EP0934076A4 - - Google Patents

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Publication number
EP0934076A4
EP0934076A4 EP97916162A EP97916162A EP0934076A4 EP 0934076 A4 EP0934076 A4 EP 0934076A4 EP 97916162 A EP97916162 A EP 97916162A EP 97916162 A EP97916162 A EP 97916162A EP 0934076 A4 EP0934076 A4 EP 0934076A4
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EP
European Patent Office
Prior art keywords
group
mmol
library
bond
groups
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EP97916162A
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EP0934076A1 (fr
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Aeolus Pharmaceuticals Inc
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
    • C07K9/005—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure containing within the molecule the substructure with m, n > 0 and m+n > 0, A, B, D, E being heteroatoms; X being a bond or a chain, e.g. muramylpeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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

Definitions

  • the present invention relates to the field of combinatorial library synthesis and drug discovery.
  • the present invention relates to the synthesis of substances and compositions that mimic inhibitors of bacterial peptidoglycan synthesis .
  • the substances and compositions of the invention are useful as potential drug candidates for the treatment of infectious disease, e.g., antibiotics.
  • the biosynthesis of bacterial peptidoglycan is thought to proceed by a complex multi-step process.
  • An early step in the biosynthetic process involves the synthesis of a lipid-linked disaccharide-peptido monomer unit . These monomer units are then linked together into a growing peptidoglycan chain by an enzyme exhibiting a transglycosylase activity. Subsequently, it is believed that the pendant pentapeptides of the peptidoglycan chain are crosslinked by the same or a separate enzyme exhibiting a transpeptidase activity.
  • the transpeptidase reaction is inhibited by -lactam antibiotics, such as penicillins or cephalosporins.
  • the structure of the naturally occurring lipid- linked glycopeptide intermediate is known.
  • This intermediate consists of an N-acetylmuramic acid whose reducing end is linked to a pyrophosphoundecaprenyl group and to the D-lactyl group of which is attached a pentapeptide, -Ala-D-Glu- (A 2 pm/ ys) -D-Ala-D-Ala.
  • a 2 pm stands for diaminopimelic acid.
  • the intermediate may be represented by the formula UDP-MurNAc-pentapeptide.
  • This intermediate is then transformed into the above- mentioned lipid-linked disaccharide-peptido monomer unit by a glycosyltransferase-mediated addition of an N-acetylglucosamine to the N-acetylmuramic acid.
  • a glycosyltransferase-mediated addition of an N-acetylglucosamine to the N-acetylmuramic acid.
  • the inhibition of the N-acetylglucosaminyl transferase that is encoded by the murG gene and which is involved in the biosynthesis of the lipid-linked disaccharide-peptido monomer unit is a promising candidate, thus.
  • Bacterial urG has been cloned and its DNA sequence determined.
  • Recombinant murG gene product is also known. It is further suspected that the N-terminal half of the gene product is responsible for binding of lipid intermediate. That portion of the enzyme, which catalyzes the transferase activity, has yet to be determined, however.
  • Flavophospholipol is a known antibiotic produced by a group of grey-green Streptomyces, which disrupts the biosynthesis of peptidoglycans by inhibiting glycosyltransferase. It is used, mainly in cattle, as a performance enhancer, i.e., it improves the fattening capacity of cattle feed. Flavophospholipol exhibits a fairly broad spectrum of activity against gram-positive and gram-negative bacteria, but mostly against gram- positive microbes. However, both gram-positive and gram-negative organisms are known to carry extrachromosomally derived resistance to antibiotics.
  • the present invention seeks to satisfy the need for new antiinfective agents, as well as the techniques for their synthesis and discovery, by providing a combinatorial approach to the preparation of a library of distinct substances having a predetermined general formula.
  • the present invention provides a solid phase technique that allows for the synthesis of a large multitude of substances and compositions each representing a potential drug or nutritional candidate.
  • An important objective of the present invention is the production of a solid phase lipoglycopeptide library comprising a plurality of distinct substances of the formula
  • the group R comprises a solid support
  • the group P comprises one or more amino acids, peptides, or polypeptides
  • the group S comprises one or more sugars
  • the group L comprises one or more lipids.
