologically active peptide, comprising:
  • N is in the range of 5 to 1000, preferably 10 to 500
  • the invention also relates to a method for identifying lead compounds for the development of mimetics of a biologically active peptide, comprising:
  • N is in the range of 10 2 to 10 9 , preferably 10 3 to 10 5 .
  • Detection of binding in step c) may be done by using a conjugate of the receptor with an enzyme that catalysises precipitation of a dye, e.g. a receptor/alkaline phosphate conjugate, which will precipitate 5-bromo-4-chloro-3-indolyl phosphate to give a torquoise color.
  • a dye e.g. a receptor/alkaline phosphate conjugate
  • Other dyes may be used, such as nitro blue tetrazolium and p-iodonitrotetrazolium (Lam et al., Immunomethods 1992, 1 , 11). The color will precipitate only on beads holding a molecule that bind the conjugate.
  • Active beads are isolated from the mixture, and the beads are analysed to reveal the active structure (Lam et al., Nature 1991 , 354, 82).
  • the invention is furthermore concerned with a method for using cholic acid as a scaffold, with 3 orthogonally protected amino acids attached to cholic acid at positions 3, 7 and 12. These positions, along with the native carboxylic acid in cholic acid position 24, can be used as starting points for build-up of peptide chains, pseudo-peptide chains or peptoid chains so as to obtain a substituted scaffold according to the invention.
  • the 4 chains are presented in a compact, globular-like form.
  • the synthetic methodology is well-suited for us in solid-phase and combinatorial chemistry, and the molecules are amenable to high-throughput screening for peptide mimetics, for lead discovery and as drugs.
  • the scaffolds necessary in providing molecules of the invention are of a structure of formula 2
  • X1 , X2 and X3 independently represent either Alloc-AA-O-, Alloc-AA-N-, Boc-AA- 0-, Boc-AA-N-, Fmoc-AA-O-, or Fmoc-AA-N-.
  • AA represents any amino acid, in L- or D-form, e.g. glycine, alanine, valine, leucine, isoleucine, methionine, threonine, serine, cysteine, histidine, phenylalanine, tyrosine, tryptophane, lysine, arginine, aspartic acid, asparagine, glutamic acid, glutamine, proline, 4- hydroxyproline, aminobutyric acid, norvaline, norleucine, 3-pyridinealanine, cyclohexyla- lanine, phenylglycin, tetrahydroisoquinoline-3-carboxylic acid, biphenylalanine, 1- naphthylalanine, 2-naphthylalanine, anthranilic acid, 3-aminobenzoic acid, 4-aminobenzoic, 4-aminobutyric acid, beta-alanine, 3-amino-1
  • X4 represent OH, for a carboxylic acid, or OR, for an active ester, such as N-hydroxy- succinimidyl, pentafluorophenyl or equivalent.
  • Carbon atoms in scaffold positions 3, 7 and 12 are substituted with either an alcohol or an amine, thereby providing attachment points for peptide, pseudo-peptide or peptoid chains, either as esters or as amides.
  • the carboxylic acid in position 24 allows attachment of a peptide, pseudo-peptide or peptoid chain as an amide.
  • the build-up of scaffolds according to the invention is exemplified by the following synthetic sequence (scheme 1):
  • the carboxylic acid of cholic acid is tert-butylated. This is done by treatment with trifluoroacetic anhydride, followed by reaction with tert-butanol and ammonia-promoted removal of trifluoroacetates from the steroid hydroxy groups protected (Bonar-Law et al., J. Chem. Soc. Perkin Trans. 1 1990, 2245).
  • the hydroxy group in position 7 is next selectively oxidized to the keton by treatment with N-bromsuccinimide in acetone/water (Fieser et al., J. Am. Chem. Soc.
