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WO2006021553A1 - Procédé pour la purification de protéines et marquage basé sur une réaction chimiosélective - Google Patents

Procédé pour la purification de protéines et marquage basé sur une réaction chimiosélective Download PDF

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WO2006021553A1
WO2006021553A1 PCT/EP2005/054114 EP2005054114W WO2006021553A1 WO 2006021553 A1 WO2006021553 A1 WO 2006021553A1 EP 2005054114 W EP2005054114 W EP 2005054114W WO 2006021553 A1 WO2006021553 A1 WO 2006021553A1
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formula
compound
carbon atoms
group
molecule
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PCT/EP2005/054114
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Maik Kindermann
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Covalys Biosciences Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

  • the present invention relates to methods and reagents for labeling a target compound and a chemoselective reaction that simultaneously cleaves and ligates different labels to a target compound.
  • proteomics aims for a functional analysis of proteins, their interactions among each other and their interaction with small organic molecules. This requires primarily the identification and characterization of all proteins involved in a complex cellular system. The isolation of unknown binding partners of a protein which is known to be part of a certain metabolic pathway is a key issue e.g. in drug discovery.
  • bio- macromolecules such as peptides
  • peptides proteins
  • Numerous strategies towards this goal are based on the construction of fusion proteins between a protein of interest and a second protein (polypeptide) with the intention to couple the high selectivity of the protein tag (second protein) for a specific probe to the protein of interest.
  • polypeptide polypeptide
  • Examples for such protein tags in fusion proteins include the 6xHis tag, glutathione S transferase, maltose binding protein, epitope tags, yeast-two hybrid system, split-ubiquitin, and green fluorescent protein (GFP).
  • Linkers and their associated strategies play a pivotal role in the successful implementation of protein labeling and purification.
  • the bond between a linker and a label e.g. a label for immobilization, is usually sensitive to certain reaction conditions leading to bond cleavage and the release of the final compound from the label.
  • the problem may arise that a molecular probe - once introduced to the fusion protein via the specific reaction of the protein tag - cannot be easily removed or changed without disruption of biological activity of the protein of interest.
  • ACP acyl carrier protein
  • ACPS holo-acyl carrier protein synthase
  • tags for in vitro protein purification comprise affinity labels that - upon contact with their counterpart - form non-covalent complexes of various stability. Elution of the protein of interest is then achieved by using a specific competitor for the binding site or a pH shift of the elution buffer: e.g. 6xHis tag with imidazole, or streptavidin (strep tagTM) with desthiobiotin.
  • Photocleavable linkers have been used for the cleavage of entirely covalent entities, with the drawback that the light of the wavelength necessary may harm some proteins, and the work has to be carried out in the dark before the cleavage event.
  • the present invention makes use of the "Staudinger reaction".
  • One of the oldest and most common transformations of azides is the reaction with trivalent phosphorous compounds to form iminophosphoranes (phospha-aza-ylides). This transformation proceeds under very mild conditions in a variety of solvents with almost quantitative yield.
  • the nucleophilic nitrogen atom in those iminophosphoranes can react with numerous electrophiles, and the simple hydrolysis of this intermediate leads to an amine and a phosphine oxide.
  • This reaction is known for almost a century as the Staudinger reaction (Staudinger et al., HeIv. Chim. Acta ⁇ S ⁇ S, 2, ⁇ 5). Bertozzi et al.
  • the invention relates to two classes of chemical compounds, to their use and their manufacture.
  • A is a group that specifically binds to or reacts with a binding partner B;
  • Ri and R 3 are, independently of each other, a linker;
  • R 2 is an electrophilic functional group able to react intramolecularly with an aza-ylide;
  • X, Y and Z are, independently of each other, aryl or heteroaryl, or an optionally substituted saturated or unsaturated alkyl, cycloalkyl or heterocyclyl group, or X and Y, X and Z or Y and Z together with the phosphorus atom represent a ring;
  • L 1 is a label, a plurality of same or different labels, a bond connecting R 3 to A forming a cyclic substrate, or a further group A; and R 3 is bound either to X or to Z;
  • the invention further relates to novel azides of formula (2)
  • L 2 is a label, a plurality of same or different labels, a group A or hydrogen, and R 4 is a linker;
  • the invention further relates to a method for detecting and/or manipulating a protein of interest, wherein the protein of interest is incorporated into a binding partner B, the binding partner B is contacted with a compound of formula (1 ) comprising a label, and the reaction product of binding partner B and the compound of formula (1 ) is detected and/or further manipulated using the label.
  • the invention relates to such a method wherein the reaction product of binding partner B and the compound of formula (1 ) is further reacted with a compound of formula (2).
  • the invention relates to two classes of chemical compounds, to their use and their manufacture.
  • the first class of compounds comprises specially designed phosphine moieties of formula (1 )
  • A is a group that specifically binds to or reacts with a binding partner B;
  • R 1 and R 3 are, independently of each other, a linker;
  • R 2 is an electrophilic functional group able to react intramolecularly with an aza-ylide;
  • X, Y and Z are, independently of each other, aryl or heteroaryl, or an optionally substituted saturated or unsaturated alkyl, cycloalkyl or heterocyclyl group, or X and Y, X and Z or Y and Z together with the phosphorus atom represent a ring; and L 1 is a label, a plurality of same or different labels, a bond connecting R 3 to A forming a cyclic substrate, or a further group A.
  • R 3 (and R 2 and the phosphorus atom further carrying Y and Z) are connected to X.
  • R 3 is connected to Z, which is one of the three ligands (X, Y, Z) of the phosphorus atom.
  • Formula (1 ) has to be understood that R 3 may be connected to either X or Z.
  • the second class of compounds comprises specially designed organic azides of the general formula (2)
  • L 2 is a label, a plurality of same or different labels, a group A or hydrogen, and R 4 is a linker.
  • A is a group that specifically binds to or reacts with a binding partner B.
  • the interaction of the group A with a binding partner B is either through a covalent bond after a chemical reaction, or through a non-covalent interaction such as a complex formation.
  • the binding partner B consists of a peptide sequence comprising a protein of interest and a protein tag.
  • A is a group recognized as a substrate by a binding partner representing a fusion protein with a suitable protein tag, e.g.
  • a substrate for an O 6 - alkylguanine-DNA alkyltransferase (AGT), for acyl carrier protein (ACP), or for a fragment of AGT or ACP or a mutant of AGT or ACP preferably a substrate for an O 6 -alkylguanine- DNA alkyltransferase (AGT) or for acyl carrier protein (ACP).
