WO2006018277A1 - Procede de synthese combinatoire rationnelle - Google Patents

Procede de synthese combinatoire rationnelle Download PDF

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Publication number
WO2006018277A1
WO2006018277A1 PCT/EP2005/008885 EP2005008885W WO2006018277A1 WO 2006018277 A1 WO2006018277 A1 WO 2006018277A1 EP 2005008885 W EP2005008885 W EP 2005008885W WO 2006018277 A1 WO2006018277 A1 WO 2006018277A1
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Prior art keywords
building blocks
building
building block
group
beads
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PCT/EP2005/008885
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English (en)
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Jean-Louis Reymond
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Universität Bern
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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
    • C07K1/04—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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C40B50/16—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support involving encoding steps
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00592—Split-and-pool, mix-and-divide processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00731—Saccharides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00736—Non-biologic macromolecules, e.g. polymeric compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof

Definitions

  • Chemically synthesized molecules are used in many different products of daily life, including drugs, food additives, dyestuffs, polymers and plastics.
  • Synthetic chemistry allows one to synthesize molecules with almost any structure by stepwise synthesis.
  • search for a more efficient method the combinatorial synthesis method which allows one to prepare large numbers of molecules simultaneously was developed. Each of these molecules is then tested for its properties and identified. This process greatly speeds up the discovery of active molecules because many more molecules can be tested for a particular property than through conventional step-by-step synthesis.
  • the molecules being synthesized are attached to a solid support.
  • the solid support generally consists of small polymer beads.
  • Combinatorial libraries of compounds are obtained by growing the molecules by stepwise attachment of building blocks through chemical reactions. IfN different building blocks are used at each step in a sequence comprising S steps where variable building blocks can be attached, the resulting number of molecules is then N in conventional synthesis, but N s for a combinatorial split-and-mix synthesis (K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, R. J. Knapp, Nature 1991, 354, 82-84). The N s molecules are then tested for a particular property either directly on the solid support, or after cleavage from this support.
  • encoding methods which include binary encoding (M. H. J. Ohlmeyer, R. N. Swanson, L. W. Dillard, J. C. Reader, G. Asouline, R. Kobayashi, M. H. Wigler, W. C. Still, Proc. Natl. Acad.
  • Another method for identifying the structure of active molecules in a combinatorial library is to carefully choose the building blocks so that each molecule has a different molecular weight.
  • determination of the molecular weight of an active molecule by mass spectrometry (MS) allows one to know its structure (WO 97/08190). This MS determination is only possible if precise rules defining the exact molecular weights of the building blocks are used in the library, which limits the diversity of building blocks that can be used.
  • the invention describes a method for preparing combinatorial libraries, characterized in that beads are equally split in n portions, each portion reacted with one building block of a first group of n building blocks, reacted beads thoroughly mixed and equally split in m portions, each portion reacted with one building block of a second group of m building blocks, the steps of thoroughly mixing, equally splitting in portions and reacting with one building block of a further group of building blocks repeated for 1 to 20 times; the resulting beads with the sequence of building blocks, or the sequence of building blocks after cleavage from the beads, analyzed for desired properties and selected based on the desired properties; selected beads with the sequence of building blocks, or the selected cleaved sequence of building blocks, cleaved into individual building blocks, the amount and type of building blocks determined, and the sequence of building blocks deduced from the amount and type of building blocks determined; wherein one building block in a group of building blocks may be absent; and wherein the building blocks in each group of building blocks are so chosen as to allow deducing the sequence of building
  • the method is particular suitable for the synthesis of combinatorial libraries of dendrimers.
  • Such combinatorial libraries are also a subject of the invention.
  • Figure 1 The known combinatorial split-and-mix vs. the classical synthesis protocol
  • Step 1) Beads are split in 4 portions and reacted with building blocks Al - A4 in 4 different vials.
  • the compounds in the vials are again treated with building blocks Al - A4 in each of steps 2) and 3), giving 4 final products (right lane).
  • Figure 2 The combinatorial group split-and-mix of the invention Different building blocks from groups A, B or C are used at each of the three steps 1), 2) and 3). In the group protocol, building blocks A1-A4, then B1-B4 and finally C1-C4 are coupled in reactions vials 1-4, respectively. This combinatorial group split-and-mix also leads to the formation of 64 different molecules.