  • the NaBH 4 reduction give selectively a hydroxy group with ⁇ -orientation.
  • Treatment with methyl sulfonyl chloride in DCM/pyridine yields the 7-mesylate, which can be reacted with sodium azide in DMF, to give the ⁇ -azide by S N 2 chemistry (Davis et al., Tetrahedron Lett. 1992, 33, 5111).
  • the azide is reduced to the amine with Ph 3 P in THF/water, and the amine is acylated with Fmoc-glycine.
  • Treatment with TFA in DCM cleaves the tert-butyl group at position 24 as well as the Boc-group from glycine at position 12.
  • the amino group of 12-glycine is Boc- reprotected by treatment with Boc-anhydride and DIEA in THF. This completes the synthesis of the scaffold.
  • the Boc-protecting groups are removed selectively by TFA-treatment and peptides are build up from the deprotected sites.
  • This branch is terminated with a Boc-amino acid.
  • the next branch is build up after selective removal of either the Fmoc- or the Alloc-protecting group (Kates et al., Anal. Biochem. 1993, 212, 303).
  • This branch is likewise terminated with a Boc-group.
  • the final branch is build up similarly.
  • the Alloc- branch can be made from Fmoc-amino acids upon initial Alloc cleavage. This is advantageous, since more Fmoc-AA's are commercially available, and since the Fmoc- deprotection procedure is simpler than the Alloc-deprotection procedure.
  • the completed resin-bound molecules are Boc-deprotected and submitted for screening. Binding activity towards a conjugate of receptor/alkaline phosphatase will lead to dye precipitation on the active bead. 5-bromo-4-chloro-3-indolyl phosphate give a torquoise color.
  • Single beads can be analyzed by standard Edman sequence analysis (Edman et al., Eur. J. Biochem. 1968, 1 , 80). When seperate packages of monomers are used on each of the 3 final branches (cholic acid positions 3, 7 and 12), the sequence analysis will unambiguously give the identity of the whole scaffolded structure.
  • Tert-butyl cholate (3) is prepared according to Bonar-Law et al.: J. Chem. Soc. 1990, 2245.
  • BocGly (21.2 g, 121 mmol) in AcOEt (70 ml) is cooled with ice and treated with dicyclohexylcarbodiimide (12.5 g, 60.6 mmol) to give (BocGly) 2 0.
  • 5 (18.3 g, 30.3 mmol) and DMAP (0.74 g, 6.06 mmol) are added and the mixture is refluxed for 1.5 h.
  • PD-MS 783 (M+Na), 762 (M+H), 706 (M-tBu).
  • PD-MS 785 (M+Na), 764 (M+H), 708 (M+H-tBu), 663 (M+H-Boc).
  • PD-MS 1064 (M+Na), 944 (M-Boc), 889 (M-tBu-Boc).
  • Amino-PEG-PS resin (130 ⁇ beads) is split in 8 portions, each coupled to either FmocAla, Cha, Gin, Leu, Phe, Pro, Trp or Val, by DIC/HOBT activation. The portions are then pooled and well mixed. The pool is deprotected with 20% piperidine/DMF and split in 8 new portions. The synthesis is continued as mix-and-split to the tetra-peptide stage.
  • the 8 individual resin portions are treated with Boc 2 0 (0.1 equiv.).
  • the scaffold (2 equiv.) is then added by DIC/HOBt chemistry on the resin pool.
  • the first scaffold branch is build-up combinatorially by using Boc-chemistry, with amino acids Pro, Trp, Gin and Leu. Deprotection are done with 50% TFA in DCM. The peptide on this branch is taken to the tripeptide stage, with the Boc-group remaining on the last AA.
  • the Alloc branch is next deprotected by (Ph 3 P) 4 Pd catalysis in CHCI 3 /AcOH/NMM (92.5/5/2.5, Kates et al., Anal. Biochem. 1993, 212, 303) and the synthesis is continued with an Alloc-amino acid (Asn, Phe, Met or Val). The branch is ended with a Boc-amino acid (Asn, Phe, Met or Val).
