WO2004031405A1 - Substrates for o6-alkylguanine-dna alkyltransferase - Google Patents
Substrates for o6-alkylguanine-dna alkyltransferase Download PDFInfo
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- WO2004031405A1 WO2004031405A1 PCT/EP2003/010889 EP0310889W WO2004031405A1 WO 2004031405 A1 WO2004031405 A1 WO 2004031405A1 EP 0310889 W EP0310889 W EP 0310889W WO 2004031405 A1 WO2004031405 A1 WO 2004031405A1
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- carbon atoms
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- 239000000758 substrate Substances 0.000 title claims abstract description 76
- 102000002226 Alkyl and Aryl Transferases Human genes 0.000 title abstract description 16
- 108010014722 Alkyl and Aryl Transferases Proteins 0.000 title abstract description 16
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 82
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 82
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 56
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 54
- 238000000034 method Methods 0.000 claims abstract description 33
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 20
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 18
- 125000000623 heterocyclic group Chemical group 0.000 claims abstract description 17
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims abstract description 5
- 150000001875 compounds Chemical class 0.000 claims description 94
- -1 1-cyclohexenyl Chemical group 0.000 claims description 83
- 125000004432 carbon atom Chemical group C* 0.000 claims description 77
- 125000005647 linker group Chemical group 0.000 claims description 38
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 34
- 239000001257 hydrogen Substances 0.000 claims description 32
- 229910052739 hydrogen Inorganic materials 0.000 claims description 32
- 229910052760 oxygen Inorganic materials 0.000 claims description 30
- 239000001301 oxygen Substances 0.000 claims description 30
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 28
- 125000000217 alkyl group Chemical group 0.000 claims description 27
- 239000007787 solid Substances 0.000 claims description 26
- 125000002947 alkylene group Chemical group 0.000 claims description 25
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 23
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 21
- 125000001072 heteroaryl group Chemical group 0.000 claims description 21
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 4
- 125000004434 sulfur atom Chemical group 0.000 claims description 4
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims description 4
- 125000004417 unsaturated alkyl group Chemical group 0.000 claims description 4
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- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical group OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 3
- 229940072107 ascorbate Drugs 0.000 claims description 3
- 235000010323 ascorbic acid Nutrition 0.000 claims description 3
- 239000011668 ascorbic acid Substances 0.000 claims description 3
- 125000002993 cycloalkylene group Chemical group 0.000 claims description 3
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- 125000005842 heteroatom Chemical group 0.000 claims description 3
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 3
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- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims description 2
- 125000000304 alkynyl group Chemical group 0.000 claims description 2
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- 125000004430 oxygen atom Chemical group O* 0.000 claims description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical class O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 abstract description 54
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 abstract description 16
- 239000000543 intermediate Substances 0.000 abstract description 13
- 238000012546 transfer Methods 0.000 abstract description 11
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- 150000003463 sulfur Chemical class 0.000 abstract description 2
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 87
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 63
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- 238000006243 chemical reaction Methods 0.000 description 35
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- XWNJMSJGJFSGRY-UHFFFAOYSA-N 2-(benzylamino)-3,7-dihydropurin-6-one Chemical group N1C=2N=CNC=2C(=O)N=C1NCC1=CC=CC=C1 XWNJMSJGJFSGRY-UHFFFAOYSA-N 0.000 description 6
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- 239000000833 heterodimer Substances 0.000 description 1
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- 125000002962 imidazol-1-yl group Chemical group [*]N1C([H])=NC([H])=C1[H] 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
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- 125000002346 iodo group Chemical group I* 0.000 description 1
- 125000006328 iso-butylcarbonyl group Chemical group [H]C([H])([H])C([H])(C(*)=O)C([H])([H])[H] 0.000 description 1
- 229960004592 isopropanol Drugs 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 125000002183 isoquinolinyl group Chemical group C1(=NC=CC2=CC=CC=C12)* 0.000 description 1
- 125000001786 isothiazolyl group Chemical group 0.000 description 1
- CTAPFRYPJLPFDF-UHFFFAOYSA-N isoxazole Chemical compound C=1C=NOC=1 CTAPFRYPJLPFDF-UHFFFAOYSA-N 0.000 description 1
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- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- YCJZWBZJSYLMPB-UHFFFAOYSA-N n-(2-chloropyrimidin-4-yl)-2,5-dimethyl-1-phenylimidazole-4-carboxamide Chemical compound CC=1N(C=2C=CC=CC=2)C(C)=NC=1C(=O)NC1=CC=NC(Cl)=N1 YCJZWBZJSYLMPB-UHFFFAOYSA-N 0.000 description 1
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- SQDFHQJTAWCFIB-UHFFFAOYSA-N n-methylidenehydroxylamine Chemical compound ON=C SQDFHQJTAWCFIB-UHFFFAOYSA-N 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 108010087904 neutravidin Proteins 0.000 description 1
- 150000002829 nitrogen Chemical class 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 125000001400 nonyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000007344 nucleophilic reaction Methods 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 125000002971 oxazolyl group Chemical group 0.000 description 1
- 150000002923 oximes Chemical class 0.000 description 1
- BPUBBGLMJRNUCC-UHFFFAOYSA-N oxygen(2-);tantalum(5+) Chemical compound [O-2].[O-2].[O-2].[O-2].[O-2].[Ta+5].[Ta+5] BPUBBGLMJRNUCC-UHFFFAOYSA-N 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
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- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
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- 229910052697 platinum Inorganic materials 0.000 description 1
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- CPTBDICYNRMXFX-UHFFFAOYSA-N procarbazine Chemical compound CNNCC1=CC=C(C(=O)NC(C)C)C=C1 CPTBDICYNRMXFX-UHFFFAOYSA-N 0.000 description 1
- 229960000624 procarbazine Drugs 0.000 description 1
- TVDSBUOJIPERQY-UHFFFAOYSA-N prop-2-yn-1-ol Chemical compound OCC#C TVDSBUOJIPERQY-UHFFFAOYSA-N 0.000 description 1
- ZJLMKPKYJBQJNH-UHFFFAOYSA-N propane-1,3-dithiol Chemical compound SCCCS ZJLMKPKYJBQJNH-UHFFFAOYSA-N 0.000 description 1
- YORCIIVHUBAYBQ-UHFFFAOYSA-N propargyl bromide Chemical compound BrCC#C YORCIIVHUBAYBQ-UHFFFAOYSA-N 0.000 description 1
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 1
- UORVCLMRJXCDCP-UHFFFAOYSA-N propynoic acid Chemical class OC(=O)C#C UORVCLMRJXCDCP-UHFFFAOYSA-N 0.000 description 1
- 238000000734 protein sequencing Methods 0.000 description 1
- 125000000246 pyrimidin-2-yl group Chemical group [H]C1=NC(*)=NC([H])=C1[H] 0.000 description 1
- 229940083082 pyrimidine derivative acting on arteriolar smooth muscle Drugs 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
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- 230000008439 repair process Effects 0.000 description 1
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- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 102220034037 rs61753995 Human genes 0.000 description 1
- 102220060558 rs80358621 Human genes 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 231100000489 sensitizer Toxicity 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 125000006253 t-butylcarbonyl group Chemical group [H]C([H])([H])C(C(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- PBCFLUZVCVVTBY-UHFFFAOYSA-N tantalum pentoxide Inorganic materials O=[Ta](=O)O[Ta](=O)=O PBCFLUZVCVVTBY-UHFFFAOYSA-N 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229960004964 temozolomide Drugs 0.000 description 1
- FGTJJHCZWOVVNH-UHFFFAOYSA-N tert-butyl-[tert-butyl(dimethyl)silyl]oxy-dimethylsilane Chemical compound CC(C)(C)[Si](C)(C)O[Si](C)(C)C(C)(C)C FGTJJHCZWOVVNH-UHFFFAOYSA-N 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 150000003577 thiophenes Chemical class 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000004408 titanium dioxide Substances 0.000 description 1
- 150000003852 triazoles Chemical class 0.000 description 1
- NQPHMXWPDCSHTE-UHFFFAOYSA-N trifluoromethanesulfonyl azide Chemical compound FC(F)(F)S(=O)(=O)N=[N+]=[N-] NQPHMXWPDCSHTE-UHFFFAOYSA-N 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N urea group Chemical group NC(=O)N XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/44—Iso-indoles; Hydrogenated iso-indoles
- C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
- C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
- C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
- C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D333/28—Halogen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
- C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
- C07D473/16—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two nitrogen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
- C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
- C07D473/18—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 one oxygen and one nitrogen atom, e.g. guanine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
Definitions
- the present invention relates to methods of transferring a label from novel substrates to O 6 - alkylguanine-DNA alkyltransferases (AGT) and O 6 -alkylguanine-DNA alkyltransferase fusion proteins, and to novel substrates suitable in such methods.