  • R comprises a solid support to which is bound the group P comprising one or more amino acids, peptides, or polypeptides, at least one member of the group P bearing the first functional group FG ⁇ capable of participating in a bond-forming reaction; (b) providing one or more groups S, L and FG 2 of the formula in which S comprises one or more sugars to at least one of which is bound a group L comprising one or more lipids, at least one sugar of the group S bearing the second functional group FG 2 capable of participating in the bond-forming reaction; (c) combining the one or more groups R, P and FG X and the one or more groups S, L and FG 2 such that the bond-forming reaction takes place to form a bond between that member of the group P, which bore the first functional group FG ⁇ to that sugar of the group S, which bore the second functional group FG 2 .
  • a solid phase lipoglycopeptide library having a plurality of distinct substances of the formula
  • R comprises a solid support to which is bound the group P comprising one or more amino acids, peptides, or polypeptides, at least one member of the group P bearing the first functional group FG X capable of participating in an initial bond-forming reaction;
  • R comprises a solid support to which is bound the group P comprising one or more amino acids, peptides, or polypeptides, at least one member of the group P bearing the first functional group FG X capable of participating in an initial bond-forming reaction;
  • S comprises one or more sugars, at least one sugar of the group S bearing the second functional group FG 2 capable of participating in the initial bond- forming reaction and the same or another sugar of the group S bearing the third functional group FG 3 capable of participating in a subsequent bond-forming reaction;
  • combining the one or more groups R, P and FG X and the one or more groups S, FG 2 and FG 3 such that the initial bond- orming reaction takes place to form a bond between that member of the group P, which bore the first functional group FG
  • the group comprises one or more lipids, at least one lipid of the group L bearing the fourth functional group FG 4 capable of participating in the subsequent bond-forming reaction; (e) combining the one or more initial bond-forming reaction products and the one or more groups L and FG 4 such that the subsequent bond-forming reaction takes place to form a bond between that member of the group S, which bore the third functional group FG 3 , to that lipid of the group , which bore the fourth functional group FG 4 .
  • Yet another objective of the present invention relates to selected compositions of the formula
  • the group P comprises one or more amino acids, peptides, or polypeptides
  • the group S comprises one or more sugars
  • the group L comprises one or more lipids.
  • synthetic compositions of the above-indicated formula, P-S-L, especially those prepared by combinatorial methods are also contemplated.
  • Fig. 1 shows a diagram of the general combinatorial approach for the conjugation of sugar- containing units to solid support-bound peptides. Each unique combination, after cleavage from the solid support, is then placed in a particular well.
  • Fig. 2 illustrates the sequence of steps making up a parallel synthetic approach in which a sugar group S to which is bound a lipid group L is coupled to a solid support-bound peptide. Conjugation is followed by the removal of protecting groups and cleavage of the resulting lipoglycopeptide from the solid support. Alternatively, the groups S and L can be coupled to the solid support-bound peptide sequentially.
  • Fig. 3 shows the structures of preferred coupling agents that facilitate the condensation reaction involving functional groups of the members of P, S and L.
  • Fig. 4 provides an illustration of some of the sugar group disaccharide building blocks used in a particular library embodiment of the invention.
  • Fig. 5, Fig. 5a and Fig. 5b provides illustrations of some of the lipid and peptide building blocks favored in a particular embodiment of the invention.
  • Fig. 6 outlines the steps in the coupling, deprotection and cleavage of a particular R-P and S-L couple.
  • Fig. 7 is a schematic of a high throughput screening method designed to reveal active members of the library produced by the combinatorial approach of the present invention.
  • Other assays can also be used, however.
  • a probe can be constructed using the binding region of the active site of glycosyltransferase. The probe can then be used to screen and detect lipoglycopeptides having an affinity for the enzyme even while the potential ligands are still bound to the solid support.
  • Fig. 8 outlines the steps of a more preferred method of coupling a S-L group to a P-R group using one of the four coupling agents, e.g., EEDQ, HATU, HBTU, or PyBOP. The deprotection and cleavage steps are also shown.
  • the present methods contemplate a combinatorial approach to the synthesis of a wide variety of potential antiinfective, nutritional and/or performance enhancing agents.
  • a parallel synthetic approach is illustrated.
  • the present methods are applicable equally to other techniques, such as the "mix and split" approach. Indeed, after design and/or selection of the desired constituents (i.e., the "building blocks") of the distinct substances, which are bound to the solid phase, an automated manner of combining the various ingredients can be readily appreciated in which a particular compartment of, for example, a planar solid surface can be assigned to a particular combination of molecular components.
  • An important objective of the present invention is the production of a solid phase lipoglycopeptide library comprising a plurality of distinct substances of the formula, R-P-S-L, as defined earlier.