  • the Fmoc branch is likewise started with an Fmoc-amino acid (lie, Phg, Cha or Ala), and ended with a Boc-amino acid (He, Phg, Cha or Ala). Fmoc- deprotections are done with 20% piperidine in DMF.
  • the completed library is deprotected with TFA, and screened against a soluble receptor, which is coupled to alkaline phosphatase. Resin beads which bind to the receptor are stained by enzymatic reaction with a dye (5-bromo-4-chloro-3-indolyl phosphate). Sequence analysis on a single bead reveals the identity of the given compound.
  • Amino-PEG-PS resin (130 ⁇ beads) is split in 8 portions, each coupled to either FmocAla, Gin, Met, Trp or Leu by DIC/HOBT activation. The portions are then pooled and well mixed. The pool is deprotected with 20% piperidine/DMF and split in 5 new portions. This procedure is continued combinatorially to the penta-peptide stage.
  • the 5 individual resin portions are treated with Boc 2 0 (0.1 equiv.).
  • the scaffold (2 equiv.) is then added by DIC/HOBt chemistry on the resin pool.
  • the first scaffold branch is build-up combinatorially by using Boc-chemistry, with amino acids Asn, Pro, Phg and lie, to the tetrapeptide stage.
  • the named four monomers are supplemented with Boc-Glu(tBu).
  • the Fmoc branch is next deprotected, and the synthesis is continued with Fmoc-amino acids Cha, Lys(Boc), Phe, Nal-2 and Thr.
  • the branch is ended at the pentapeptide stage with a Boc- amino acid (Cha, Lys(Boc), Phe, Nal-2 or Thr).
  • the Alloc branch is deprotected and buildup from Fmoc-amino acids (Val, Tyr, Gly, Asp and Ser).
  • Scaffolded library example 3. including hit evaluation and synthesis of single compounds.
  • Amino-PEG-PS resin (130 ⁇ beads) is split in 5 portions, each coupled to either FmocAla, Gin, Met, Trp or Leu by DIC/HOBT activation. The portions are then pooled and well mixed. The pool is deprotected with 20% piperidine/DMF and split in 5 new portions. This procedure is continued combinatorially to the dipeptide stage.
  • the 5 individual resin portions are treated with BocVal (0.1 equiv.) for capping.
  • the scaffold (2 equiv.) is then added by DIC/HOBt chemistry on the resin pool.
  • the first scaffold branch is build-up combinatorially by using Boc-chemistry, with amino acids Asn, Pro, Phg and lie, to the tripeptide stage (also counting the amino acid build into the scaffold).
  • Boc-Glu(tBu) For the last amino acid on this branch, the named four monomers are supplemented with Boc-Glu(tBu).
  • the Fmoc branch is next deprotected, and the synthesis is continued with Fmoc-amino acids Cha, Lys(Boc), Phe, Nal-2 and Ser(tBu).
  • the branch is ended at the tripeptide stage with a Boc-amino acid (Cha, Lys(Boc), Phe, Nal-2 or Thr(tBu).
  • the Alloc branch is deprotected and build-up from Fmoc-amino acids (Val, Tyr(tBu), Gly, Asp(tBu) and Ser(tBu)).
  • the synthesis of this branch is ended at the tripeptide stage, with Boc-AA's (Val, Tyr, Gly, Asp and Ser).
  • the completed library is deprotected with 95% TFA, and screened against solubilized insulin receptor conjugated to alkaline phosphatase.
  • Resin beads which bind to the receptor are stained by enzymatic reaction with a dye. In the present example only about 10 beads are colored, some more than others.
  • the 4 most intensely colored beads are isolated and subjected to sequence analysis with the following results (notated as sequenced, from the N terminal):
  • Beadl lie Lys Gly Phe Phg Thr
  • Bead3 lie Lys Gly
  • Bead4 lie Lys Leu