- AGT O 6 - alkylguanine-DNA alkyltransferases
- a 6 -alkylguanine-DNA alkyltransferase fusion proteins and to novel substrates suitable in such methods.
- AGT O 6 -alkylguanine-DNA alkyltransferase
- AGT a nucleophilic reaction of the S N 2 type which explains why not only methyl groups, but also benzylic groups are easily transferred.
- overexpression of AGT in tumour cells is the main reason for resistance to alkylating drugs such as procarbazine, dacarbazine, temozolomide and bis-2- chloroethyl-N-nitrosourea
- inhibitors of AGT have been proposed for use as sensitisers in chemotherapy (Pegg etal, Prog Nucleic Acid Res Mol Biol 51: 167-223, 1995).
- DE 199 03 895 discloses an assay for measuring levels of AGT which relies on the reaction between biotinylated O 6 -aIkylguanine derivatives and AGT which leads to biotinylation of the AGT. This in turn allows the separation of the AGT on a streptavidin coated plate and its detection, e.g. in an ELISA assay. The assay is suggested for monitoring the level of AGT in tumour tissue and for use in screening for AGT inhibitors.
- WO 01/85221 proposes the use of radiolabelled fluoro- or iodo-substituted O 6 -benzyl- guanines for detection of AGT and monitoring the level of AGT.
- PCT/GB02/01636 discloses a method for detecting and/or manipulating a protein of interest wherein the protein is fused to AGT and the AGT fusion protein contacted with an AGT substrate carrying a label, and the AGT fusion protein detected and optionally further manipulated using the label.
- AGT fusion proteins to be used, general structural principles of the AGT substrate and a broad variety of labels and methods to detect the label useful in the method are described.
- the invention relates to a method for detecting and/or manipulating a protein of interest, wherein the protein of interest is incorporated into an AGT fusion protein, the AGT fusion protein is contacted with particular AGT substrates carrying a label, and the AGT fusion protein is detected and optionally further manipulated using the label in a system designed for recognising and/or handling the label.
- the particular AGT substrates used in the method of the invention are O 6 -substituted guanine derivatives or related nitrogen containing hydroxy-heterocycles and their sulfur analogs wherein the O 6 -substituent is an activated methyl derivative suitable for transfer from guanine or the corresponding heterocycle to AGT, and further carrying a label.
- the label may consist of a plurality of same or different labels.
- Activated methyl derivatives are e.g. arylmethyl derivatives suitably substituted in the aryl ring, heteroarylmethyl derivatives suitably substituted in the heteroaryl ring, and allyl type derivatives suitably substituted at the double bond.
- Suitable substituents of the aryl ring, heteroaryl ring or allylic double bond are linkers connecting a label to the aryl ring, heteroaryl ring or allyl group, preferably linkers which may undergo further modification or cleavage, and also linkers which give rise to dimeric or cyclised AGT substrates.
- the invention relates also to the novel AGT substrates as such, to methods of manufacture of such novel substrates, and to intermediates useful in the synthesis of such novel AGT substrates.
- Figure 1A Western Blot of an anti-biotin conjugate coupled to HRP (Pierce).
- Lanes 1 and 2 PGEA hAGT-GST (an AGT fusion protein) after reaction with compound 11 (a compound of formula 1 wherein R 3 is triazolylmethyl) and then with a biotinylated O 6 -benzyl- guanine oligonucleotide known to be an AGT substrate.
- Lane marked “C” control: The reaction product of PGEA hAGT-GST and the biotinylated O 6 -benzylguanine oligonucleotide.
- Figure 1 B Western Blot of a goat anti-fluorescein antibody (Rockland) and a HRP conjugated anti-goat antibody (Sigma).
- Lanes 1 and 2 show the reaction product of compound 60 bearing a fluorescein label and a biotin label (a compound of formula 1 wherein L is a plurality of labels) with PGEA hAGT-GST.
- the lane on the left side shows molecular weight markers of 47 kDa and 79 kDa.
- Figure 2 Sensogram of a surface plasmon resonance of the reaction product of PGEA hAGT- GST (an AGT fusion protein) with compound 54 (compound of formula 1 wherein L is biotin) applied to a Streptavidin Biacore chip.
- the chip is further treated with a solution of an ⁇ GST antibody.
- C Addition of ⁇ GST antibody.
- Figure 3A Western Blot with an anti-hAGT antibody and an anti-mouse-HRP conjugate as a second antibody.
- the ratio of compound 43 (a compound of formula 1 wherein L is a further -R 3 -CH 2 -X-R ⁇ -R 2 residue, i.e. a dimerizing compound) to PGEG hAGT-GST is shown.
- Figure 3B Western Blot with a goat anti-hAGT and an anti-goat IgG of the in vivo reaction product of dimerizing compound 61 with fusion protein HA- 160 hAGT in E. coli.
- different concentrations of 61 are indicated.
- Figure 4 Schematic representation of hAGT-based three-hybrid system. On reaction of the hAGT-LexA fusion protein with the compound of formula 1 wherein L is methotrexate (M) in the presence of a fusion protein DHFR-B42, hAGT-LexA and DHFR-B42 are coupled and transcription started.
- L methotrexate
- a protein or peptide of interest is fused to an O 6 -alkylguanine-DNA alkyltransferase (AGT).
- AGT O 6 -alkylguanine-DNA alkyltransferase
- 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. Examples of such protein or peptide of interest are provided below, and are e.g.
- the protein or peptide of interest is preferably fused to AGT by way of a linker which may be cleaved by an enzyme, e.g. at the DNA stage by suitable restriction enzymes, e.g. AGATCT cleavable by Bgl II, and/or linkers cleavable by suitable enzymes at the protein stage, e.g. tobacco etch virus Nla (TEV) protease.
- Fusion proteins may be expressed in prokaryotic hosts, preferably E. coli, or eukaryotic host, e.g. yeast, insect or mammalian cells.
- the O 6 -alkylguanine-DNA alkyltransferase has the property of transferring a label present on a substrate to one of the cysteine residues of the AGT forming part of a fusion protein.
- the AGT is a known human O 6 -alkylguanine-DNA alkyltransferase, hAGT.
- Murine or rat forms of the enzyme are also considered provided they have similar properties in reacting with a substrate like human AGT.
- O 6 -alkylguanine-DNA alkyltransferase also includes variants of a wild-type AGT which may differ by virtue of one or more amino acid substitutions, deletions or additions, but which still retain the property of transferring a label present on a substrate to the AGT part of the fusion protein.
- AGT variants may be obtained by chemical modification using techniques well known to those skilled in the art.
- AGT variants may preferably be produced using protein engineering techniques known to the skilled person and/or using molecular evolution to generate and select new O 6 -aIkylguanine-DNA alkyltransferases. Such techniques are e.g. saturation mutagenesis, error prone PCR to introduce variations anywhere in the sequence, DNA shuffling used after saturation mutagenesis and/or error prone PCR, or family shuffling using genes from several species.
- the fusion protein comprising protein of interest and an O 6 -alkylguanine-DNA alkyl- transferase (AGT) is contacted with a particular substrate having a label.
- Conditions of reaction are selected such that the AGT reacts with the substrate and transfers the label of the substrate.
- Usual conditions are a buffer solution at around pH 7 at room temperature, e.g. around 25°C.
- AGT reacts also under a variety of other conditions, and those conditions mentioned here are not limiting the scope of the invention.
- AGT irreversibly transfers the alkyl group from its substrate, O 6 -alkylguanine-DNA, to one of its cysteine residues.
- a substrate analogue that rapidly reacts with hAGT is O 6 -benzyl- guanine, the second order rate constant being approximately 10 3 sec "1 M "1 .
- Substitutions of O 6 -benzyIguanine at the C-4 of the benzyl ring do not significantly affect the reactivity of hAGT against O 6 -benzylguanine derivatives, and this property has been used to transfer a label attached to the C-4 of the benzyl ring to AGT.