  • Particular groups A recognized as a substrate by an optionally modified O 6 -alkylguanine- DNA alkyltransferase (AGT) or a fragment thereof are those disclosed in International Patent Application WO 2004/031405.
  • a particular group is, e.g., a para-substituted O 6 -benzylguanine residue of the formula (3)
  • a recognized as a substrate by an acyl carrier protein (ACP) in the presence of a holo-acyl carrier protein synthase (ACPS) are coenzyme A type substrates described in International Patent Application WO 2004/104588, for example of the formula (4).
  • A is a haloalkane, reacting specifically with an active-site variant of dehalogenase (DhaA).
  • Haloalkane dehalogenases are enzymes that hydrolyze carbon- halogen bonds in a broad range of substrates. In the catalytic mechanism for dehalogenation, a nucleophilic attack of an Asp (aspartic acid) residue on the halogen- substituted carbon atom of the substrate occurs forming a covalent alkyl-enzyme intermediate. In wild-type dehalogenases, this intermediate is subsequently hydrolyzed by a water molecule which is activated by His and Asp residues (T. Bosma et al.,
  • Particular groups A recognized as a substrate by a dehalogenase mutant are omega-haloalkanes with a carbon chain of 1 -20, preferably 2-8, carbon atoms.
  • Binding partner B may then be a fusion protein of such a dehalogenase mutant and a protein of interest (WO 2004/072232).
  • A is a biotin moiety that can be captured by a binding partner B representing an avidin derivative, e.g. streptavidin or a protein of interest carrying streptavidin.
  • A is a synthetic ligand (SLF ' ) which interacts with a binding partner B comprising FKBP12(F36V) in the nM range as disclosed in K. M. Marks et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9982-9987.
  • SPF ' synthetic ligand
  • A is an activity based chemical probe that targets a binding partner B representing proteins of interest fused to specific classes of enzymes, for example a fluorophosphonate reacting specifically with members of the serine hydrolase superfamily (D. Kidd, B.F. Cravatt, Biochemistry, 2001 , 40, 4005-4015), an (acyloxy)- methyl ketone reacting with cysteine proteases (N. A. Thornberry et al., Biochemistry, 1994, 33, 3934-3940), or a 4-fluoromethyl-1 -phosphaphenyl group reacting with tyrosine phosphatases (J. K. Meyers et al., Science, 1993, 262, 1451 -1453).
  • a fluorophosphonate reacting specifically with members of the serine hydrolase superfamily
  • an (acyloxy)- methyl ketone reacting with cysteine proteases
  • A is a bis-arsenical dye reacting specifically with a binding partner B representing a tetracysteine-tagged protein (S. R. Adams, R. Y. Tsien et al., J. Am. Chem. Soc. 2002, 124, 6063-6076).
  • A is an O 6 -benzylguanine residue of formula (3) or a coenzyme A type substrate of formula (4).
  • R 1 , R 3 and R 4 are, independently of each other, a linker.
  • a linker R 1 is a linker connecting group A to the electrophilic functional group R 2 bound to ligand X of phosphine PXYZ.
  • a linker R 3 is a linker connecting a label L 1 (or L 1 with another meaning) to the X group bearing R 2 (in the compound of formula (1 A)), or to another phosphine ligand Z (in the compound of formula (1 B)).
  • a linker R 4 is a linker connecting a label L 2 (or L 2 with another meaning) to the azide function -N 3 .
  • Linkers R 1 , R 3 and R 4 are preferably flexible linkers.
  • Linker units R 1 and R 3 are chosen in the context of the envisioned application, i.e. the reaction with a binding partner B, such as the transfer of the group bound to A to a fusion protein comprising AGT or ACP, or the reaction with a derivative of a dehalogenase mutant, of avidin, of FKBP12(F36V), of members of the serine hydrolase superfamily, of cysteine proteases, of tyrosine phosphatases, or reaction with tetracysteine-tagged proteins, respectively.
  • linker R 4 is chosen in the context of the envisioned application, i.e. the reaction of the compound of formula (2) with a compound of formula (1 ) connected to a binding partner B.
  • the linkers are also supposed to increase the solubility of the substrates in the appropriate solvent.
  • the linkers used are chemically stable under the conditions of the actual application.
  • the linkers R 1 , R 3 and R 4 do not interfere with the interaction of group A with the binding partner B nor with the detection of the labels L 1 and L 2 , respectively, but may be constructed such as to be cleaved at some point in time after the reaction of the compound of formula (1 ) with the binding partner B or after the reaction of compound of formula (2) with compound (1 ) carrying the binding partner B, respectively.
  • Linkers R 1 , R 3 and R 4 considered are those disclosed in International Patent Application WO 2004/031405 (as a linker R 4 ).
  • a linker R 4 is e.g. a straight or branched chain alkylene group with 1 to 300 carbon atoms, wherein optionally (a) one or more carbon atoms are replaced by oxygen, in particular wherein every third carbon atom is replaced by oxygen, e.g. a poylethyleneoxy group with 1 to 100 ethyleneoxy units;
  • one or more carbon atoms are replaced by a phenylene, a saturated or unsaturated cycloalkylene, a saturated or unsaturated bicycloalkylene, a bridging heteroaromatic or a bridging saturated or unsaturated heterocyclyl group;
  • a particularly preferred linker R 1 , R 3 and R 4 is a straight chain alkylene group of 10 to 40 carbon atoms wherein 3 to 12 carbon atoms are replaced by oxygen, and optionally one carbon atom is replaced by a 1 ,4-phenylene unit.
  • Another particularly preferred linker R 1 , R 3 and R 4 is a straight chain alkylene group of 10 to 40 carbon atoms optionally substituted by oxo wherein 3 to 12 carbon atoms are replaced by oxygen and one or two carbon atoms are replaced by nitrogen.
  • the Staudinger ligation e.g. the reaction corresponding to the reaction of compound of formula (1 ) with compound of formula (2), proceeds without detectable racemization (M. B. Soellner et al. J. Org. Chem. 2002, 67, 4993-4996).
  • the central subunit The central subunit
  • X, Y and Z are, independently of each other, aryl or heteroaryl, or an optionally substituted saturated or unsaturated alkyl, cycloalkyl or heterocyclyl group.
  • Y and Z differ from X in that X is not only bound to phosphorus, but further to the electrophilic functional group R 2 and, in compounds of formula (1 A), to the linker R 3 .
  • Z differs further from Y in that Z is bound to the linker R 3 .
  • this ring may have 4, 5, 6, 7 or 8 ring members selected from carbon, nitrogen and oxygen atoms, preferably carbon atoms.