  • a solid phase lipoglycopeptide library comprising a plurality of distinct substances of the formula, R-P-S-L, as defined earlier.
  • more than one copy of each distinct substance may be present. Hence, multiple hits may be observed on screening of the library.
  • Suitable solid supports include most synthetic polymer resins, preferably in the form of sheets, beads, or resins, such as polystyrene, polyolefins, polymethyl methacrylates and the like, derivatives thereof and copolymers thereof. Polymers having varying degrees of crosslinking are also useful.
  • a preferred solid support is a Merrifield resin, which is a 1% divinylbenzene copolymer of polystyrene.
  • suitable polymer supports are insoluble in most organic solvents but swellable in some.
  • Still other solid supports may be comprised of glass, ceramic, or metallic substances and their surfaces.
  • any solid support contain functional groups that can participate in clndensation reactions, so that the molecular residues of choice may be bound or attached to the surface of the solid support.
  • Such functional groups will generally involve halides, unsaturated groups, carboxylic acids, hydroxyls, amines, esters, thiols, siloxy, aza, oxo and the like.
  • linker groups may be used.
  • Such linkers are well known in the art and may include, but are not limited to, polyamino, polycarboxylic, polyester, polyhalo, polyhydroxy, polyunsaturated groups, or combinations thereof.
  • the linker is preferably labile under a given set of conditions that do not adversely affect the compounds attached to the library or the reagents used in their preparation or manipulation. More preferably, the linker is acid labile or is photolabile. Desirable linkers include a halotrityl moiety (e.g., a chlorotrityl moiety) linking at least one member of the group P to the solid support or an alpha-halo (e.g., bromo) alpha-methylphenacyl moiety. Linkers, of course, may be used to covalently bind the various members of one group to each other or to members of other groups present in a given library.
  • a halotrityl moiety e.g., a chlorotrityl moiety
  • alpha-halo e.g., bromo
  • covalent attachment may utilize functional groups comprising one or more amine, ether, thioether, ester, thioester, amide, acetamide, phosphate, phosphonate, phosphinate, or sulfate bonds.
  • functional groups comprising one or more amine, ether, thioether, ester, thioester, amide, acetamide, phosphate, phosphonate, phosphinate, or sulfate bonds.
  • the library contains sugar moieties, the presence of one or more glycosidic bonds is expected, though not required.
  • at least one member of each group of the formula is covalently attached to at least one member of an adjacent group.
  • the same sugar of the group S is covalently attached to at least one member of the group P and to at least one member of the group L.
  • the covalent bond attaching the lipid member of the group L to a sugar member of the group S may comprise a glycosidic ether bond, phosphate, pyrophosphate, phosphinyl, phosphanyl, phosphonate, phosphonyl, phosphono, phosphino, phosphanoacetate, phosphonyl formate, phosphoramidyl phosphorothioate, phosphonylsulfonate, phonphonylsulfonate bond, or the like. Examples of some glycosidic bonds are shown on Figure 5b.
  • At least one sugar of the group S may also be desirable to have at least one sugar of the group S to be covalently attached to a lipid of the group L through an anomeric alpha-hydroxy acetamide bond. More particularly, at least one sugar of the group S is covalently attached to a member of the group P through a C-3 hydroxy alpha-acetamido or alpha- propionamido moiety.
  • amino acid residues can be used in the present library and its method of preparation.
  • the amino acid, peptide, or polypeptide of the group P may be comprised exclusively or predominantly of hydrophilic amino acid residues, however. It is also possible that the members of the group P comprise exclusively or predominantly of hydrophobic amino acid residues.
  • Various combinations of amino acids, peptides and polypeptides, including both stereoisomers, analogs, or homologs thereof are provided in Fig. 5.
  • the group S may comprise one or more monosaccharides, disaccharides, or polysaccharides, as illustrated in the exemplary sugar group of Fig. 4.
  • a monosaccharide of the invention may be a hexose, pentose, deoxy analog thereof, dideoxy analog thereof, azido-substituted analog thereof, or amino- substituted analog thereof.
  • a disaccharide or polysaccharide of the invention may include hexoses, pentoses, deoxy analogs thereof, dideoxy analogs thereof, azido-substituted analogs thereof, amino- substituted analogs thereof, or combinations thereof.
  • alpha or beta stereochemical configurations may be present, as are cis-1,2 or trans-1,2 arrangements.
  • the bonds between the sugars may be (1,6) , (1,3) , (1,4) and the like, including the possible stereochemical configurations.