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Abstract

L'invention concerne un procédé en phase solide permettant d'effectuer la synthèse entre plusieurs échafaudages à base d'acide cholique à substitution différente et un large éventail de substituants à chaîne peptidique, pseudo-peptidique ou peptoïde, constituant des produits initiaux pour l'obtention de composés qui peuvent présenter un intérêt thérapeutique. Les acides choliques substitués sont préparés par une synthèse initiale en solution d'un échafaudage soutenant des acides aminés avec trois groupes protecteurs orthogonaux. Ensuite, la synthèse en phase solide débouche sur la construction des molécules cibles, sous forme discrète ou en combinaison. Il est possible de cribler les molécules sur la résine ou d'en obtenir le clivage à partir de la résine et de les cribler ensuite en solution. Le procédé décrit permet ainsi d'accéder facilement et rapidement à des composés extrêmement divers, à des structures peptidomimétiques potentielles et à des produits initiaux potentiels pour l'obtention de composés qui présentent un intérêt thérapeutique. Le procédé en question se prête à l'automatisation.
PCT/DK1998/000547 1997-12-12 1998-12-11 Echafaudages a base d'acide cholique pour la presentation moleculaire multidimensionnelle de peptides WO1999031124A1 (fr)

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Application Number Priority Date Filing Date Title
AU15560/99A AU1556099A (en) 1997-12-12 1998-12-11 Cholic acid-based scaffolds for multidimensional molecular presentation of peptides

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DK145097 1997-12-12
DK1450/97 1997-12-12
US6807397P 1997-12-18 1997-12-18
US60/068,073 1997-12-18

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WO1999031124A1 true WO1999031124A1 (fr) 1999-06-24

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US6350738B1 (en) 1998-03-06 2002-02-26 Brigham Young University Steroid derived antibiotics
EP1311531A1 (fr) * 2000-08-15 2003-05-21 Brigham Young University Antibiotiques derives de steroides
US6767904B2 (en) 1998-03-06 2004-07-27 Bringham Young University Steroid derived antibiotics
CN101448512A (zh) * 2005-12-14 2009-06-03 Ambrx公司 含有非天然氨基酸和多肽的组合物、涉及非天然氨基酸和多肽的方法以及非天然氨基酸和多肽的用途
CN106366193A (zh) * 2016-08-25 2017-02-01 广东工业大学 一种以胆酸为原料制备甘氨胆酸多克隆抗体的方法

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6350738B1 (en) 1998-03-06 2002-02-26 Brigham Young University Steroid derived antibiotics
US6486148B2 (en) 1998-03-06 2002-11-26 Brigham Young University Steroid derived antibiotics
US6767904B2 (en) 1998-03-06 2004-07-27 Bringham Young University Steroid derived antibiotics
US7598234B2 (en) 1998-03-06 2009-10-06 Brigham Young University Steroid derived antibiotics
EP1311531A1 (fr) * 2000-08-15 2003-05-21 Brigham Young University Antibiotiques derives de steroides
JP2004506645A (ja) * 2000-08-15 2004-03-04 ブリガム　ヤング　ユニバーシティ ステロイド由来抗生物質
EP1311531A4 (fr) * 2000-08-15 2009-10-28 Univ Brigham Young Antibiotiques derives de steroides
CN101448512A (zh) * 2005-12-14 2009-06-03 Ambrx公司 含有非天然氨基酸和多肽的组合物、涉及非天然氨基酸和多肽的方法以及非天然氨基酸和多肽的用途
CN101448512B (zh) * 2005-12-14 2015-11-25 Ambrx公司 含有非天然氨基酸和多肽的组合物、涉及非天然氨基酸和多肽的方法以及非天然氨基酸和多肽的用途
CN105384807A (zh) * 2005-12-14 2016-03-09 Ambrx公司 含有非天然氨基酸和多肽的组合物、涉及非天然氨基酸和多肽的方法以及非天然氨基酸和多肽的用途
CN106366193A (zh) * 2016-08-25 2017-02-01 广东工业大学 一种以胆酸为原料制备甘氨胆酸多克隆抗体的方法

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