  • the ring members may be substituted by the substituents listed below under alkyl, and may be annealed to an aromatic ring, e.g. phenyl.
  • Particular ring residues bound to phosphorous considered are e.g. 1 ,4-butylene, 1 ,5-pentylene, 1 ,6-hexylene, or o-phenylene-dimethylene.
  • X, Y and Z are preferably aryl, in particular optionally substituted phenyl, wherein the substituents preferably have the meanings listed below under aryl.
  • the rate of reaction of the Staudinger ligation depends on the electronic properties of X, Y and Z, and substituents are chosen to take this into account. Most preferably, X, Y and Z are unsubstituted phenyl.
  • phosphines wherein X is an optionally substituted methyl group, in particular methyl or benzyl, or imidazolyl, and Y and Z represent aryl, preferably phenyl.
  • R 2 is an electrophilic functional group able to react intramolecularly with an aza-ylide, in particular with an iminophosphorane.
  • R 2 is bound to linker R 1 and to group X, and is preferably separated by two carbon atoms from -PYZ, e.g. located in the ortho position relative to PYZ if X is aryl (e.g. phenyl) or heteroaryl, or by one carbon atom from -PYZ, e.g. bound to the methyl group in an optionally substituted methyl (e.g. benzyl) group X.
  • An electrophilic functional group R 2 is, for example, a derivative of carboxylic acid such as an ester, thioester or carboxamide, or a sulfonic acid ester, preferably a carboxylic acid ester.
  • This electrophilic functional group R 2 is bound to X in such a way that, on reaction with a nucleophile, X represents the leaving group, i.e. the sequence -R 1 -R 2 -X- corresponds to partial formula -R 1 -CO-O-X-, -R 1 -CO-S-X-, -R 1 -CO-NH-X-, and -R 1 -SO 2 -O-X-, respectively.
  • a further partial formula corresponding to this definition is
  • Aryl is an aromatic group comprising 6 to 10 carbon atoms, and is preferably phenyl or naphthyl, in particular phenyl.
  • Aryl may be (further) substituted by lower alkyl, such as methyl, lower alkoxy, such as methoxy or ethoxy, halogen, e.g. chlorine, bromine or fluorine, halogenated lower alkyl, such as trifluoromethyl, or hydroxy.
  • Heteroaryl is mono- or bicyclic heteroaryl comprising zero, one, two, three or four ring nitrogen atoms and zero or one oxygen atom and zero or one sulfur atom, with the proviso that at least one ring carbon atom is replaced by a nitrogen, oxygen or sulfur atom, and which has 5 to 12, preferably 5 or 6 ring atoms; and which may be (further) substituted by lower alkyl, such as methyl, lower alkoxy, such as methoxy or ethoxy, halogen, e.g. chlorine, bromine or fluorine, halogenated lower alkyl, such as trifluoromethyl, or hydroxy.
  • lower alkyl such as methyl, lower alkoxy, such as methoxy or ethoxy
  • halogen e.g. chlorine, bromine or fluorine
  • halogenated lower alkyl such as trifluoromethyl, or hydroxy.
  • heteroaryl is pyrrolyl, imidazolyl, benzimidazolyl, pyridyl, pyrimidinyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, thiophenyl, or furanyl.
  • Alkyl has preferably 1 to 10 carbon atoms, is linear or branched, and includes lower alkyl of 1 to 7 carbon atoms, in particular 1 to 4 carbon atoms, e.g.
  • Substituents considered are aryl, heteroaryl, cycloalkyl, lower alkoxy, such as methoxy or ethoxy, halogen, e.g. chlorine, bromine or fluorine, hydroxy, lower acyloxy, amino, e.g.
  • Substituted alkyl is preferably aryl-lower alkyl, in particular arylmethyl, such as benzyl.
  • Unsaturated alkyl has preferably 2 to 10 carbons and corresponds to the definitions given for alkyl, further containing one, two or three double or triple bonds.
  • Unsaturated alkyl is e.g. 1 -alkenyl or 1 -alkynyl.
  • Substituents considered are for example aryl, e.g. phenyl, lower alkoxy, e.g. methoxy, lower acyloxy, e.g. acetoxy, or halogen, e.g. chloro.
  • Cycloalkyl has preferably 3 to 7 carbon atoms, and is e.g. cyclopropyl, cyclopentyl, cyclohexyl or cycloheptyl, in particular cyclohexyl, optionally substituted by lower alkyl, e.g. methyl, lower alkoxy, e.g. methoxy, lower acyloxy, e.g. acetoxy, or halogen, e.g. chloro.
  • Unsaturated cycloalkyl corresponds to the definitions given for cycloalkyl, further containing one or two double bonds.
  • Unsaturated cycloalkyl is e.g. cyclopentenyl or cyclohexenyl, being optionally substituted by substituents listed under cycloalkyl.
  • Heterocyclyl has preferably 3 to 12 atoms comprising 1 to 5 hetero atoms selected from nitrogen, oxygen and sulfur, and is, for example, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, morpholinyl or dioxolanyl.
  • Substituents considered are e.g. lower alkyl, e.g. methyl, benzyl, lower alkoxy, e.g. methoxy, lower acyloxy, e.g. acetoxy, or halogen, e.g. chloro.
  • Lower alkyl or benzyl substituents may be connected to a ring nitrogen atom.
  • Unsaturated heterocyclyl corresponds to the definitions given for heterocyclyl, further containing one or two double bonds. Unsaturated heterocyclyl is e.g. dihydro- or tetrahydropyridyl, and is optionally substituted by substituents listed under heterocyclyl.
  • L 1 is a label, a plurality of same or different labels, a bond connecting R 3 to A forming a cyclic substrate, or a further group A.
  • L 2 is a label, a plurality of same or different labels, a group A, or hydrogen, preferably a label, a plurality of same or different labels, or a group A.
  • Labels L 1 and L 2 can be chosen by those skilled in the art dependent on the application for which the compounds of formula (1 ) and (2), respectively, are intended.
  • the labels may be e.g. such that the binding partner B after reaction with the compound of formula (1 ) then carrying label L 1 is easily detected or separated from its environment.
  • Other labels considered are those which are capable of sensing and inducing changes in the environment of the labeled binding partner B of compound (1 ), or labels which aid in manipulating the binding partner B of compound (1 ) by the physical and/or chemical properties of the compound of formula (1 ) carrying the label.
  • label L 1 and L 2 can be chosen such that, e.g., after reaction of binding partner B with a compound of formula (1 ) the label L 1 is used to isolate and purify B, and then on reaction with a compound of formula (2) the label L 2 replacing label L 1 is used to further manipulate B, as will be described in more detail hereinbelow.