  • lipids may be simple or quite complex.
  • the lipid may be saturated, unsaturated, or polyunsaturated. It may be linear, branched or cyclic. It may be made up exclusively or partially of aliphatic group comprising 2-60 carbon atoms, preferably 5-55, more preferably 10-40, most preferably 15-30. Simple fatty acid chains may be useful, as are cholesterol-inspired structures.
  • the lipids may be branched or unbranched alkyl or alkenyl (optionally substituted by one or more lower alkoxy, lower acyloxy, halogen, or aryl) ; branched or unbranched acyl (optionally substituted by one or more lower alkoxy, lower acyloxy, halogen, or aryl) ; lower alkoxycarbonyl, optionally substituted by one or more lower alkenyl, lower alkoxy, lower acyloxy, halogen, aryl, or 9-fluorenyl; or silicon, substituted by one or more lower alkyl, lower alkoxy, or aryl.
  • simple or complex aromatic or heteroaromatic structures may also be useful.
  • aromatic structures include, but are not limited to, aromatic carboxylic acids such as, subsituted or unsubtituted hydroxyphenylacetic acid, hydroxyphenylproprionic acid, methylbenzylate, hydroxynaphthoic acid, hydroxy-biphenylcoaboxylic acid, hvdroxy-fluorenecarboxylic acid, hydroxybenzamide;
  • aromatic carboxylic acids such as, subsituted or unsubtituted hydroxyphenylacetic acid, hydroxyphenylproprionic acid, methylbenzylate, hydroxynaphthoic acid, hydroxy-biphenylcoaboxylic acid, hvdroxy-fluorenecarboxylic acid, hydroxybenzamide;
  • FIG. 5a An important aspect of the invention is the method by which the solid phase lipoglycopeptide library is prepared. The method includes providing one or more groups R, P and FG : of the formula, R- P-FG 1 , as defined above.
  • This material is allowed to react with a second unit having the formula, FG 2 -S-L, also as defined above, such that the bond-forming reaction takes place to form a bond between that member of the group P, which bore the first functional group FG,, to that sugar of the group S, which bore the second functional group FG 2 .
  • the resin is first soaked with an organic solvent that swells the resin prior to the combination step. If the functional group FG X of the resin-bound component is protected, it is understood that the protecting group, such as fluorenylmethyloxycarbonyl (Fmoc) , must first be removed. Thus, the method may entail the removal of any protecting groups present on groups P, S, or L.
  • the protecting group such as fluorenylmethyloxycarbonyl (Fmoc)
  • the combination step in which the respective functional groups are allowed to react will preferably be facilitated by the addition of activating or coupling agents, which are well known to those of ordinary skill. Such agents assist in the formation of a bond between, e.g., a carboxylic acid and an amine group.
  • the resulting solid support-bound library of lipoglycopeptides can then be released from the solid support or screened while still solid support bound.
  • the resulting lipoglycopeptide library be screened for one or more active substances using one or more probes, receptors, affinity binders, enzymes or whole cells. If the compounds are to be released from the solid support, the group P is cleaved from the group R to provide the structural unit P-S-L, which is then recovered.
  • one or more groups S, L and FG 2 are combined with the one or more groups R, P and FG 2 at room temperature in a solvent in the presence of an activating agent.
  • the functional group FG 2 may comprise a pentafluorophenyl ester or simply a free carboxylic acid, especially when allowed to react with an amino group in the presence of an activating agent, such as EEDQ, HATU, HBTU, PyBOP, or the like.
  • the various functional groups may be located anywhere in the molecular unit, including the ends or on the side chain, especially of a member of the group P.
  • certain compounds of the general formula may differ from natural counterparts in the number of amino acid residues present in the group P.
  • Preferred compounds comprises two or more amino acids in sequence, more preferably, four or more, most preferably six or more.
  • the group P may further comprise one or more peptides or polypeptides, which may be joined in sequence or separated by non-peptide moieties, including linker groups.
  • Still other polypeptide components may possess three or more amino acids in sequence or two or more sugars, preferably three or more sugars.
  • the lipid portion of the compounds of the instant library can vary widely in structure. Typically, however, the lipid group will comprise 4-60 carbon atoms, preferably 5-50 and more preferably 10-30.
  • the various structural units which can be prepared by the present methods, may or may not correspond with the structure of a naturally occurring lipid linked glycopeptide intermediate.
  • the group L if comprising a single lipid, does not include an undecaprenyl group.