- the label part of the substrate can be chosen by those skilled in the art dependent on the application for which the fusion protein is intended. After contacting the fusion protein comprising AGT with the substrate, the label is covalently bonded to the fusion protein. The labelled AGT fusion protein is then further manipulated and/or detected by virtue of the transferred label.
- the label may consist of a plurality of same or different labels. If the substrate contains more than one label, the corresponding labelled AGT fusion protein will also comprise more than one label which gives more options for further manipulating and/or detecting the labelled fusion protein.
- the particular AGT substrates are compounds of the formula 1
- R R 2 is a group recognized by AGT as a substrate;
- X is oxygen or sulfur;
- R 3 is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to CH 2 ;
- R 4 is a linker
- L is a label, a plurality of same or different labels, a bond connecting R to Ri forming a cyclic substrate, or a further group -R 3 -CH 2 -X-R R2.
- the residue R-i is preferably a heteroaromatic group containing 1 to 5 nitrogen atoms, recognized by AGT as a substrate.
- a heteroaromatic group R ⁇ is mono- or bicyclic and has 5 to 12, preferably 6 or 9 or 10 ring atoms; which in addition to carrying a substituent R 2 may be unsubstituted or substituted by one or more, especially one, two or three further substituents selected from the group consisting of lower alkyl, such as methyl, lower alkoxy, such as methoxy or ethoxy, hydroxy, oxo, amino, lower alkylamino, di-lower alkylamino, acylamino, halogen, such as chlorine or bromine, halogenated lower alkyl, such as trifluoromethyl, carboxy, lower alkoxycarbonyl, carbamoyl, lower alkylcarbamoyl, or lower alkylcarbonyl.
- lower alkyl such as methyl, lower alkoxy, such as methoxy or ethoxy, hydroxy, oxo, amino, lower alkylamino, di-lower alkylamino
- Lower alkyl is preferably alkyl with 1 to 7, preferably from 1 to 4 C atoms, and is linear or branched; preferably, lower alkyl is butyl, such as n-butyl, sec-butyl, isobutyl, tert-butyl, propyl, such as n-propyl or isopropyl, ethyl or methyl. Preferably lower alkyl is methyl.
- lower alkoxy the lower alkyl group is as defined hereinbefore.
- Lower alkoxy denotes preferably n-butoxy, tert-butoxy, iso-propoxy, ethoxy, or methoxy, in particular methoxy.
- the mono- or bicyclic heteroaromatic group Ri is selected from 2H-pyrrolyl, pyrrolyl, imidazolyl, benzimidazolyl, pyrazolyl, indazolyl, purinyl, 8-azapurinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, quinolinyl, pteridinyl, indolizinyl, 3H-indolyl, indolyl, isoindolyl, triazolyl, tetrazolyl, or benzo[d]pyrazolyl.
- the mono- or bicyclic heteroaromatic group R 1 is selected from the group consisting purinyl, 8-azapurinyl, pyridyl, pyrazinyl, pyrimidinyl, and pyridazinyl.
- R R 2 may be a purine radical of formula 2
- R 2 is hydrogen, alkyl of 1 to 10 carbon atoms, or a saccharide moiety
- R- is hydrogen, halogen, e.g. chloro or bromo, trifluoromethyl, or hydroxy
- R 6 is hydrogen, hydroxy or unsubstituted or substituted amino.
- R 5 or R 6 is hydroxy, the purine radical is predominantly present in its tautomeric form wherein a nitrogen adjacent to the carbon atom bearing R 5 or R 6 carries a hydrogen atom, the double bond between this nitrogen atom and the carbon atom bearing R 5 or R 6 is a single bond, and R 5 or R 6 is double bonded oxygen, respectively.
- a substituted amino group R 6 is lower alkylamino of 1 to 4 carbon atoms or acylamino, wherein the acyl group is lower alkylcarbonyl with 1 to 5 carbon atoms, e.g. acetyl, propionyl, n- or isopropylcarbonyl, or n-, iso- or tert-butylcarbonyl, or arylcarbonyl, e.g. benzoyl.
- R 6 is unsubstituted or substituted amino and the residue X connected to the bond of the purine radical is oxygen, the residue of formula 2 is a guanine derivative.
- R 2 as alkyl of 1 to 10 carbon atoms is linear or branched and includes lower alkyl of 1 to 4 carbon atoms, e.g. methyl, ethyl, butyl, such as n-butyl, sec-butyl, isobutyl or tert-butyl, and propyl, such as n-propyl or isopropyl.
- R 2 as alkyl may also be pentyl, hexyl, heptyl, octyl, nonyl, or decyl, e.g. n-hexyl.
- a saccharide moiety R 2 is a saccharide monomer or oligomer connected with a spacer of variable length to the N 9 position of the guanine base.
- a saccharide moiety R 2 further includes a ⁇ -D-2'-deoxyribosyl, or a ⁇ -D-2'-deoxyribosyl being incorporated into a single stranded oligodeoxyribonucleotide having a length of 2 to 99 nucleotides, wherein the guanine derivative Ri occupies any position within the oligonucleotide sequence.
- R R 2 is a purine radical of formula 2, R 2 is hydrogen, R 5 is hydrogen, R 6 is unsubstituted amino, and X is oxygen, i.e. unsubstituted guanine derivatives.
- the group R R 2 is a 8-azapurine radical of formula 3
- the group R R 2 is a pyrimidine radical of formula 4
- R 2 has the meaning as defined under formula 2, and is preferably hydrogen; and R 7 and Re are both independently of one another hydrogen, halogen, e.g. chloro or bromo, lower alkyl with 1 to 4 carbon atoms, e.g. methyl, amino, or nitro.
- R 7 and Re are both independently of one another hydrogen, halogen, e.g. chloro or bromo, lower alkyl with 1 to 4 carbon atoms, e.g. methyl, amino, or nitro.
- X is preferably oxygen.
- R 3 as an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group is a group sterically and electronically accepted by AGT (in accordance with its reaction mechanism) which allows the covalent transfer of the R 3 -R -L unit to the fusion protein.
- AGT in accordance with its reaction mechanism
- R 4 -L may also have the meaning of a plurality of same or different linkers R 4 carrying a plurality of same or different labels L.
- R 3 as an aromatic group is preferably phenyl or naphthyl, in particular phenyl, e.g. phenyl substituted by R 4 in para or meta position.
- a heteroaromatic group R 3 is a mono- or bicyclic heteroaryl group 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 in addition to carrying a substituent l ⁇ may be unsubstituted or substituted by one or more, especially one, further substituents selected from the group consisting of lower alkyl, such as methyl, lower alkoxy, such as methoxy or ethoxy, halogen, e.g.
- the mono- or bicyclic heteroaryl group Rs is selected from 2H-pyrrolyl, pyrrolyl, imidazolyl, benzimidazolyl, pyrazolyl, indazolyl, purinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, quinolinyl, pteridinyl, indolizinyl, 3H-indolyl, indolyl, isoindolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazoly
- the mono- or bicyclic heteroaryl group is selected from the group consisting of pyrrolyl, imidazolyl, such as 1H-imidazol-1-yl, benzimidazolyl, such as 1- benzimidazolyl, indazolyl, especially 5-indazolyl, pyridyl, e.g.
- the heteroaryl group R 3 is triazolyl, especially 1 -triazolyl, carrying the further substituent R 4 in the 4- or 5-position, tetrazolyl, especially 1 -tetrazolyl, carrying the further substituent R in the 4- or 5-position, or 2- tetrazolyl carrying the further substituent R 4 in 5-position, isoxazolyl, especially 3-isoxazolyl carrying the further substituent R* in 5-position, or 5-isoxazolyl, carrying the further substituent R 4 in 3-position, or thienyl, especially 2-thienyl, carrying the further substituent R in 3-, 4- or 5-position, preferably 4-position, or 3-thienyl, carrying the further substituent R 4 in 4-position.
- heteroaryl group R 3 as triazolyl, carrying the substituent j in 4- or 5- position, and also R 3 as 2-thienyl carrying the substituent l ⁇ in 4- or 5-position.
- An optionally substituted unsaturated alkyl group l ⁇ is 1-alkenyl carrying the further substituent R in 1- or 2-position, preferably in 2-position, or 1-alkynyl.