  • Labels L 1 and L 2 considered are those disclosed in International Patent Application WO 2004/031405 (as a label L).
  • labels L 1 and L 2 include a spectroscopic probe such as a fluorophore, a chromophore, a magnetic probe or a contrast reagent; a radioactively labelled molecule; a molecule which is one part of a specific binding pair which is capable of specifically binding to a partner; a molecule that is suspected to interact with other biomolecules; a library of molecules that are suspected to interact with other biomolecules; a molecule which is capable of crosslinking to other molecules; a molecule which is capable of generating hydroxyl radicals upon exposure to H 2 O 2 and ascorbate, such as a tethered metal-chelate; a molecule which is capable of generating reactive radicals upon irradiation with light, such as malachite green; a molecule covalently attached to a solid support, where the support may be a glass slide, a microtiter plate or any polymeric structure known to those proficient in the art; a nucleic acid or a derivative thereof
  • Preferred labels L 1 are spectroscopic probes, molecules which are one part of a specific binding pair which is capable of specifically binding to a partner, so- called affinity labels, and molecules covalently attached to a solid support.
  • Preferred labels L 2 are spectroscopic probes, and molecules which are one part of a specific binding pair which is capable of specifically binding to a partner.
  • label L 1 or L 2 is a fluorophore, a chromophore, a magnetic label, a radioactive label or the like
  • detection is by standard means adapted to the label and whether the method is used in vitro or in vivo.
  • the method can be compared to the applications of the green fluorescent protein (GFP) which is genetically fused to a protein of interest and allows protein investigation in the living cell.
  • GFP green fluorescent protein
  • Particular examples of labels L 1 and L 2 are also boron compounds displaying non-linear optical properties, or a member of a FRET pair which changes its spectroscopic properties on reaction of the compound of formula (1 ) with the binding partner B.
  • the binding partner B may be bound to a solid support.
  • the label L 1 or L 2 may already be attached to a solid support when entering into reaction with binding partner B, or with binding partner B bound to the compound of formula (1 ), respectively, or may subsequently, i.e. after the corresponding reaction, be used to attach the binding partner B to a solid support.
  • the label may be one member of a specific binding pair, the other member of which is attached or attachable to the solid support, either covalently or by any other means.
  • a specific binding pair considered is e.g. biotin and avidin or streptavidin. Either member of the specific binding pair may be the label L 1 or L 2 , the other being attached to the solid support.
  • labels allowing convenient binding to a solid support are e.g. maltose binding protein, glycoproteins, FLAG tags, or reactive substituents allowing chemoselective reaction between such substituent with a complementary functional group on the surface of the solid support.
  • pairs of reactive substituents and complementary functional group are e.g. amine and activated carboxy group forming an amide, azide and a propiolic acid derivative undergoing a 1 ,3-dipolar cycloaddition reaction, amine and another amine functional group reacting with an added bifunctional linker reagent of the type of activated dicarboxylic acid derivative giving rise to two amide bonds, or other combinations known in the art.
  • Examples of a convenient solid support are e.g. glass surfaces such as glass slides, microtiter plates, and suitable sensor elements, in particular functionalized polymers (e.g. in the form of beads), chemically modified oxidic surfaces, e.g. silicon dioxide, tantalum pentoxide or titanium dioxide, or also chemically modified metal surfaces, e.g. noble metal surfaces such as gold or silver surfaces. Irreversibly attaching and/or spotting a compound of formula (1 ) may then be used to attach binding partners B in a spatially resolved manner, particularly through spotting, on the solid support representing protein microarrays, DNA microarrays or arrays of small molecules.
  • suitable sensor elements e.g. glass surfaces such as glass slides, microtiter plates, and suitable sensor elements, in particular functionalized polymers (e.g. in the form of beads), chemically modified oxidic surfaces, e.g. silicon dioxide, tantalum pentoxide or titanium dioxide, or also chemically modified metal surfaces, e.g. noble metal surfaces such
  • the label L 1 or L 2 is capable of generating reactive radicals, such as hydroxyl radicals, upon exposure to an external stimulus
  • the generated radicals can then inactivate the binding partner B, e.g. an AGT fusion protein, as well as those proteins that are in close proximity of such AGT fusion protein, allowing to study the role of these proteins.
  • the binding partner B e.g. an AGT fusion protein
  • examples of such labels are tethered metal-chelate complexes that produce hydroxyl radicals upon exposure to H 2 O 2 and ascorbate, and chromophores such as malachite green that produce hydroxyl radicals upon laser irradiation.
  • chromophores and lasers to generate hydroxyl radicals is also known in the art as chromophore assisted laser induced inactivation (CALI).
  • CALI chromophore assisted laser induced inactivation
  • labels considered are for example fullerenes, boranes for neutron capture treatment, nucleotides or oligonucleotides, e.g. for self-addressing chips, peptide nucleic acids, and metal chelates, e.g. platinum chelates that bind specifically to DNA.
  • L 1 or L 2 represent two or more labels, these labels may be identical or different. Particular preferred combinations are two different affinity labels, or one affinity label and one chromophore label, in particular one affinity label and one fluorophore label.
  • L 1 or L 2 representing a plurality of labels may consist of up to 100 same or different labels, preferably up to 5 same or different labels, and may also comprise appropriately functionalized dendritic structures, where the outer sphere of such a dendritic structure carries the same or different label molecules.
  • L 1 or L 2 is a group A
  • the reaction with a binding partner B leads to dimerization of such binding partner B.
  • the chemical structure of such dimers may be either symmetrical (homodimers) or unsymmetrical (heterodimers).
  • the compound of formula (2) represents a simple azide N 3 -R 4 -H.
  • this compound serves as a scavenger reagent, cleaving the entity from the group A and the binding partner B without introducing another label, as will be described in more detail hereinbelow.
  • the present invention relates also to novel azides of formula (2)
  • L 2 is a label, a plurality of same or different labels, or a group A
  • R 4 is a linker.
  • novel azides are e.g. compounds of formula (2) wherein L 2 is a group A, in particular a group A which is recognized as a substrate by an optionally modified O 6 - alkylguanine-DNA alkyltransferase or by an acyl carrier protein, such as the substituted benzylguanine (47) described below.
  • Other novel azides are e.g. compounds of formula (2) with a particular combination of linker R 4 and label L 2 , for example compounds (40), (42) and (44) described below.
  • the present invention provides a new method and reagents for labeling proteins of interest and for chemoselective cleavage and re-labeling of labeled proteins of interest, leaving the proteins of interest in their native state.