  • L 1 comprises a lipid group
  • P 1 comprises one or more amino acids, peptides or polypeptides
  • G 1 , G 2 , G 3 , G* , or G 5 can each independently be a substituted or unsubstituted, branched or unbranched alkyl, alkoxy, alkenyl, C1-C8 acyl, acetyl, alkoxycarbonyl, hydroxyalkyl, carboxyalkyl group or a substituted or unsubstituted aromatic or heteroaromatic group, or hydrogen;
  • X 1 , X 2 , X 5 , X 6 or X 7 can each independently be a functional group comprising an oxyalkyl, amine, ether, thioether, ester, thioester, amide, acyl, acetamido, phosphate, phosphinate, pyrophosphate, sulfate, azido, hydroxy group or hydrogen, provided that if the functional group is azido, hydroxy, or hydrogen the attached G group is not present;
  • X 3 or X 4 can each independently be a functional group comprising an amine, ether, thioether, ester, thioester, amide, acetamide, phosphate, phosphinate, pyrophosphate, sulfate group; y is 0, 1, 2, or 3; m, n, v, w, or z can independently be 0, 1, 2, or 3; q or u can independently be 1, 2, or 3 provided that the sum of q and u is not greater than 5, provided that such compounds do not include: 2-N-Acetyl-1- ⁇ -O- allyl-4, 6-O-isopropylidenemuramyl-L-alanyl-D-glutamine benzylester; (2R) -Benzyl 2- [N- (2' -N-Acetyl-1' -a- O- allyl-4' , 6' -O-acetylmuramyl-L-alanyl)amino] -4- cyan
  • the compound of formula (I) comprises lipoglycopeptides in which groups X ⁇ 1 , X 5 G 3 , X 6 G 5 , or X 7 G 4 can be a hydroxyl, HOCH 3 CH 2 -, acetamide, phthalimido, benzoyl, alkoxybenzoyl, alkoxycarbonylalkyl, carboxyalkyl, pivaloyl group or any substituents commonly found on carbohydrates.
  • the X ⁇ 1 or X 2 G 2 groups are preferably located on position 2 or 5 of the ring, group X 3 P preferably located on position 3, and group X 4 L 1 preferably located on position 1 of the ring.
  • the compound of formula (I) further comprises one or more peptides or polypeptides, and one or more lipids.
  • each peptide or polypeptide comprises three or more amino acids in sequence and each lipid comprises one or more saturated, unsaturated, or polyunsaturated linear, branched or cyclic aliphatic group comprising 2-60 carbon atoms, or one or more substituted or unsubstituted aromatic or heteroaromatic groups.
  • the lipid and sugar can be linked via covalent attachment of X 4 including a glycosidic ether bond, phosphate, pyrophosphate, phosphinyl, phosphanyl, phosphonate, phosphonyl, phosphono, phosphino, phosphanoacetate, phosphonyl formate, phosphoramidyl phosphorothioate, phosphonylsulfonate, or phonphonylsulfonate bond.
  • X 4 including a glycosidic ether bond, phosphate, pyrophosphate, phosphinyl, phosphanyl, phosphonate, phosphonyl, phosphono, phosphino, phosphanoacetate, phosphonyl formate, phosphoramidyl phosphorothioate, phosphonylsulfonate, or phonphonylsulfonate bond.
  • MS FAB 759 (M+Na) , 782 (M+2Na) * .
  • FAB 745(M+Na) ⁇ 767 (M+2Na-H) + .
  • Trifluoromethanesulfonyl anhydride (13.3 mL, 79 mmol) is added dropwise to a stirred solution of anhydrous pyridine (6.7 mL, 83 mmol) in anhydrous dichloromethane (250 mL) at -40 °C to give a white suspension.
  • a solution of methyl-2(S)- hydroxypropiolate (8.2 g, 79 mmol) in dichloromethane (15 mL) is then added to the reaction mixture at -40 °C, and the stirred reaction mixture is allowed to warm to room temperature over 1 h.
  • the white precipitate is filtered off, and the filtrate is concentrated below 30 °C on a rotary evaporator to give a brown liquid.