  • Figure 3 The combinatorial group split-and-mix with one "no-building-block"
  • FIG. 4 Dendrimer architecture suitable for group split-and-mix procedure A combinatorial split-and-mix library of 65'536 different peptide dendrimers with the structure (((A8-A7) 2 B-A6-A5) 2 B-A4-A3) 2 B-A2-Al-spacer-su ⁇ port.
  • Al to A8 denote variable ⁇ -amino acid positions, dots • (shown with three bonds) indicate a branching unit B (e.g. a diaminoalkanoic acid), spacer is e.g. an co-aminoalkanoic acid allowing undisturbed peptide coupling, and the support is a bead suitable for peptide coupling.
  • branching unit B e.g. a diaminoalkanoic acid
  • spacer is e.g. an co-aminoalkanoic acid allowing undisturbed peptide coupling
  • the support is a bead suitable for peptide coupling.
  • the present invention describes a method for preparing combinatorial libraries where the building block information determines the structure.
  • the molecules are designed in advance in such a manner that the structure of each molecule in the library can be determined by quantitative analysis of the building blocks used in the synthesis.
  • the structure of a product can be determined by analyzing the original building block composition and by assigning the found compounds to the specific position in the molecule design.
  • One of the advantages of this method is that combinatorial libraries can be prepared without the need to attach an extra molecule for encoding the steps or being restricted by the need to use molecules of different molecular weight. In this manner, the synthesis can be simplified and accelerated. For example, the use of combinatorial libraries for the discovery of active molecules of which the structure can be determined is greatly improved.
  • the concept can be used for any type of library and any type of analysis method and is not restricted to the examples described in the following.
  • Computer libraries are collections of macromolecules of related structure comprising sequences of building blocks.
  • Beads are solid carriers in shapes convenient for handling in chemical reactions, e.g. balls, rods or the like. Solid means that the beads are solid under the usual conditions of reactions with building blocks, i.e. do not dissolve in aqueous solvents or organic solvents such as hydrocarbons, chlorinated hydrocarbons, ethers, alcohols, ketones, and, in particular, dipolar aprotic solvents such as dimethylformamide, dimethylacetamide, N- methylpyrrolidone, dimethyl sulfoxide or hexamethyl phosphoric acid triamide. Beads may be of different sizes, preferably having an average diameter of between 0.05 mm and 5 mm, depending on the reaction vessels contemplated. Preferred as solid carrier material are polystyrene cross-linked with 1-2% divinylbenzene, and polystyrene-polyethylene glycol copolymer, but any other type of resin suitable for solid phase synthesis may be used.
  • Building blocks are small organic compounds that can be attached in a chemical reaction to a functionalized solid carrier, and are preferably such building blocks that are conventionally used in solid phase synthesis. Examples of such building blocks are nucleotides, carbohydrates and amino acids, but also other di-and polyfunctional compounds attachable to a functionalized solid carrier. Preferred building blocks of the invention are carbohydrates and amino acids, in particular amino acids.
  • Amino acids may be naturally occurring (L)- ⁇ -amino acids such as Ala, Arg, Asn, Asp, Cys, GIn, GIu, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and VaI, but also corresponding (D)- ⁇ -amino acids or other (e.g. homologous) ⁇ -amino acids, ⁇ -amino acids, ⁇ -amino acids, ⁇ - amino acids, ⁇ -amino acids, di-, tri-, tetra- and penta-amino acids, and the like.
  • Reacting with a building block comprises all steps required in solid phase synthesis to attach the building block to the growing sequence of building blocks on a bead. Such reaction steps and reaction conditions are well known in the art and depend on the type of bead, the type of building block, and the length of sequence on the bead.
  • Particular reaction steps that may be necessary are activating a functional group on the bead or the growing sequence on the bead by mixing with an activating reagent, mixing beads with building blocks in protected or partially protected form, shaking or stirring reactions mixtures containing beads and reagents in particular solvents for a predetermined amount of time at a particular reaction temperature or a reaction temperature gradient, filtering or otherwise separating beads from reagents and solvents, washing beads by shaking or stirring in particular solvents or wash solutions, cleaving or otherwise manipulating protecting groups on building blocks connected to the beads using particular reagents, and the like.
  • Micromolecule refers to a compound synthesized by a series of steps wherein building blocks are attached in sequence to a growing structure.