- Substituents considered in 1-alkenyl are e.g. lower alkyl, e.g. methyl, lower alkoxy, e.g. methoxy, lower acyloxy, e.g. acetoxy, or halogenyl, e.g. chloro.
- R 3 is 1-alkynyl.
- An optionally substituted unsaturated cycloalkyl group is a cycloalkyl group with 3 to 7 carbon atoms unsaturated in 1-position, e.g. 1-cyclopentyl or 1-cyclohexyl, carrying the further substituent R 4 in any position.
- Substituents considered are e.g. lower alkyl, e.g. methyl, lower alkoxy, e.g. methoxy, lower acyloxy, e.g. acetoxy, or halogenyl, e.g. chloro.
- An optionally substituted unsaturated heterocyclyl group has 3 to 12 atoms, 1 to 5 heteroatoms selected from nitrogen, oxygen and sulfur, and a double bond in the position connecting the heterocyclyl group to methylene CH 2 .
- Substituents considered are e.g. lower alkyl, e.g. methyl, lower alkoxy, e.g. methoxy, lower acyloxy, e.g. acetoxy, or halogenyl, e.g. chloro.
- an optionally substituted unsaturated heterocyclyl group is a partially saturated heteroaromatic group as defined hereinbefore for a heteroaromatic group R 3 .
- An example of such a heterocyclyl group is isoxazolidinyl, especially 3-isoxazolidinyl carrying the further substituent in 5-position, or 5-isoxazoIidinyl, carrying the further substituent in 3-position.
- a linker group R-i is preferably a flexible linker connecting a label L or a plurality of same or different labels L to the substrate.
- Linker units are chosen in the context of the envisioned application, i.e. in the transfer of the substrate to a fusion protein comprising AGT. They also increase the solubility of the substrate in the appropriate solvent.
- the linkers used are chemically stable under the conditions of the actual application. The linker does not interfere with the reaction with AGT nor with the detection of the label L, 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 fusion protein comprising AGT.
- a linker R is a straight or branched chain alkylene group with 1 to 300 carbon atoms, wherein optionally
- 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;
- substituents considered are e.g. those obtained when an ⁇ -amino acid, in particular a naturally occurring ⁇ -amino acid, is incorporated in the linker F wherein carbon atoms are replaced by amide functions-NH-CO- as defined under (b).
- part of the carbon chain of the alkylene group l ⁇ is replaced by a group -(NH-CHR-CO) n - wherein n is between 1 and 100 and R represents a varying residue of an ⁇ -amino acid.
- a further substituent is one which leads to a photocleavable linker R ⁇ e.g. an o-nitrophenyl group.
- this substituent o-nitrophenyl is located at a carbon atom adjacent to an amide bond, e.g. in a group -NH-CO-CH 2 -CH(o-nitrophenyl)-NH-CO-, or as a substituent in a polyethylene glycol chain, e.g. in a group -O-CH 2 -CH(o-nitrophenyl)-O-.
- Other photocleavable linkers considered are, e.g. phenacyl, alkoxybenzoin, benzylthioether and pivaloyl glycol derivatives.
- a phenylene group replacing carbon atoms as defined under (e) hereinbefore is e.g. 1,2-, 1 ,3-, or preferably 1 ,4-phenylene.
- the phenylene group is further substituted by a nitro group, and, combined with other replacements as mentioned above under (a), (b), (c), (d), and (f), represents a photocleavable group, and is e.g.
- 4-nitro-1 ,3- phenylene such as in -CO-NH-CH 2 -4-nitro-1,3-phenylene-CH(CH 3 )-O-CO-, or 2-methoxy-5- nitro-1 ,4-phenylene, such as in -CH 2 -O-2-methoxy-5-nitro-1,4-phenylene-CH(CH 3 )-O-.
- Other particular embodiments representing photocleavable linkers are e.g.
- a saturated or unsaturated cycloalkylene group replacing carbon atoms as defined under (e) hereinbefore is derived from cycloalkyl with 3 to 7 carbon atoms, preferably from cyclopentyl or cyclohexyl, and is e.g.
- 1,2- or 1,3-cyclopentylene 1,2-, 1,3-, or preferably 1,4-cyclo- hexylene, or also 1 ,4-cyclohexylene being unsaturated e.g. in 1- or in 2-position.
- a saturated or unsaturated bicycloalkylene group replacing carbon atoms as defined under (e) hereinbefore is derived from bicycloalkyl with 7 or 8 carbon atoms, and is e.g.
- bicyclo[2.2.1] heptylene or bicyclo[2.2.2]octylene preferably 1,4-bicyclo[2.2.1]heptylene optionally unsaturated in 2-position or doubly unsaturated in 2- and 5-position, and 1 ,4-bicyclo[2.2.2] octylene optionally unsaturated in 2-position or doubly unsaturated in 2- and 5-position.
- a bridging heteroaromatic group replacing carbon atoms as defined under (e) hereinbefore is e.g. triazolidene, preferably 1 ,4-triazolidene, or isoxazolidene, preferably 3,5-isoxazolidene.
- a bridging saturated or unsaturated heterocyclyl group replacing carbon atoms as defined under (e) hereinbefore is e.g. derived from an unsaturated heterocyclyl group as defined under R 3 above, e.g. isoxazolidinene, preferably 3,5-isoxazolidinene, or a fully saturated heterocyclyl group with 3 to 12 atoms, 1 to 3 of which are heteroatoms selected from nitrogen, oxygen and sulfur, e.g.
- pyrrolidinediyl piperidinediyl, tetrahydrofuranediyl, dioxanediyl, morpholinediyl or tetrahydrothiophenediyl, preferably 2,5-tetrahydrofuranediyl or 2,5-dioxanediyI.
- a particular heterocyclyl group considered is a saccharide moiety, e.g. an ⁇ - or ⁇ -furanosyl or ⁇ - or ⁇ -pyranosyl moiety.
- Cyclic substructures in a linker * reduce the molecular flexibility as measured by the number of rotatable bonds within R 4 , which leads to a better membrane permeation rate, important for all in vivo labelling applications.
- a particularly preferred linker R is a straight chain alkylene group of 10 to 40 carbon atoms wherein 3 to 12 carbon atoms are replaced by oxygen, one or two carbon atoms are replaced by one or two 1 ,4-triazolidene units, respectively, and optionally one carbon atom is replaced by a 1 ,4-phenylene unit.
- Another particularly preferred linker 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.
- a linker Rj may carry one or more same or different labels, e.g. 1 to 100 same or different labels, in particular 1 to 5, preferably one, two or three, in particular one or two same or different labels.
- the label part L of the substrate can be chosen by those skilled in the art dependent on the application for which the fusion protein is intended. Labels may be e.g. such that the labelled fusion protein 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 labelled fusion protein and/or labels which aid in manipulating the fusion protein by the physical and/or chemical properties specifically introduced by the label to the fusion protein.
- labels L 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 polymer known to those proficient in the art; a nucleic acid or a derivative thereof capable of undergoing base
- labels L are labels L as mentioned hereinbefore with the exception of a radioactively labelled molecule.
- labels L are spectroscopic probes, and molecules which are one part of a specific binding pair which is capable of specifically binding to a partner, so-called affinity labels.
- the label L 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 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 labelled substrate with the AGT fusion protein.
- the fusion protein comprising protein of interest and AGT may be bound to a solid support.
- the label of the substrate reacting with the fusion protein comprising AGT may already be attached to a solid support when entering into reaction with AGT, or may subsequently, i.e. after transfer to AGT, be used to attach the AGT fusion protein 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 binding pair may be the label L of the substrate, the other being attached to the solid support.
- Further examples of labels allowing convenient binding to a solid support are e.g.
- Such 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 bis-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 AGT substrates may then be used to attach AGT fusion proteins in a spatially resolved manner, particularly through spotting, on the solid support representing protein microarrays, DNA microarrays or arrays of small molecules.
- the label L When the label L is capable of generating reactive radicals, such as hydroxyl radicals, upon exposure to an external stimulus, the generated radicals can then inactivate the AGT fusion proteins as well as those proteins that are in close proximity of the AGT fusion protein, allowing to study the role of these proteins.
- 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).
- labelling AGT fusion proteins with chromophores such as malachite green and subsequent laser irradiation inactivates the AGT fusion protein as well as those proteins that interact with the AGT fusion protein in a time-controlled and spatially-resolved manner.