  • the crude product is purified by flash chromatography on silica (250 g) using a gradient elution 10 ⁇ 20% ethyl acetate-dichloromethane to give firstly allyl-4, 6-0-benzylidene-3-0- [1' (R) - (methylcarboxy) ethyl] -2-deoxy-2-acetamido- ⁇ -D- glucopyranoside (5a) (6.1 g, 14 mmol, yield 54%) and then allyl-4, 6-0-benzylidene-3-0- [1' (S) - (methylcarboxy) ethyl] -2-deoxy-2-acetamido- ⁇ -D- glucopyranoside (5b) (1.65 g, 3.8 mmol, yield 14.5%), both as white solids.
  • the carboxylic acid (26) is prepared in a similar manner to compound (10) by the reaction of compound
  • Alcohol (3) (40.5 g, 106.7 mmol) is azeotroped with toluene (100 mL) then dissolved in anhydrous DMF
  • Benzylidene acetal (31) (42.33 g, 96.4 mmol) is azeotroped once with toluene (150 mL) then dried under vacuum in a 2L flask.
  • 3A powdered molecular sieves (65 g) , sodium cyanoborohydride (54.5 g, 868 mmol) and anhydrous THF (800 mL) are added under argon.
  • the stirred suspension is cooled to 0 °C and IN HCl in diethyl ether (800 mL) is added by cannula over 40 min.
  • the milky solution is stirred at r.t. until the reaction is complete by TLC (10% MeOH-CH 2 Cl 2 ) at 2 h.
  • TMSOTf 35ml is added to the stirred suspension followed by a second 10ml portion after 30 min and a further 10 mL portion at 4 h.
  • Benzoyl chloride (0.75 mL, 6.5 mmol) is added to a stirred solution of compound (38) (2.8 g, 3.61 mmol) and DMAP (0.2 g) in (4:1) pyridine-dichloromethane (100 mL) at -20 °C.
  • the reaction mixture is allowed to warm up to r.t. over 1 h.
  • the reaction mixture is then recooled to -20 °C, and DMF (20 mL) is added, causing the milky solution to become clear and bright yellow in colour.
  • the reaction is allowed to warm up to r.t.
  • Solvent is removed by evaporation. The residue is dissolved in ethyl acetate (150 mL) , washed with cold aqueous HCl (IM) to pH 2, washed with saturated NaHC0 3 , dried (Na 2 S0 4 ) and concentrated. The residue (70 g) and thiophenol (13 mL, 130 mmol) are dissolved in methylene chloride (200 mL) . To the ice bath cooled solution is added dropwise BF 3 .Et 2 0 (34 g, 240 mmol) .
  • the suspension is filtered.
  • the filtrate is concentrated and purified with a short silica column
  • a suspension of DL17 (500 mg, 0.5 mmol) and palladium on carbon (10%, 300 mg) in ethanol (10 mL) is subjected to hydrogenolysis at 50 psi for 48 h.
  • the suspension is filtered and concentrated to give 400 mg
  • Tetrabutylammonium iodide (5.24 g, 14.2 mmol) and benzyl bromide (2.2 mL, 18.5 mmol) are added to the reaction mixture and stirred at 80 °C for 32 h. Ethyl acetate (150 mL) is added. The reaction mixture is washed with water (2 x 150 mL) and brine (2 x 150 mL) , dried (Na 2 S0 4 ) and concentrated. The residue is purified by flash chromatography (EtOAc:Hexane 3:7 to
  • reaction mixture is stirred for 2 h at room temperature.
  • Ethyl acetate (100 mL) and 2M hydrochloric acid (100 mL) are added.
  • the reaction mixture is stirred for 1 h at room temperature.
  • the organic layer is separated, washed with water (50 mL) and saturated brine
  • An Fmoc-protected resin-bound peptide building block, R-P is first swollen in DMF (2 mL, 380 rpm, 0.5 h) .
  • the Fmoc group is cleaved off (2 mL of 20% piperidine in DMF, 380 rpm, 0.5 h) .
  • the deprotected R-P is washed with fresh DMF four times (2 mL, 380 rpm, 5 min/wash) .
  • the R-P (1.0 equiv) is then treated with the free acid of an S-L building block (1.0- 3.0 equiv) in the presence of an activating or coupling agent with agitation for about 4 h.
  • Coupled agents Any of four different coupling agent-solvent combinations (“coupling cocktails") are used: EEDQ-DCM, HATU-DIPEA/DMF, HBTU- DIPEA/DMF, or PyBOP-DIPEA/DMF. (See, Fig. 3 for the structures of the activating agents.)
  • the coupling cocktail is filtered off and the resin is washed four times with fresh 2 mL portions of DMF (380 rpm, 5 min/wash) .