  • Preferred examples of macromolecules are those composed of the preferred building blocks defined above, and are e.g. polynucleotides, carbohydrate chains or polypeptides, linear or branched, or also combinations thereof such as polypeptides bearing carbohydrate groups, and polypeptides comprising artificial building blocks.
  • Variable position refers to a substructure of a macromolecule in a combinatorial library constituted of one copy or multiple copies of a building block attached to the growing macromolecule chain during a defined step of its synthesis, wherein a mixture of different building blocks are used for that step creating such substructure (position) during library synthesis.
  • Consed position refers to a substructure of a macromolecule in a combinatorial library constituted of one copy or multiple copies of a building block attached to the growing macromolecule chain during a defined step of its synthesis, wherein a single building block is used for that step creating such substructure (position) during library synthesis.
  • Copy number refers to the number of copies of a building block attached at a given "position" of a macromolecule in a combinatorial library during its synthesis.
  • beads are split in equal portions, e.g. n or m portions.
  • the number of portions may be any number between and including 2 and 20, preferably between 2 and 6, for example 3 or 4 portions.
  • the number of portions may be the same or different in any step of "mixing / equally spitting / reacting with building block" as defined hereinbefore.
  • “Groups of building blocks” contain corresponding numbers of building blocks, i.e. between and including 2 and 20, preferably between 2 and 6, for example 3 or 4 building blocks, creating a
  • variable position as defined hereinbefore.
  • One building block within a group of building blocks may be absent, i.e. the actual number of building blocks in a group may be one lower than indicated above, and as a consequence one portion of beads may not react with a building block in a particular step of "mixing / equally spitting / reacting with building block".
  • the beads may be reacted with a single building block in place of a group of building blocks, creating a "conserved position".
  • Analysis for desired properties and selection of members of a combinatorial library based on such an analysis is well known in the art, and is applied accordingly.
  • the analysis may be performed directly on the bead carrying a member of the combinatorial library, or also on the macromolecules cleaved from the bead, i.e. the sequence of building blocks after cleavage, taking into account that all macromolecules cleaved from a bead have an identical structure.
  • the present invention is based on the following two basic conditions:
  • the building blocks should be partitioned into separate groups in such a way that when a building block and its copy number are identified by analysis after cleavage, the position of the building block in the sequence is unequivocally deducible (within the accuracy of the quantification possible by the analysis method chosen).
  • the invention describes a method of distributing a given series of building blocks to a series of variable positions in a macromolecule synthesis such that the structure of the macromolecule can be deduced from knowing the synthesis sequence, the list of building blocks used at each position, and a quantitative analysis of the building blocks that have been used at the variable positions.
  • the invention describes such a method wherein any building block used for variable positions can be used at more than one variable position, in particular 2 or 3 positions, during the synthesis.
  • the invention further concerns combinatorial libraries obtainable by the method of the invention, in particular a combinatorial library of dendritic compounds as defined hereinbelow. Such a combinatorial library is e.g.
  • a building block in Al may be identical with a building block in A3 (or A4); and/or identical with a building block in A5 (or A6); and/or identical with a building block in A7 (or A8); likewise a building block in A3 (or A4) may be identical with a building block in A5 (or A6); and/or identical with a building block in A7 (or A8); and likewise a building block in A5 (or A6) may be identical with a building block in A7 (or A8).
  • Such building blocks are e.g. amino acids, for example naturally occurring ⁇ -amino acids.
  • B is e.g. a diamino acid.
  • the combinatorial split-and-mix and group split-and-mix syntheses described below comprise variable steps creating a variable position, in which different building blocks are used, and conserved steps creating a conserved position, where common operations are carried out and/or common building blocks are attached to the molecule undergoing synthesis.
  • the description focuses on the variable steps where different building blocks are used.
  • the split-and-mix protocol thus allows the rapid preparation of many different compounds in a separated manner.
  • the structure of each compound on each synthesis bead is not known since the beads are not identified during the synthesis but simply mixed randomly.
  • analyzing the composition of the bead in terms of building block does not give the structure unequivocally. For example, if a composition analysis indicates one copy of each Al, A2 and A3 as building blocks for a product on one of the beads, there are six different possible structures for this product: A1-A2-A3, A1-A3-A2, A2-A3- Al, A2-A1-A3, A3-A1-A2, A3-A2-A1.