- This method can be applied both in vivo or in vitro.
- proteins which are in close proximity of the AGT fusion protein can be identified as such by either detecting fragments of that protein by a specific antibody, by the disappearance of those proteins on a high-resolution 2D- electrophoresis gels or by identification of the cleaved protein fragments via separation and sequencing techniques such as mass spectrometry or protein sequencing by N-terminal degradation.
- the label L is a molecule that can cross-link to other proteins, e.g. a molecule containing functional groups such as maleimides, active esters or azides and others known to those proficient in the art
- contacting such labelled AGT substrates with AGT fusion proteins that interact with other proteins leads to the covalent cross-linking of the AGT fusion protein with its interacting protein via the label.
- Labels L for photo cross- linking are e.g. benzophenones.
- the label L is a molecule which is itself an AGT substrate leading to dimerization of the AGT fusion protein.
- the chemical structure of such dimers may be either symmetrical (homodimers) or unsymmetrical (heterodimers).
- labels L 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.
- Methotrexate is a tight-binding inhibitor of the enzyme dihydrofolate reductase (DHFR).
- DHFR dihydrofolate reductase
- CIDs chemical inducers of dimerization
- fusion proteins of hAGT with the DNA-binding domain LexA and adding DHFR with the transcriptional activation domain B42 to the in vivo labeling of the hAGT fusion protein with a compound of formula 1 wherein L is methotrexate induces the coupling ("dimerization") of the hAGT-LexA fusion protein and DHFR-B42 fusion protein, leading to spatial proximity of LexA and B42 and subsequent stimulation of transcription as shown in Figure 4.
- the substrate carries 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.
- the present invention further provides a method to label AGT fusion proteins both in vivo as well as in vitro.
- the term in vivo labelling of a AGT fusion protein includes labelling in all compartments of a cell as well as of AGT fusion proteins pointing to the extracellular space. If the labelling of the AGT fusion protein is done in vivo and the protein fused to the AGT is a membrane protein, more specifically a plasma membrane protein, the AGT part of the fusion protein can be attached to either side of the membrane, e.g. attached to the cytoplasmic or the extracellular side of the plasma membrane.
- the labelling of the fusion protein can be either performed in cell extracts or with purified or enriched forms of the AGT fusion protein.
- the labelling of the endogenous AGT of the host is advantageously taken into account. If the endogenous AGT of the host does not accept O 6 -alkylguanine derivatives or related compounds as a substrate, the labelling of the fusion protein is specific. In mammalian cells, e.g. in human, murine, or rat cells, labelling of endogenous AGT is possible. In those experiments where the simultaneous labelling of the endogenous AGT as well as of the AGT fusion protein poses a problem, known AGT- deficient cell lines can be used.
- the present invention provides a method of determining the interaction of a candidate compound or library of candidate compounds and a target protein or library of target proteins.
- candidate compounds and target proteins include ligands and proteins, drugs and targets of the drug, or small molecules and proteins.
- the protein of interest fused to the AGT comprises a DNA binding domain of a transcription factor or an activation domain of a transcription factor.
- the putative protein target of the substances or library of proteins is linked to either of the DNA binding domain or the activation domain of the transcription factor in a way a functional transcription factor can be formed, and the label L of the AGT substrate according to the invention is a candidate compound or library of candidate compounds suspected of interacting with the target substance or substances.
- the candidate compound or library of candidate compounds being part of the substrate is then transferred to the AGT fusion protein.
- the AGT fusion protein(s) comprising the target substance(s) now are labelled with the candidate compound(s).
- the interaction of a candidate compound joined to the AGT fusion protein with the target protein fused to either the DNA binding domain or the activation domain leads to the formation of a functional transcription factor.
- the activated transcription factor can then drive the expression of a reporter which, if the method is carried out in cells, can be detected if the expression of the reporter confers a selective advantage on the cells.
- the method may involve one or more further steps such as detecting, isolating, identifying or characterising the candidate compound(s) or target substance(s).
- the label L is a drug or a biological active small molecule that binds to a yet unidentified protein Y.
- a cDNA library of the organism which is expected to express the unknown target protein Y is fused to the activation domain of a transcription factor, and the AGT is fused to the DNA binding domain of a transcription factor.
- Adding the AGT substrate of the invention comprising such a label L leads to the formation of a functional transcription factor and gene expression only in the case where this molecule binds to its target protein Y present in the cDNA library and fused to the activation domain. If gene expression is coupled to a selective advantage, the corresponding host carrying the plasmid with the gene coding for the target protein Y of the drug or bioactive molecule can be identified.
- the label L is a library of chemical molecules.
- the library is expected to contain yet unidentified compounds that bind to a known drug target protein Y under in vivo conditions.
- the target protein Y is fused to the activation domain of a transcription factor and the AGT is fused to the DNA binding domain of a transcription factor.
- Adding the substrate carrying the library of chemical compounds leads to the formation of a functional transcription factor and gene expression only in the case where the label (i.e. a compound in the chemical library) binds to its target protein Y fused to the activation domain. If gene expression is coupled to a selective advantage, those molecules of the library leading to the growth of the host can be identified.
- a preferred compound is the cyclic substrate wherein the bond from R 4 to R ⁇ is a bond connecting the linker R to an amino group R 6 as defined under formula 2.
- R 2 is preferably an oligonucleotide, i.e. a ⁇ -D-2'-deoxyribosyl being incorporated into a single stranded oligodeoxyribonucleotide having a length of 2 to 99 nucleotides as detailed above.
- This oligonucleotide may be further chemically modified so that it can be detected and functions therefore as a label. The chemical modification of substituents might be of the same nature as mentioned above for the label L.
- the substrate is a dimeric compound leading to a dimerised fusion protein on reaction with a fusion protein comprising AGT.
- the subunit L as a residue -R 3 -CH 2 -X-RrR 2.
- R 1f R 2 , R 3 and X may be identical with the corresponding meaning in the other group R2-RrX-CH 2 -R 3 -, representing a homodimer, or different, representing a heterodimer.
- the invention concerns a method wherein a compound of the formula R 2 -RrY, wherein Y is a reactive leaving group, e.g. an ammonium salt, is treated with a compound of formula HX-CH 2 -R 3 -R -L or a precursor thereof, and the resulting compound is further manipulated, e.g. by elaboration of the linker l ⁇ and/or the introduction of a label L.
- Y is a reactive leaving group, e.g. an ammonium salt
- a compound of the formula R 2 -R XH is alkylated with a compound of the formula Y-CH 2 -R 3 -R - L, wherein Y is a leaving group, e.g. halogen, or a precursor thereof, and the resulting compound is further manipulated, e.g. by elaboration of the residue R 3 and/or the linker R 4 , and/or the introduction of a label L.
- Y is a leaving group, e.g. halogen, or a precursor thereof
- the invention concerns methods of manufacture of novel substrates carrying more than one label.
- the synthesis proceeds through a core compound of the formula R 2 -R ⁇ -X-CH 2 -R 3 -R 4 wherein R 4 is a polyfunctional residue having two or more reactive nucleophilic or electrophilic groups.
- groups include hydroxy, amino, sulfhydryl, halo or carboxy.
- Further examples of reactive groups are Michael acceptors such as maleimides or vinylsulfones.
- functional groups which can take part in cycloaddition reactions are also included, such as dienes or dienophiles, azides, nitrones, nitriloxides, acetylenes, and alkenes.
- Examples of functional groups which can be provided on the outer sphere of a branched residue l ⁇ include fluoro, chloro, bromo, cyano, nitro, amino, alkylcarbonylamino, arylcarbonylamino, alkoxycarbonylamino, aryloxycarbonylamino, carboxy, carbamoyl, alkoxycarbonyl, aryloxycarbonyl, carbaldehyde, hydroxy, alkoxy, aryloxy, alkylcarbonyloxy, arylcarbonyloxy, a carbon-carbon double bond, a carbon-carbon triple bond, and the like.
- Most preferable examples include amino, hydroxy, cyano (a latent carboxy function), carbamoyl, carbaldehyde, or a carbon-carbon double bond.
- the preferred synthesis of a branched residue l ⁇ carrying a plurality of labels L makes use of orthogonally protected functional groups.
- Such a choice of protective groups allows for a separate deprotection so that each released functionality in turn can be further chemically manipulated either to attach a label to it or for the introduction of further extension of linker R .