  • This approach is particular useful for in vitro protein purification and labeling.
  • the reaction can be carried out under a wide range of mild reaction conditions, making this application particularly suitable for the chemical modification of protein tags and fusion proteins with proteins of interest.
  • the method features the specific transfer of a "molecular probe" (L 1 ) to a target peptide sequence (peptide of interest incorporated in binding partner B).
  • the interaction between the molecular probe L 1 and the target peptide sequence is non- covalent, i.e. when the interaction of group A with the binding partner B is non-covalent.
  • Most preferred is the specific and covalent introduction of the molecular probe L 1 to an AGT or ACP fusion protein with the protein of interest.
  • the molecular probe L 1 can be cleaved off and replaced by a new (the same or different) label L 2 by a chemoselective ligation/cleavage reaction that can be carried out under a variety of different mild reaction conditions, which do not interfere with the native state of the protein of interest.
  • the reagent used to re-label or cleave the first label employs the functional group of an azide. Azides (e.g. compounds of formula (2)) do not appear in biomolecules and are therefore termed "bio-orthogonal". Since they have a high intrinsic reactivity they are perfect reagents for the chemoselective reaction in the Staudinger ligation as described in this invention.
  • the present invention provides a possibility for covalent labeling of AGT or ACP fusion proteins (as the binding partner B) and to exchange the first label L 1 by a second label L 2 in a highly specific manner, and at the same time conserving the covalent nature of the bond between fusion protein and label.
  • the invention relates to a method for detecting and/or manipulating a protein of interest, wherein the protein of interest is incorporated into a binding partner B, most preferably an AGT or ACP fusion protein, the binding partner B is contacted with a compound of formula (1 ), i.e. a particular substrate for binding partner B carrying a properly substituted phosphine entity and a label as described hereinbefore, and the reaction product of binding partner B and the compound of formula (1 ) is detected and/or further manipulated using the label.
  • a compound of formula (1 ) i.e. a particular substrate for binding partner B carrying a properly substituted phosphine entity and a label as described hereinbefore
  • the reaction product of binding partner B and the compound of formula (1 ) is detected and/or further manipulated using the label.
  • manipulation any physical or chemical treatment is understood.
  • manipulation may mean isolation from cells, purification, quantification, reaction with a compound of formula (2), and/or reaction with a reaction partner for the label.
  • detection may be before or after manipulation, or may occur during manipulation as defined herein.
  • binding partner B with the compound of formula (1 ), and optionally also with a compound of formula (2) can generally be performed in vitro, either in cell extracts or with already purified or enriched forms of binding partner B.
  • a protein or peptide of interest is fused to a tag that recognizes (reacts) specifically with the group A of compound (1 ).
  • Binding partner B as defined hereinbefore then corresponds to the entity "protein of interest — tag” (Schemel ).
  • the "tags” are preferably mutants of O 6 -alkylguanine-DNA alkyltransferase (AGT) or an acyl-carrier protein (ACP).
  • AGT O 6 -alkylguanine-DNA alkyltransferase
  • ACP acyl-carrier protein
  • the protein or peptide of interest may be of any length and both with and without secondary, tertiary or quaternary structure, and preferably consists of at least twelve amino acids and up to 2000 amino acids, preferably between 50 and 1000 amino acids.
  • group A may still be present or may be displaced as a result of the reaction with the binding partner B. This is the case, for example, in the preferred method wherein group A is a purine type group of formula (3) and the "tag" is AGT, or wherein group A is a coenzyme A type group of formula (4) and the "tag” is ACP.
  • the next two steps in Scheme 1 demonstrate the reaction of compound (6) with a compound of formula (2) corresponding to the Staudinger ligation.
  • the intermediate formed in the reaction of the phosphine with the azide under loss of nitrogen, the iminophosphorane, is further converted to the final product of formula (7) now carrying the label L 2 connected to the protein of interest, simultaneously cleaving off a phospine oxide together with label L 1 , originally bound to the protein of interest.
  • the original electrophilic functional group R 2 is changed to a related functional group now carrying an amino residue. If L 2 is hydrogen, the conversion of compound (6) to compound (7) corresponds to a simple cleavage of label L 1 from the binding partner B.
  • This reaction with an azide of formula (2) is accomplished in aqueous buffered solution.
  • Usual conditions are again a buffer solution in a pH range of 5 to 9, and a temperature between 4° and 5O 0 C, i.e. conditions under which the protein of interest remains unchanged.
  • the buffer used depends upon the properties of the protein of interest incorporated in the binding partner B.
  • Preferred concentration of the compound of formula (2) is in the range of 1 ⁇ M up to 100 mM.
  • the reaction time can be varied between 1 min and 24 hours.
  • Methods of manufacture of novel substrates and intermediates are also an object of this invention. These methods are generally known in the art, are chosen as to best produce the preferred substrates of the invention, and are exemplified hereinbelow. Particular methods for the synthesis of the A group of compound (1 ), wherein A is a substrate for AGT, and of combinations of A with a linker, of labels combined with a linker, and the use of such intermediates are disclosed in International Patent Application WO 2004/031405.
  • compounds of formula (1 ) are manufactured by reacting A) a compound of formula (1 C)
  • Triarylphosphines containing diverse substituted aromatic substituents are accessible via palladium-catalyzed P-C coupling reactions between appropriate phosphines and aromatic iodo- or bromo-aryl compounds.
  • DJ. Brauer et al. J. Organomet. Chem. 2002, 645, 14-26 In most cases, no protecting groups are required.
  • the commercially available diamine (11 ) is coupled to (10) under standard peptide coupling conditions using benzotriazol-1 -yloxy-tripyrrolidino- phosphonium hexafluorophosphate (PyBOP) in dimethyl formamide (DMF) as an activation reagent for the phosphine-carboxylic acid (10).
  • the building block (12) is subsequently coupled to a carboxylic acid N-succinimide ester activated solid support of formula (13).
  • the benzylguanine derivative (15) is activated in situ (PyBOP, triethylamine; DMF) and coupled to the phenolic functional group in (14) to yield the appropriate functionalized surface (16) for the immobilization of AGT fusion proteins.
  • Scheme 2 The synthesis of an intermediate useful in the preparation of compounds of formula (1 ) wherein R 2 is a carbamate group (i.e. an ester group bound to nitrogen) is summarized in Scheme 3.
  • the phenolic group of the functionalized solid support (14) is activated by the reaction with 4-nitrophenyl chloroformate (17) to yield the carbonate (18).