  • the structure of the product must be A1-B2-C3 as based on the original design. It is the only product containing building blocks Al, B2 and C3 in the library.
  • the preparation of length- variable libraries by using a "no-building-block" variable at each variable step as shown in Figure 3 can be performed (three building blocks per group and an omission).
  • the synthesis with three groups of three building blocks A, B and C results in a combinatorial group split-and-mix library of 63 different compounds which can be identified by composition analysis as described above.
  • the invention is directed to the preparation of structures in which certain variable positions occur in multiple copies, meaning that one of the coupling steps attaches precisely one, two, or more copies of a building block to the molecule.
  • Such a synthesis results in ramified molecules, i.e. dendrimers.
  • Table 1 lists the combinations of copy numbers acceptable according to the invention, i.e. the combinations which still allow to deduce the correct structure of the sequence from the cleaved building blocks.
  • One building block of different groups of building blocks can be used for the synthesis at several different variable positions (with different copy numbers), provided that all possible sum combinations of copy numbers of these different variable positions are different from the copy number used in the synthesis.
  • the key of the invention is that the analysis by quantification of building blocks allows to know how much of a building block is present, and one can assign it then to two different positions with different copy numbers, for example 1 and 2 copies, because all possible sums are unique. This is useful for dendritic sequences.
  • One given building block may be used more than once, in particular 2 times or 3 times, if the copy number of the corresponding positions are as described in Table 1.
  • the general protocol for applying group split-and-mix synthesis for making a library of molecules is described in the Flowchart.
  • the library and synthetic schemes are designed for a group split-and-mix library synthesis (steps 1, 2 and 3 in the Flowchart).
  • the split- and-mix synthesis is then carried out (step 4 in the Flowchart) and the library is tested for active molecules (step 5 in the Flowchart).
  • the sample of an active molecule which is either the polymer bead with the molecule attached to it, or a solution containing this molecule, is then analyzed for identification and quantification of the building blocks (step 6 and 7 in the Flowchart).
  • Knowledge of the building block composition then allows to deduce the actual structure of the molecule by taking the library design into consideration (step 8 in the Flowchart).
  • the known molecule can then be resynthesized in pure form and its activity confirmed and investigated in more detail.
  • peptide dendrimers bearing surface histidine residues catalyze ester hydrolysis in water.
  • a combinatorial approach to peptide dendrimers based on group split-and-mix synthesis and on-bead screening is used.
  • the method is exemplified by the discovery of catalytic and binding peptide dendrimers in a 65'536-member library.
  • a split-and-mix library of peptide dendrimers is designed by distributing sixteen proteinogenic amino acids into four groups of four amino acids each.
  • the resulting 65'536-member library requires splitting in four portions at each variable sequence step, whereby each group of four amino acids would be used at two positions in different branches of the dendrimer.
  • the single-bead sequencing problem is solved by taking advantage of the dendrimeric structure in which amino acids are present in one, two, four or eight copies depending on their placement in the different branches. Since each amino acid is present at most at two positions in different branches, the sequence can be unambiguously assigned by quantitative amino acid analysis of the dendrimer.
  • Example 1 Synthesis of a peptide dendrimer library of a structure as shown in Figure 4 using the group split-and-mix protocol.
  • a combinatorial split-and-mix library of 65'536 different peptide dendrimers is synthesized on solid support with the structure (((AcNH-A8-A7) 2 B-A6-A5) 2 B-A4- A3) 2 B-A2-Al-spacer-support, where Al to A8 denote variable ⁇ -amino acid positions, the branching unit B is (5)-2,3-diaminopropanoic acid, the spacer is 6-aminohexanoyl- glycine, and the support is Tentagel bearing the photolabile 4-(4-hydroxymethyl-2- methoxy-5-nitrophenoxy)butanoic acid at 0.28 mmol/g loading.
  • the peptide dendrimer library is prepared starting with a 400 mg resin batch, which ensures that each sequence would be present in approximately 15 beads.
  • Aromatic, hydrophobic, positively charged, negatively charged and small amino acids are distributed evenly among the inner positions A1-A4 and the outer positions A5-A8.
  • Histidine is placed in the outermost layer (A6 and A8) since surface placement of this residue is known to favor catalysis.