- Appropriate protecting groups for the envisioned functionalities can be chosen by those skilled in the art, and are e.g. summarized in T.W. Greene and P.G.M. Wuts in "Protective Groups in Organic Synthesis", John Wiley & Sons, New York 1991.
- Preferred intermediates according to the invention are compounds of formula 1 wherein R R 2 is a radical of formula 2 wherein R 2 is hydrogen, R 5 is hydrogen and R 6 is unsubstituted amino; and X is oxygen; R 3 is triazolyl, tetrazolyl, isoxazolyl, thienyl, isoxazolidinyl or alkynyl; R is a linker; and L is amino or azido, in particular amino.
- R R 2 is a radical of formula 2 wherein R 2 is hydrogen, R 5 is hydrogen and R 6 is unsubstituted amino; and X is oxygen; R 3 is 1 ,4-phenylene; R is a straight chain alkylene group of 10 to 40 carbon atoms optionally substituted by oxo wherein up to 12 carbon atoms are replaced by oxygen and zero, one or two carbon atoms are replaced by nitrogen; and L is amino or azido, in particular amino.
- the azido compound 7 is prepared from commercially available tetraethylene glycol 5 by mesylation (methanesulfonyl chloride, triethylamine) followed by reaction with sodium azide in ethanol. 7 is again mesylated and subjected to a Gabriel amine synthesis to give azido- amine 9 (Carolay etal, J. Org. Chem. 56: 4326-4329, 1991).
- the Cu(l)-catalyzed 1,3- dipolar cycloaddition between azide 9 and the acetylene derivative 10 (Griffin et al, J. Med. Chem. 43: 4071 -4083, 2000) yields the 1 ,4-substituted triazole 11.
- the azide 9 and the cyano derivative 12 react under Lewis acid catalysis (ZnBr 2 ) to form tetrazole 13 (Scheme 1).
- Scheme 1 Scheme 1
- Azide 7 is transformed to the central building block, the aldehyde 14, by means of a Swerns oxidation (oxalyl chloride, DMSO, triethylamine).
- the reaction of 14 with a hydroxylamine derivative yields the nitrone 17, which upon reaction with the acetylene derivative 10 forms the class of isoxazolidines 18.
- the oxime 15 is formed as an equimolar mixture of isomers.
- the corresponding nitrile-oxide is formed in situ by oxidation with sodium hypochlorite followed by reaction with 10 to yield the isoxazole 16 (Scheme 2).
- the commercially available tetraethylene glycol 5 is monofunctionalized through the reaction with one equivalent of allyl iodide under strongly basic conditions to yield 22 which is further dimethoxytrityl (DMT)-protected to 23.
- DMT dimethoxytrityl
- ⁇ -N-Fmoc-lysine 47 is converted to the corresponding ⁇ -azido- ⁇ -N-Fmoc-lysine 48 by the diazo transfer method (Lundquist et al, J. Org. Lett. 3: 781-783, 2001). Condensation of ⁇ -azido- ⁇ -N-Fmoc-lysine with an appropriate functionalized amino-benzylguanine derivative (e.g. 49) under standard peptide coupling conditions leads to the fully protected molecular scaffold 50.
- an appropriate functionalized amino-benzylguanine derivative e.g. 49
- the ⁇ -azido group is reduced to the corresponding amine 51 under neutral conditions with trimethylphosphine in aqueous 1 ,4-dioxane, giving a compound suitable for condensation with a variety of different building blocks (containing e.g. affinity labels).
- a compound suitable for condensation with a variety of different building blocks containing e.g. affinity labels.
- coupling with N-(+)-biotin-6-amino- caproic acid N-succinimidyl ester to give 52 followed by Fmoc-deprotection with diethylamine yields intermediate 53 with a free amino group which can be used to couple further labels to the central unit.
- Digoxygenin-3-O-methylcarbonyl- ⁇ -aminocaproic acid-N-hydroxy- succinimide ester is coupled to the free amino group of 53 leading to the double labelled substrate 54 (Scheme 9).
- Scheme 11 shows the synthesis of two more examples of benzylguanine derivatives bearing a different combination of labels (two affinity labels biotin and dinitrophenyl in 59, or an affinity label combined with a fluorophore in 60).
- two affinity labels biotin and dinitrophenyl in 59 or an affinity label combined with a fluorophore in 60.
- the coupling of 2,4-dinitrofluorbenzene to the free amino group yields 59.
- Coupling of the free amino group in 53 with fluorescein-5(6)-carboxamidocaproic acid N-succinimidyl ester gives 60.
- Example 1 Preparation of glass slides for the covalent attachment of AGT substrates and subsequent covalent immobilization of AGT fusion proteins for the preparation of protein microarrays
- a commercially available microscope glass slide (SiO 2 ) is cleaned thoroughly with dichloromethane, acetone, H 2 O 2 /H 2 SO 4 in an ultrasonic bath, and bi-distilled water. It is aminosilylated using 3-aminopropyltriethoxysilane in a solvent mixture ethanol/water (95:5) for 1 h following a published procedure, then treated with a solution of disuccinimidyl glutarate (10 mM) in dichloromethane/diisopropylethylamine (100:1) for 2 h under argon at room temperature. The surface is washed several times with dichloromethane.
- the glass surface bearing activated carboxy functions is incubated for 4 h with a solution of a compound of formula 1 bearing a free amino group instead of label L, such as compound 11, 13, 16, 18, 28, 34, 49, 68 or 73, in methanol in a concentration of 10 mM, and supplemented with triethylamine.
- the slides are washed at least three times with methanol to yield a surface with the corresponding AGT-substrate covalently attached by an amide bond.
- all unreacted succinimidyl groups are quenched by addition of 6-aminohexanol (100 mM in DMF).
- Example 2 11-Azido-3.6.9-trioxaundecanol (7) and 1.11-diazido-3,6.9-trioxaundecane (36)
- a solution of 50.0 g (260 mM) tetraethylene glycol and 50 mL triethylamine in 200 mL dry diethyl ether is cooled to 0°C under an argon atmosphere, and 15.0 g (130 mM) methane- sulfonyl chloride is added over a 3 h period and stirred at room temperature for 20 min.
- the solvent is removed in vacuo, and 300 mL 95% ethanol and 18.0 g (280 mM) sodium azide are added.
- the product is purified by silica gel chromatography (gradient: petrol ether/ethyl acetate 10:1 ⁇ ethyl acetate), yielding 2.41 g 22 (10.3 mmol, 27%) as a colorless liquid.
- Example 6 1-(2-Amino-7H-purin-6-yl)-1-methyl-pyrrolidinium chloride (27) 1.0 g (5.9 mmol) 6-Chloroguanine is dissolved in 40 mL DMF at 40°C. After cooling to room temperature, 1.4 mL 1-methylpyrrolidine (13.2 mmol) is added, and the reaction mixture is stirred for 18 h. 2 mL of acetone are added to complete precipitation. The solid is filtered, washed with ether and dried in vacuo, yielding 1.03 g 27 (3.9 mmol, 66%).
- Example 7 O 6 -(4-Aminomethyl-benzyl)guanine (32) a) 4-Aminomethyl-benzyl alcohol: 2.83 g LiAIH 4 (74.5 mmol) are suspended in 150 mL dry ether, and 1.9 mL H 2 SO (100 %, 37.2 mmol) are added dropwise and under cooling. The mixture is stirred for 1 h at room temperature, followed by dropwise addition of 2.0 g (12.4 mmol) 4-cyanobenzoate in 12 mL ether. After 2 h of refluxing the reaction is quenched with 20 mL water followed by 7.4 g NaOH in 60 mL water.
- Example 10 4-(Prop-2-ynyloxymethyl)benzyl alcohol (39) and 1 ,4-bis-(prop-2-vnyloxy- methyl)benzene (40)
- Example 12 1-f(tert-Butyldimethylsilyloxy)methyl1-4-(iodomethyl)benzene (45) 9.15 g (34.88 mmol) Triphenylphosphine and 3.2 g (44.5 mmol) imidazole are dissolved in a 3:1 mixture of diethyl ether/acetonitrile (30 mL). 8.85 g (34.9 mmol) iodine is added under vigorous stirring until a yellow suspension has formed. A solution of 6.1 g (23.25 mmol) of the monoprotected benzyl alcohol 44 in 20 mL of the same solvent mixture is added, and the mixture is stirred for 2 h.