  • the sugar protecting groups e.g., acetyl groups, pivaloyl groups
  • the sugar protecting groups are removed by treating the resin with a solution of sodium methoxide in a solvent mixture of methanol-DMF (1:1, v/v) with agitation (380 rpm) for ca. 15-24 h.
  • the resin is washed four times with fresh 2 mL portions of the methanol-DMF solvent mixture (380 rpm, 5 min/wash) , followed by four 2 mL portions of DCM (380 rpm, 5 min/wash) .
  • the dichloromethane is removed, and the sugar- deprotected resin is next treated with a 90% solution of TFA in water (2 mL, 380 rpm, ca. 2 h) .
  • the desired 97 product is obtained from the product solution.
  • Residual resin may be treated a second time with 90% TFA-H 2 0 (1 mL, 380 rpm, 15 min) .
  • a second product solution may hence be obtained, which is combined with the first product solution.
  • the trifluoroacetic acid-water solvent is removed by evaporation in a block heater at 40 °C using an air stream.
  • the solid support-bound peptides are prepared using known methods using, for example, a Merrifield resin (polystyrene/1% divinylbenzene copolymer) and an acid labile chlorotrityl linker group.
  • the building blocks may be obtained from commercial sources, such as Advanced ChemTech (Kentucky, U.S.A.) .
  • the following illustrates a specific example of coupling a support-bearing peptide to a lipid-bearing sugar, deprotection and cleavage.
  • the solid phase synthesis of peptidoglycan monomer analogous 52 and 53 is illustrated in Fig. 8.
  • the lipid- bearing disaccharide sugar 8 is coupled to a tripeptide bound to polystyrene resin via a chlorotrityl linker, AKA- (Clt) - (PS) .
  • the lipid-bearing monosaccharide sugar 51 is coupled to AKA- (Clt) - (PS) as well as to a tripeptide bound to polyethylene glycol grafted 98 polystyrene resin, AKA- (PEG) -(PS) .
  • Solid phase coupling reactions are carried out with two equivalents of the glycocarboxylic acid, 8 or 51, and two equivalents of the coupling reagent. The reaction mixtures are then shaken for four hours at room temperature.
  • the resin-bound monosaccharide coupledproducts, 51- AKA- (Clt) - (PS) and 51-AKA- (PEG)- (PS) are treated with 90% aqueous trifluoracetic acid to complete the cleavage and global deprotection process.
  • the monosaccharide peptidoglycan monomer 52 is obtained by precipitation with diethyl ether [Fab MS: 819 (M-H + 2Na)*].
  • the disaccharide coupled product 8-AZA-(Clt) -(PS) , is first treated with sodium methoxide to remove the acetate protecting groups. It is followed by the 90% TFA cleavage and peptide deprotection step. The disaccharide peptidoglycan monomer 53 is finally precipitated with diethyl ether [Fab MS: 991 (M-H + 2Na) ⁇ ] .
  • the coupling yield of each combination of solid support and coupling reagent is determined by RP-HPLC analysis of the isolated products.
  • FIG. 6 Another method for the synthesis of peptidogylcan monomers is seen in Fig. 6.
  • An Fmoc-protected peptide building block, Fmoc- [L-Ala] - [L-Lys] - [D-Ala] - (R) is deprotected in the manner described in the general procedures.
  • the deprotectd peptide is coupled to the pentafluorophenyl (Pfp) ester of the lipid-sugar by Dhbt- OH.
  • Pfp pentafluorophenyl
  • the deprotection and cleavage steps are as described in the general procedure to yield 54.
  • a peptidoglycan polymerization assay is adapted from one described by Mirelman et al. , in Biochemistry (1976) 15:1781-1790 and modified by Allen et al. , in FEMS Microbiol . Lett . (1992) 98:109-116. Briefly, E. coli (ATCC Cat. No. 23226) are permeabilized with ether to permit exogenously added radiolabeled and non- radiolabeled cell wall precursors to penetrate the bacterial cell wall.
  • UDP muramyl-pentapeptide UDP muramyl-pentapeptide
  • UDP-N-acetylmuramyl-L-Ala-D-Glu- /neso-diaminopimelyl-D-Ala-D-Ala UDP muramyl-pentapeptide
  • ATCC Cat. No. 11778 B. cereus
  • Bacterial protein is determined by the method of Bradford, in Anal. Biochem. (1976) 72:248.
  • Polymerization assays are conducted in 96-well filter-bottom plates (Millipore GF/C - Cat. No. MAFC NOB 10) .