  • the sequence starts with an 6-aminohexanoyl-glycine spacer to minimize interactions with the solid support. All coupling steps are quantitative, as indicated by the negative TNBS-staining test. Side-chain protecting groups are removed with trifluoroacetic acid after Fmoc deprotection, resulting in a functional dendrimer library on beads.
  • N-termini were acetylated.
  • Al and A3 Asp, Ala, Thr, VaI A2 and A4: Lys, Phe, He, Tyr A5 and A7: Pro, Ser, Arg, Leu A6 and A8: His, GIu, Trp, GIy
  • the resin was washed and swelled inside the reactor (e.g. polypropylene syringes from which the resin beads could be easily washed out for the mixing operation) with dichloromethane (DCM, 2x5 ml) and dimethylformamide (DMF, 1x5 ml).
  • DCM dichloromethane
  • DMF dimethylformamide
  • the Tentagel resin (0.23 mmol/g) was acylated with 2.5 equivalents of JV- Fmoc amino acid in the presence of 2.5 equivalents of benzotriazol-1-yl-oxy-tris- (dimethylamino)-phosphoniumhexafluorophosphate (BOP, Novabiochem) and 6 equivalents of JV,JV'-diisopropylethylamine (DIEA) in DMF. After 30 min (first generation), 1 h (second generation), 2 h (third generation) the resin was washed (3> ⁇ each) with DMF, DCM and MeOH and controlled with the trinitrobenzenesulfonic acid (TNBS) test. Double coupling and longer coupling times were necessary for completion toward the end of the synthesis, in particular after addition of the third branching diamino acid.
  • BOP benzotriazol-1-yl-oxy-tris- (dimethylamino)-phosphoniumhexafluorophosphate
  • Fmoc-amino acids were purchased from Novabiochem (Fmoc-Cys(Trt)-OH), Bachem (Fmoc-His(l -Boc)-OH, Fmoc-Gly-OH, Fmoc-DAP(Fmoc)-OH) or Senn Chemicals (Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Phe- OH, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-As ⁇ (OtBu)- OH, Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-
  • Fmoc protecting group was removed with 5 ml of a solution of piperidine in DMF (1 :4) for 10 min. After filtration, the procedure was repeated and then washed (3x each) with DMF, DCM and MeOH.
  • the resin was acetylated with a solution of acetic anhydride in DCM (1:1) for 30 min, washed with DCM, DMF and methanol. The resin was dried under vacuum and stored at -20°C.
  • TFA cleavage of protecting group was carried out using trifluoroacetic acid / 1, 2-ethanedithiol / H 2 O/ triisopropylsilane 94/2/2/2 solution for 3 h. Then the library was washed with water, methanol and DMF, dried under vacuum and stored at -2O 0 C.
  • Example 2 Screening of the dendrimer library for catalytic hydrolysis of the fluorogenic ester 8-butyryloxypyrene-l , 3, 6-trisulfonate.
  • Dendrimer 10 which did not contain a histidine residue, was not active and represented a false positive. All three histidine-containing dendrimers 8, Cl 1 and C12 showed enzyme-like catalysis with the substrate, with substrate binding ZM ⁇ 0.1 mM and catalytic rate constant & cat ⁇ 0.1 min "1 and specific rate acceleration k c jk un cai ⁇ 2 1 OOO- lO'OOO. Catalysis was proportional to dendrimer concentration, and showed multiple turnovers. There was no detectable product inhibition or catalyst inactivation upon reaction, as evidenced by the fact that addition of fresh substrate after completion of a dendrimer catalyzed reaction resulted in further hydrolysis of the substrate at the same initial rate as in the first reaction.

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Abstract

Cette invention concerne un procédé servant à préparer des bibliothèques combinatoires en utilisant une modification de la procédure de division et de mélange, les blocs constitutifs étant choisis de façon à permettre de déduire la séquence des blocs constitutifs à partir de la détermination de la quantité et du type de blocs constitutifs après clivage total. Ce procédé est particulièrement adapté pour la synthèse de bibliothèques combinatoires de dendrimères.
PCT/EP2005/008885 2004-08-17 2005-08-16 Procede de synthese combinatoire rationnelle WO2006018277A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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WO2001056955A1 (fr) * 2000-02-03 2001-08-09 Nanoscale Combinatorial Synthesis, Inc. Synthese non redondante partage/melange de bibliotheques combinatoires

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