  • the subsequent reaction of (18) with the amino-benzylguanine (19) forms the desired carbamate of formula (20).
  • Scheme 3 provides a method for the synthesis of an intermediate useful in the context of a compound of formula (1 ) wherein X is a substituted benzyl group.
  • the synthesis is based on the addition of diphenylphosphine to 4-formylbenzoic acid (21 ) to form alcohol (22).
  • This building block is extended with the polyethylene glycol linker (1 1 ), and the resulting compound (23) coupled to the solid support (13), followed by esterification with benzylguanine-carboxylic acid derivative (15). This leads to the desired modified solid support of formula (24).
  • Scheme 4 The synthesis of an intermediate useful in the preparation of compounds of formula (1 ) wherein A is phosphopantetheine (a derivative of coenzyme A) is shown in Scheme 5.
  • Esterification of the phenolic hydroxy group in phosphine (10) with the maleimido- carboxylic acid (25) and subsequent immobilization of the resulting compound (26) to an amino-functionalized solid support (27) yields a thiol reactive surface (28).
  • the free thiol group of a suitable coenzyme A derivative is added to the double bond of maleimide (28) to give (29), a compound of formula (1 ) representing a CoA covered surface suitable for reaction with an ACP fusion protein.
  • Scheme 5 The synthesis of a compound of formula (1 ) wherein R 2 is an ester group and L 1 is biotin is summarized in Scheme 6.
  • Phospine (10) and the benzylguanine-carboxylic acid derivative (15) are coupled by in situ activation of (15) to yield ester (30).
  • This compound is coupled to amine (31 ) (E. Saxon, CR. Bertozzi, Science, 2000, 287, 2007-2010) using 1 -hydroxybenztriazole, 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide or PyBOP.
  • the alcohol group in the phosphine-borane complex (35) is activated by methanesulfonyl chloride (MsCI) and converted to the protected thiol (37) with thioacetic acid (using triethylamine as a base).
  • MsCI methanesulfonyl chloride
  • thioacetic acid using triethylamine as a base.
  • the phosphinothiol (37) may be further elaborated to a compound of formula (1 ) in analogy to the reactions in Schemes 2 to 6.
  • Scheme 7 Examples for the synthesis of compounds of formula (2) are shown in Schemes 8 to 10.
  • a useful intermediate is 11 -azido-3,6,9-trioxaundecanamine (38) prepared according to A.W. Schwabacher et al., J. Org. Chem. 1998, 63, 1727-1729.
  • the synthesis of several compounds of formula (2) is summarized in Scheme 8. (38) is coupled with N-succinimidyl esters of formula (39), (41 ) and (43) bearing a biotin, digoxigenin or fluorophore label, respectively, to give azides of formula (40), (42) and (44), respectively.
  • Scheme 8 A compound of formula (2) wherein L 2 is a group A, namely a benzylguanine type compound useful for reaction with AGT, is shown in Scheme 9.
  • Aminomethyl-benzyl- guanine (45) is acylated with chloroacetic anhydride in methanol to give the intermediate (46).
  • Displacement of the halide with sodium azide provides the desired compound (47).
  • Example 1 O 6 -r4-(13-f4-r4-(13-Biotinylamido-4,7,10-trioxa-tridecyl-aminocarbonyl)-2- diphenylphosphino-phenyloxycarbonyl1-butanoylamino)-2, 5,8,1 1 -tetraoxa-tridecvD- benzyli ⁇ uanine (32)
  • N- (13-biotinylamido-4,7,10-trioxa-tridecyl)-3-diphenylphosphino-4-hydroxybenzamide (Example 1 b, 67 mg, 0.09 mmol) is added and the reaction mixture heated to 5O 0 C for 1 min, then stirred at room temperature for 8 h.
  • the crude product is precipitated in 50 ml diethyl ether, all organic solvents decanted and the precipitate dried in vacuo. The precipitate is further purified via reversed phase medium pressure liquid chromatography (MPLC).
  • MPLC reversed phase medium pressure liquid chromatography
  • Example 1 a O 6 -r4-(13-Glutarylamido-2, 5,8,1 1 -tetraoxa-tridecyl)-benzyl1 ⁇ uanine (15) (- ⁇ - ⁇ -(IS-Amino ⁇ S ⁇ H -tetraoxa-tridecyO-benzylJguanine (491 mg, 1.10 mmol) is dissolved in 10 ml dimethylacetamide by heating the solution to 8O 0 C for 5 min.
  • Example 1 b N-(13-Biotinylamido-4,7,10-trioxa-tridecyl)-3-diphenylphosphino-4-hydroxy- benzamide
  • N-(13-Amino-4,7,10-trioxa-tridecyl)-3-diphenylphosphino-4-hydroxy-benzamide (Example 1c, 53 mg, 0.1 mmol) is dissolved in 1 ml DMF followed by the addition of N-biotinyl- succinimide (Biotin-NHS, 38 mg, 0.1 1 mmol) and triethylamine (15 ⁇ ). The reaction mixture is stirred at room temperature over night and the product purified via reversed phase MPLC.
  • 3-Diphenylphosphino-4-hydroxy-benzoic acid (Example 1 d, 100 mg, 0.31 mmol) and PyBOP (177 mg, 3.41 mmol) are dissolved in 5 ml DMF/CH 3 CN (1 :1 ) and stirred for 5 min at room temperature. 4,7,10-Trioxa-1 ,13-tridecanediamine (136 mg, 6.2 mmol) and triethylamine (46 Dl) are added and the reaction mixture stirred at room temperature for 48 h.
  • Example 1 d 3-Diphenylphosphino-4-hvdroxy-benzoic acid (10) 4-Hydroxy-3-iodo-benzoic acid (Example 1 e, 264 mg, 1 mmol) and Pd(OAc) 2 (2.2 mg) are suspended in 4 ml dry acentonitrile, and 0.5 ml triethylamine are added. The mixture is degassed in an ultrasonic bath while passing dry argon through it for 30 min. Diphenylphosphine (0.175 ml, 1 mmol) is added and the mixture heated to reflux for 48 h. After cooling to room temperature all volatiles are removed in vacuo.
  • 3-Amino-4-hydroxybenzoic acid (1.0 g, 6.53 mmol) is dissolved in 10 ml HCI cone, and cooled to O 0 C in an ice bath. Under vigorous stirring NaNO 2 (540 mg, 7.84 mmol) dissolved in 2 ml of water is added drop wise and the mixture stirred at room temperature for 40 min. The mixture is subsequently filtered through glass-wool into a solution of Kl (10.82 g, 65 mmol) in 14 ml H 2 O. The solution is stirred for 1.5 h diluted with CH 2 CI 2 (150 ml) and washed twice with 50 ml Na 2 SO 3 sat., 50 ml water, and 50 ml of brine.