  • each well contains: 50 mM Tris - HCl (pH 8.3) ; 50 mM NH 4 C1 ; 20 mM MgS0 4 * 7 H 2 0; 10 mM ATP (disodium salt) ; 0.5 mM ⁇ - mercaptoethanol; 0.15 mM D-aspartic acid; 0.001 mM UDP-N- acetyl [ 14 C-] -D-glucosamine (DuPont/N.E.N. - 307 mCi/mmol) ; 0.01 mM UDP-MurNAc-pentapeptide, 100 ⁇ g/mL 100 tetracycline and 10 ⁇ g/well ether-treated bacterial protein.
  • Novel test compounds are solubilized in distilled water and screened at a final assay concentration of 100 ⁇ g/mL. With the exception of radiolabel and isolated native pentapeptide, all remaining biochemicals are available from Sigma.
  • Reactions are begun by adding 10 ⁇ L aliquots of bacterial protein in assay buffer into wells containing all remaining reagents. Plates are covered, mixed for 30 seconds, then incubated at 37 °C for 2 hr. Ice cold 20% TCA (100 ⁇ L) is added to each well. Plates are gently mixed (60 sec) , then refrigerated (4 °C) for 30 min to assure precipitation of all peptidoglycan.
  • the peptide portion of the three compounds exhibiting inhibitory activity are: L-S- (L-Ala-D-iso-Gln- L-Lys-D-Ala-OH) , L-S- (L-Lys-D-Ala-D-Ala-OH) andL-S- (Gly- D-iso-Gln-L-Lys-D-Ala-D-Ala-OH) .

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Abstract

L'invention a pour objet une bibliothèque de substances et de compositions distinctes, de formules générales R-P-S-L et P-S-L, représentant respectivement des lipoglycopeptides liés par un support solide et des lipoglycopeptides libres. L'invention se rapporte également à des procédés combinatoires de préparation de différentes bibliothèques et à un test servant à déterminer l'activité biologique de membres sélectionnés dans cette bibliothèque. Un mode de réalisation spécifique concerne des inhibiteurs de la synthèse de peptidoglycane bactérien.
EP97916162A 1996-03-21 1997-03-21 Bibliotheque de lipoglycopeptides en phase solide, compositions et procedes Withdrawn EP0934076A1 (fr)

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PCT/US1997/004637 WO1997034623A1 (fr) 1996-03-21 1997-03-21 Bibliotheque de lipoglycopeptides en phase solide, compositions et procedes

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DE19624345B4 (de) * 1996-06-19 2004-12-23 Südzucker AG Mannheim/Ochsenfurt Acylierte Kohlenhydrate mit mindestens einer mit dem Kohlenhydrat veretherten Carboxyalkyl-Gruppe, Verfahren zu deren Herstellung und deren Verwendung in Waschmitteln
DE19642751A1 (de) * 1996-10-16 1998-04-23 Deutsches Krebsforsch Saccharid-Bibliothek
CN1249081C (zh) 1998-12-23 2006-04-05 施万制药 糖肽衍生物或含有它们的药物组合物
US20010051349A1 (en) 2000-02-17 2001-12-13 Glycominds Ltd. Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof
US7138531B2 (en) * 2001-10-15 2006-11-21 Kemin Pharma B.V.B.A. Preparation and use of carbohydrate-based bicyclic ring structures with antimicrobial and cytostatic activity
AUPS213802A0 (en) * 2002-05-03 2002-06-06 Alchemia Pty Ltd Disaccharides for drug discovery
AU2002952121A0 (en) 2002-10-17 2002-10-31 Alchemia Limited Novel carbohydrate based anti-bacterials
JP5174672B2 (ja) * 2005-10-04 2013-04-03 アルケミア リミティッド 薬物設計の方法
PL2465542T3 (pl) * 2008-12-16 2015-06-30 Genzyme Corp Koniugaty białko-oligosacharyd
CN113200935B (zh) * 2021-04-26 2022-07-08 威海海洋生物医药产业技术研究院有限公司 一种用于次氯酸检测的荧光探针及其制备方法

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WO1995018971A1 (fr) * 1994-01-11 1995-07-13 Affymax Technologies N.V. Procede de synthese chimique en phase gazeuse de glycoconjugues

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WO1995018971A1 (fr) * 1994-01-11 1995-07-13 Affymax Technologies N.V. Procede de synthese chimique en phase gazeuse de glycoconjugues

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