- Example 14 O 6 -[4-(13-Azido-2,5,8,11-tetraoxatridecyl)-benzyl]guanine (41) 0.974 g (2.87 mmol) 4-(13-azido-2,5,8,11-tetraoxatridecyl)-benzyl alcohol (46) is dissolved in 5 mL dry DMF, and 1.3 g (11.5 mmol) potassium tert-butoxide are added. 0.731 g (2.87 mmol) 1-(2-amino-7H-purin-6-yl)-1-methyl-pyrrolidinium chloride is added and the solution stirred for 22 h. After removal of the solvent in vacuo the crude product is purified by flash column chromatography (methanol/dichloromethane 5:95). Yield: 0.675 g (50%).
- 1-Azido-11-phtalimido-3,6,9-trioxaundecane (8, Example 3, 2.0 g, 5.74 mmol), is treated with 1.0 ml 55% hydrazine hydrate in 50 ml abs. methanol and heated to reflux for 2 h. After cooling to room temperature, the mixture is concentrated in vacuo, diluted with 50 ml of water and treated with 5 ml cone. HCI. All precipitate is filtered off, the aqueous filtrate neutralized with NaOH, and the solvent removed in vacuo. The residue is dissolved in dichloromethane, and washed with 3 N NaOH and water. The organic phase is dried over MgSO 4 and concentrated in vacuo yielding 0.63 g (2.9 mmol, 51%) of a colourless oil, which is used in the next step without further purification.
- Example 18 O 6 -(1-[11-Amino-3.6.9-trioxaundecvn-1.2.3-triazolyl-4-methyl)-guanine (11)
- O 6 -Propargyl-guanine 10 75 mg, 0.4 mmol
- azide 9 44 mg, 2 mmol
- Cu-wire (5 mg) and Cu 2 l 2 (2 mg) are added, and the reaction mixture stirred at room temperature for 48 h. All insoluble material is filtered off, and the solvents evaporated in vacuo. Flash chromatography (dichloromethane/methanol 20:1) yields 35 mg 11 (21%).
- Example 20 O 6 -[4-(13-Amino-2.5.8.11-tetraoxatridecyl)-benzyl1guanine (49) O 6 -[4-(13-Azido-2,5,8,11-tetraoxatridecyl)-benzyl]guanine (41, 0.21 g, 0.44 mmol) is dissolved in 8 ml dry methanol by heating. Triethylamine (410 ⁇ L, 2.2 mmol) and 1,3- propanedithiol (225 ⁇ L, 2.2 mmol) are added under an argon atmosphere. The reaction mixture is heated at 40°C and stirred for 48 h.
- Example 21 O 6 -Benzylguanine-PEG-amino- ⁇ -azido- ⁇ -N-Fmoc-lysine 50 O 6 -Benzy!guanine-PEG-amine 49 (76 mg, 0.17 mmol) and ⁇ -azido- ⁇ -N-Fmoc-lysine 48
- Example 23 O 6 -Benzylguanine-PEG-amino- ⁇ -N-(biotinyl-6-aminocaproyl)- ⁇ -N-Fmoc-lysine 52
- Example 25 O 6 -Benzylquanine-PEG-amino- ⁇ -N-(biotinyl-6-aminocaproyl)- ⁇ -N-([diqoxiqenin- 3-oxymethylcarbonyl1-6-aminocaproyl)-lysine 54
- O 6 -benzylguanine-PEG-amino- ⁇ -N-(biotinyl-6-aminocaproyl)-lysine 53 (6.93 mg, 0.0075 mmol) and 10 ⁇ L triethylamine in 0.4 mL dry DMF, (digoxygenin-3-oxymethyl- carbonyl)-6-aminocaproic acid N-hydroxysuccinimide ester (5 mg, 0.0075 mmol) is added, and the reaction mixture stirred at 40°C for 2 h.
- Example 26 O 6 -Benzylguanine-PEG-amino- ⁇ -N-Fmoc- ⁇ -N-Dde-lysine 56
- O 6 -BenzyIguanine-PEG-amine 49 50 mg, 0.11 mmol
- ⁇ -N-Fmoc- ⁇ -N-Dde-lysine 55 58 mg, 0.11 mmol
- DMF 600 ⁇ L
- HOBT 280 ⁇ L of a 1 M solution in NMP, 0.28 mmol
- 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC, 54 mg, 0.28 mmol
- Example 28 O 6 -Benzylquanine-PEG-amino- ⁇ -N-(biotinyl-6-aminocaproyl)- ⁇ -N-Dde-lysine 58 O 6 -Benzylguanine-PEG-amino- ⁇ -N-Dde-lysine 57 and N-(+)-biotinyl-6-aminocaproic acid N- succinimidyl ester (31 mg, 68.6 ⁇ mol) are dissolved in DMF (700 ⁇ L). Triethylamine (30 ⁇ L) is added, and the reaction stirred for 2 h. Diethyl ether is added to the solution to precipitate the product, and the supernatant is removed via pipette. Residual solvent is evaporated in vacuo. Purification of the product is achieved by column chromatography on silica gel (gradient of 2 to 3 % methanol in chloroform; yield: 37 % over 2 steps).
- Example 29 O 6 -Benzylguanine-PEG-amino- ⁇ -N-(biotinyl-6-aminocaproyl)-lysine 53
- O 6 -Benzylguanine-PEG-amino- ⁇ -N-(biotinyl-6-aminocaproyl)- ⁇ -N-Dde-lysine 58 (27 mg, 25.0 ⁇ mol) is stirred for 20 min in a solution of 2 % hydrazine in DMF (250 ⁇ L). Acetone is added to the reaction mixture, and the mixture stirred for another 3 min. Ethyl ether is added to the solution to precipitate the product, and the supernatant is removed via pipette.
- Example 30 O 6 -Benzylquanine-PEG-amino- ⁇ -N-(biotinyl-6-aminocaproyl)- ⁇ -N-(2.4- dinitrobenzoyP-lysine 59
- Example 31 O 6 -Benzylguanine-PEG-amino- ⁇ -N-(biotinyl-6-aminocaproyl)- ⁇ -N-(6- [fluorescein-5-carbonyl]-aminocaproyl)-lysine 60
- Triethylamine (15 ⁇ l) is added to a solution of O 6 -[4-(13-amino-2,5,8,11-tetraoxatridecyl)- benzyl]-guanine (49, 30 mg, 67 ⁇ mol) and N,N ' -disuccinimidylcarbonat (8.6 mg, 33 ⁇ mol) in 0.8 ml dry DMF, and the reaction mixture stirred at room temperature for 24 h.
- the crude product is precipitated in diethyl ether (10 ml), adsorbed on SiO 2 and purified by flash column chromatography (methanol/dichloromethane 5:1). Yield: 8.5 mg (9.2 ⁇ mol, 27%).
- Example 33 1-(tert-Butyldimethylsilyloxy)but-2-yn-4-ol (63) tert-Butyldimethylsilyl chloride (8.0 g, 53 mmol) is dissolved in dry dichloromethane, and added dropwise to a solution of 2-butyne-1 ,4-diol (13.71 g, 159.2 mmol, 3 equ.), 4-dimethyl- aminopyridine (1.29 g, 10.6 mmol, 0.2 equ.) and triethylamine (6.9 mL) in a solvent mixture of dichloromethane (200 mL) and THF (100 mL).
- Example 35 4-(11-Azido-3,6,9-trioxaundecyloxy)-1-(tert-butyldimethylsilyloxy)-2-butyne (65) 1 -Azido-3,6,9-trioxaundecanol (7, 3.79 g, 17.02 mmol) is dissolved in dry THF, and sodium hydride (1.02 g, 42.45 mmol) is added in small portions. After stirring at room temperature for 40 min, 1-(tert-butyldimethylsilyloxy)-4-iodobut-2-yne (64, 4.4 g, 14.18 mmol) is added, and the reaction mixture stirred for additional 15 h.
- Example 36 (13-Azido-2.5.8.11-tetraoxatridecyl)-proparqyl alcohol (66)
- Tert-butyldimethylsilyl ether 65 (1.5 g, 3.37 mmol) is dissolved in 25 mL THF with 0.25 mL acetic acid, and treated with tetrabutylammonium fluoride (4.1 mL, 1 M in THF). The reaction mixture is stirred for 2 h, and the amount of solvent reduced in vacuo to about 30%. After addition of ethyl acetate (50 mL), the organic phase is washed with sat. sodium bicarbonate (20 mL) and brine (30 mL), and dried over MgSO 4 . The product is used in the next step without further purification. Yield: 0.774 g (80%).