  • O 6 -(4-Glutarylamidomethyl-benzyl)guanine (Example 2a, 54 mg, 0.14 mmol) is suspended in 3 ml dimethylacetamide, and PyBOP (84 mg, 0.16 mmol) and diisopropylethylamine (50 ml) are added. After 5 min a clear solution is formed.
  • N-(13-biotinylamido-4,7,10-trioxa-tridecyl)-3-diphenylphosphino-4-hydroxy- benzamide (Example 1 b, 105 mg, 14 mmol) is added and the reaction mixture heated to 5O 0 C for 1 min, then stirred at room temperature for 8 h.
  • the crude product is precipitated in 50 ml diethyl ether, all organic solvents decanted and the precipitate dried in vacuo. The precipitate is further purified with reversed phase MPLC.
  • O 6 -(4-Aminomethyl-benzyl)guanine (300 mg, 1.10 mmol) is dissolved in 7 ml dimethyl ⁇ acetamide by heating the solution to 8O 0 C for 5 min. After cooling to room temperature, glutaric anhydride (126 mg, 1.10 mmol), N,N-dimethylaminopyridine (DMAP, 60 mg, 0.48 mmol) and diisopropylethylamine (170 ⁇ ) are added and the reaction mixture stirred at room temperature for 20 h. The reaction mixture is poured into 70 ml water resulting in a clear solution. The pH is adjusted to 4-5 by 0.5 N HCI and the white precipitate isolated by filtration.
  • DMAP N,N-dimethylaminopyridine
  • Example 3a Immobilized N-(13-amino-4,7,10-trioxa-tridecyl)-3-diphenylphosphino-4- hvdroxy-benzamide (14)
  • NHS-Sepharose4FastFlow (GE-Amersham) is modified with N-(13-amino-4,7,10-trioxa- tridecyl)-3-diphenylphosphino-4-hydroxy-benzamide (Example 1 c) at 1 mM according to the manufacturer's instructions (coupling at 1 mM, blocking with 0.5 M ethanolamine, washing). An appropriate quantity of the resulting resin is dried by brief centrifugation. This and all following steps are performed on Biorad Microbiospin columns.
  • N PC-activated solid support (18, Example 4a) is treated with a freshly prepared solution of (- ⁇ - ⁇ -(IS-amino ⁇ SAH -tetraoxa-tridecyO-benzylt ⁇ uanine (5 mM) and DMAP (5 mM) in 1 ml anhydrous DMF for 14 h under vigorous shaking. Excess reagent is removed by washing twice with DMF for 5 min. Residual p-nitrophenyl carbonate groups are subsequently quenched by treatment with ethanolamine.
  • the resin is immersed in a 0.5 M solution of ethanolamine in anhydrous DMF for 15 min at room temperature under vigorous shaking, rinsed with anhydrous DMF, washed in anhydrous DMF for 5 min, rinsed extensively with deionized water, and finally washed in deionized water twice for 5 min.
  • Phosphine modified solid support (14, Example 3a) is activated by applying a freshly prepared mixture of p-nitrophenyl chloroformate (NPC, 1 mM) and triethylamine (1 mM) in anhydrous THF for 1 h at room temperature under vigorous shaking. Excess NPC is removed from the solid support by several washings with THF, water and again THF. The resulting resin is dried by brief centrifugation under a flow of nitrogen, then stored in a nitrogen atmosphere until used for further functionalization.
  • NPC p-nitrophenyl chloroformate
  • Coenzyme A disodium salt (5 mg, 0.006 mmol) in 200 ⁇ DMF and 50 ⁇ 50 mM Tris Cl pH 7.5 are added to an appropriate quantity of modified beads (28) of Example 5a. The mixture is shaken for 4 hours at room temperature. It is washed several times with CH 3 CN / H 2 O 1 :4, and finally washed in deionized water twice for 5 min.
  • Example 5a Immobilized 3-diphenylphosphino-4-r4-(N-maleimidomethyl)-cvclohexyl- carbonyloxyi-benzoic acid (28)
  • EAH Sepharose 4B (GE-Amersham) is modified with 3-diphenylphosphino-4-[4-(N- maleimidomethyl)-cyclohexyl-carbonyloxy]-benzoic acid (26, Example 5b) at 1 mM according to the manufacturer's instructions (coupling at 1 mM, blocking, washing). An appropriate quantity of the resulting resin is dried by brief centrifugation. This and all following steps are performed on Biorad Microbiospin columns.
  • Example 5b 3-Diphenylphosphino-4-[4-(N-maleimidomethyl)-cvclohexyl-carbonyloxy1- benzoic acid (26)
  • Example 6 1 1 -Azido-1 -(6-biotinylamido-caproylamino)-3,6,9-trioxa-undecane
  • 6-Biotinylamido-caproic acid N-succinimide 250 mg, 0.55 mmol
  • 1 -amino-11 -azido- 3,6,9-trioxa-undecane 240 mg,1 .1 mmol
  • the reaction mixture is stirred at room temperature over night, than all volatiles are removed in vacuo.
  • the crude product is adsorbed on silica gel and purified by flash- column chromatography (gradient CH 2 CI 2 :Me0H 50:1 to 10:1 ) yielding 210 mg (0.37 mmol, 68%) of the title compound.
  • MS(ESI) m/z. 558.4 [M+H] + .
  • Example 7 11 -Azido-1 -(fluoresceine-5(6)-carboxamido)-3,6,9-trioxa-undecane
  • Example 8 Immobilization and release of AGT from a modified resin Resin (16) (100 Dl, Example 3) is dried by brief centrifugation on a Biorad Microbiospin column.
  • a solution of AGT (100 Dl, 25 DM, a recombinantly expressed variant of human alkylguanine-DNA-alkyltransferase genetically optimized for reactivity, available from Covalys under the tradename SNAP26) is loaded onto the resin and left on the resin at room temperature for 1 h. Subsequently the solution is separated from the resin and the resin is washed 2 times with buffer (100 Dl). The washout and the two rinsing solutions are combined. The protein content of the combined washout solutions is compared with an identical protein sample pretreated for 30 min with 100 DM benzylguanine. The protein content indicates that at least 20% of the AGT is bound to the resin.
  • the resin is mixed with 100 Dl of 0.5 mM 1 -azido-3,6,9-trioxadecane, (CJ. Hawker et al., J. Org. Chem. 1994, 59, 3503) and incubated for 1 hour at 25 0 C.
  • the solution is removed from the column by centrifugation and combined with 2 times 100 Dl wash solution.
  • the protein content is analyzed from an aliquot. At least 20% of the immobilized protein is removed from the column.
  • eluted protein is removed from low molecular weight labeling compounds by sequential separation over two NAP5 column (GE-Amersham).
  • Example 9 Binding and release with attachment of fluorescent group Resin (16) (100 Dl, Example 3) is loaded with the AGT (SNAP26 as described in Example 8) under identical conditions.
  • the resin is mixed with 100 Cl of 0.5 mM fluorescein-PEG-azide of Example 7.
  • the eluted protein is sequentially purified over two NAP5-columns to reduce the level of free fluorescein label.
  • the protein solution is loaded onto the resin, afterwards buffer is added to a total volume of 700 ⁇ l. Protein is subsequently eluted by further adding buffer and collecting the eluate. From the first column 500 ⁇ l of eluate are used for the second purification step.
  • a sample of the resin not loaded with protein is treated in the same way to establish a background value. Fluorescence is read on a plate reader instrument (Tecan, Safire; fluorescein settings). The protein level is estimated after background subtraction to be greater than 10 nM by comparison with fluorescein solutions.
  • Example 10 Binding and release with attachment of affinity group Example 8 is repeated, however AGT is added at 100 ⁇ M concentration. Subsequently AGT is eluted with 100 d of 0.5 mM biotin-PEG-azide of Example 6. Again at least 4% of the protein initially loaded to the resin are recovered. About 1 Dg of the protein are loaded in duplicates to an SDS-gel as described below (Example 12). Separation, western blotting, and detection are done as described below. The resulting protein bands are clearly biotin-labeled, while a control with preblocked protein shows no significant bands on the western blot.
  • Example 11 Docking of AGT with fluorescein
  • a solution of AGT (100 Cl, 20 DM) is labeled with the benzylguanine-phosphine-biotin compound (32) (Example 1 , 30 CM) for 1 hour at 25 0 C.
  • the resulting mixture is purified over a Biorad Microbiospin column.
  • a fraction of the eluate is mixed with 0.5 mM fluorescein-PEG-azide of Example 7 and incubated for 1 hour at 25 0 C.
  • the resulting solution is again separated sequentially over 2 Biorad Microbiospin columns to reduce the concentration of free fluorescein-PEG-azide.
  • AGT preblocked with 100 DM benzylguanine for 30 min at 25 0 C.
  • ACP (20 DM) is loaded with phosphine-CoA compound (29) (Example 5, 30 DM) by coincubating with ACP-Synthase (ACPS, 5 DM) for 1 h. Labeled protein is separated over a BioRad Microbiospin column to remove phosphine-CoA not bound to protein.
  • Fluorescein-PEG-azide of Example 7 is used for the modification of the protein.
  • a sample of ACP is incubated with non-modified coenzyme A (50 DM) and ACPS (5 DM) at 25 0 C for 1 hour. Again unreacted substrate is removed by spin-dialysis.
  • the undiluted samples and a sample diluted 1 :5 are loaded on an SDS-gel. After separation analysis is done on a UV-transilluminator, followed by Coomassie Blue staining. Protein bands are clearly visible for control and for the sample not preblocked and treated with the fluorescein-PEG-azide, while only the sample not preblocked and treated with the fluorescein-PEG-azide shows significant green fluorescence.
  • Example 14 Docking of ACP with biotin All steps of the reaction are carried out as given above in Example 13, but the fluorescein- PEG-azide is replaced with biotin-PEG-azide of Example 6. All samples are loaded in duplicate. After separation the gel is cut in half and one half is stained with Coomassie Blue. The other half is transferred to a semi-dry blotting system (Biorad), proteins are transferred to nitrocellulose and subsequently a western blot detection is performed with streptavidin-horse radish peroxidase conjugate and chromogenic substrate (all materials from Pierce; all conditions as published by Pierce). In the Coomassie Blue stain all bands are clearly visible, while on the western blot only the bands of non blocked protein incubated with the biotin-PEG-azide derivative give a clear signal.
  • Biorad semi-dry blotting system
  • Example 15 Binding and release of ACP-ta ⁇ to phosphine-modified beads
  • Binding and release of an ACP-tagged protein is done at the same concentrations as in Example 14 before.
  • Resin material offering a phosphine-CoA substrate (29) is used.
  • ACP-fusion protein (25 DM) and ACP-Synthase (5DM) are loaded onto the resin and left on the resin for 1 h. At least 20% of the ACP are retained on the resin.
  • the resin is mixed with 1 -azido-3,6,9-trioxadecane (100 Dl, 0.5 mM) and incubated for 1 hour at 25 0 C.
  • the solution is removed from the column by centrifugation and combined with 2 times 100 Dl wash solution.
  • the protein content is analyzed from an aliquot. During that step at least 20% of the immobilized protein is removed from the column.
  • eluted protein is removed from the low molecular weight labeling compounds by sequential separation over two NAP5 columns (GE-Amersham) as described in Example 9.

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Abstract

La présente invention concerne des procédés et des réactifs pour marquer un composé cible et une réaction chimiosélective clivant et ligaturant simultanément différents traceurs sur un composé cible. Ces réactifs sont des phosphines de formule (1) dans laquelle A est un groupe qui se lie spécifiquement à ou qui réagit avec un partenaire de liaison B comprenant une protéine cible d’intérêt, R1 et R3 sont des liants, R2 est un groupe fonctionnel électrophile, X, Y et Z sont un groupe aryle ou un autre groupe, L1 est un traceur ou un autre groupe, et R3 est lié soit à X soit à Z, et des azides de formule (2) N3-R4-L2 (2) dans laquelle L2 est un traceur ou un autre groupe et R4 est un liant.
PCT/EP2005/054114 2004-08-23 2005-08-22 Procédé pour la purification de protéines et marquage basé sur une réaction chimiosélective WO2006021553A1 (fr)

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US9045739B2 (en) 2004-01-16 2015-06-02 Fraunhofer-Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Immunokinases
US8829178B2 (en) 2005-12-21 2014-09-09 Fraunhofer-Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Immuno-RNA-constructs
US7869712B2 (en) 2006-12-05 2011-01-11 Electronics And Telecommunications Research Institute Method and apparatus for increasing transmission capacity in optical transport network
WO2009013359A3 (fr) * 2007-07-25 2009-03-19 Fraunhofer Ges Forschung Protéines de fusion d'anticorps recombinantes à auto-couplage
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