- Example 37 O 6 -[(13-Azido-2,5.8,11-tetraoxatridecyl)-proparqy ⁇ -guanine (67)
- Propargyl alcohol 66 (724 mg, 2.5 mmol) is dissolved in dry DMF (1 mL), and sodium hydride (181 mg, 7.55 mmol) is added in two portions. After stirring at room temperature for 30 min, 1-(2-amino-7H-purin-6-yl)-1-methyl-pyrrolidinium chloride (27, 641 mg, 2.5 mmol) and 4- dimethylaminopyridine (100 mg, 0.81 mmol) are added.
- Azide 67 400 mg, 0.951 mmol
- triphenylphosphine 748 mg, 2.85 mmol
- the solvent is removed in vacuo, and the residue purified via flash column chromatography (dichloromethane/methanol 10:1) yielding 312 mg (83%).
- Example 40 O 6 -[(13-(Digoxigenin-3-oxymethylcarbonyl-6-aminocaproyl-amino)-2,5,8,11- tetraoxatridecyl)-propargyl]-guanine 70
- Example 41 O 6 -[4-(12-Fmoc-amino-dodecanoyl)aminomethyllbenzyl guanine 72 12-Fmoc-aminododecanoic acid (147 mg, 0.33 mmol) and 1-benzotriazolyloxy-tris- pyrrolidino-phosphonium hexafluorophosphate (PyBOP,193 mg, 0.37 mmol) are dissolved in 11 ml DMF. After stirring at room temperature for 45 min O 6 -4-aminomethyl-benzyl guanine (71, 100 mg, 0.37 mmol) in 5 mL DMF are added.
- reaction mixture is stirred at 50°C for 5 min, then at room temperature for 1 h. After evaporation of the solvent in vacuo the product is purified by flash column chromatography (gradient methanol/dichloromethane from 1 :50 to 1:10) to yield 185 mg (0.27 mmol, 79 %) of white solid product.
- R f 0.33 (methanol/ dichloromethane 1:10).
- Example 42 O 6 -f4-(12-Aminododecanoyl)-aminomethv ⁇ -benzyl guanine (73)
- Fmoc-protected derivative 72 (180 mg, 0.26 mmol) is dissolved in 3.2 mL DMF containing 20% (v/v, 6.5 mmol) piperidine. After stirring at room temperature for 30 min the solvent is removed in vacuo and the product purified by flash column chromatography (gradient: methanol / dichloromethane from 1 :20 to 1:5 containing 1% triethylamine) to obtain 95 mg (0.20 mmol, 78%) of a pale yellow solid.
- L-Glutamic acid 1-benzylester (75, 114 mg, 0.48 mmol) and K 2 CO 3 (66.4 mg, 0.48 mmol) are added to 3 mL DMSO and sonicated at 60°C for 1 h.
- This mixture is added to a solution of 4- (N-[2,4-diamino-6-pteridinylmethyl]-methylamino)-benzoic acid 74 (150 mg, 0.46 mmol), diisopropylethylamine (112 ⁇ L, 0.65 mmol) and PyBOP (250 mg, 0.48 mmol) in 1.3 ml DMSO which had been stirred for 1 h.
- the reaction mixture is stirred at 50°C for 10 min and then at room temperature for 2.5 h. After quenching with 0.5 mL 1 M triethylamine the crude product is purified by HPLC with a linear gradient water/0.1% trifluoracetic acid (TFA) : acetonitrile/ 0.08% TFA from 100:0 to 20:80. The solution is adjusted to pH 7, and the solvents are removed in vacuo to yield 50 mg (0.09 mmol, 20%) of an orange solid.
- TFA trifluoracetic acid
- Example 44 O 6 -4-([12-N-(4- ⁇ 4-[N-(2.4-Diamino-6-pteridinylmethyl)-methylaminol-benzoyl- amino)-4-benzylo ⁇ ycarbonylbutanoyl)-aminododecanoyl]-aminomethyl)-benzyl guanine (77) (Benzotriazol-l-yloxy)tripyrrolidino-phosphonium hexafluorophosphate (PyBOP, 152 mg, 0.29 mmol) is dissolved in a solution of 1-hydroxybenzotriazole (HOBT) (146 ⁇ L of 1 M solution in 1-methyl-2-pyrrolidone (NMP), 0.15 mmol) and added to benzyl ester 76 (380 ⁇ L of 0.154 M solution in NMP, 0.06 mmol).
- HOBT 1-hydroxybenzotriazole
- NMP 1-methyl-2-pyrrolidone
- Example 46 O 6 -4-[13-N-(4- ⁇ 4-[N-(2.4-Diamino-6-pteridinylmethyl)-methylaminol-benzoyl- amino ⁇ -4-carboxybutanoyl)-amino-2.5.8,11-tetraoxatridecyl]-benzyl guanine (79) PyBOP (201 mg, 0.39 mmol) and HOBT (194 ⁇ l of a 1 M solution in NMP, 0.19 mmol) are added to benzyl ester 76 (21 mg, 0.04 mmol), and the mixture shaken at room temperature for 30 min.
- Example 47 4-Allyloxymethyl-1-(tert-butyldimethylsilyloxymethyl)-benzene (80) To a solution of 4-(tert-butyldimethylsilyloxymethyl)-benzyl alcohol 44 (2.85 g, 11.31 mmol) in 15 mL dry THF, sodium hydride (326 mg, 13.57 mmol) is added in small portions under an argon atmosphere, and the reaction mixture stirred at room temperature for 30 min. A solution of allyl iodide (3.80 g, 22.62 mmol) in 5 mL dry THF is added, and the resulting mixture stirred for 17 h.
- reaction is quenched with water, the solvent reduced to about 30% of its volume, and the residue extracted with ethyl acetate (3 times 20 mL).
- organic phase is dried over MgSO 4 , the solvent removed in vacuo, and the product purified by flash column chromatography (petrol ether/ethyl acetate 100:1), yield 1.83 g (6.25 mmol, 59%).
- Example 48 1.4-Di-[4-(tert-butyldimethylsilyloxymethyl)-benzyloxy1-2-butene (81) Benzylidene-bis(tricyclohexylphosphine)-dichlororuthenium (420 mg, 0.513 mmol, 15 mol%) is added under an argon atmosphere to a solution of 4-allyloxymethyl-1-(tert-butyldimethyl- silyloxymethyl)-benzene (80, 1.0 g, 3.24 mmol) in 70 mL dry dichloromethane. The reaction mixture is stirred at room temperature for 15 h, and additional catalyst (200 mg) is added and the reaction mixture heated to reflux for 5 h. The solvent is removed in vacuo, and the product purified by column chromatography (petrol ether/ethyl acetate 50:1) to yield 520 mg (0.93 mmol, 55%) of the title compound.
- Example 49 Bis-1.4-(O 6 -benzylguanine-methoxy)-2-butene 82 TBDMS-dimer 81 (351 mg, 0.63 mmol) is dissolved in 5 mL dry THF, and tetrabutylammonium fluoride (3.78 mmol) and acetic acid (3.78 mmol) are added. The reaction mixture is stirred at room temperature for 15 h. After evaporation of the solvent, the product is purified by column chromatography using a solvent gradient (petrol ether / ethyl acetate 1:1 to 1:3) to yield 150 mg (0.45 mmol, 73%) of a colourless solid.
- dimer 82 as a colourless solid (30 mg, 0.05 mmol, 16%).
- the product is purified by column chromatography using a solvent gradient petrol ether/ethyl acetate 10:1 to ethyl acetate 100% yielding a colourless oil (2.41 g, 10.3 mmol, 27%).
- Example 51 1-(tert-Butyldimethylsilyloxymethyl)-4-(13-allyloxy-2.5.8.11-tetraoxa-tridecyD- benzene (84)
- Example 52 TBDMS protected Bis-1.4-(benzyl alcohol-PEG)-2-butene 85 Benzylidene-bis(tricyclohexylphosphine)-dichlororuthenium (263 mg, 0.32 mmol, 15 mol%) is added under an argon atmosphere to a solution of TBDMS protected allyloxy derivative 84 (1.0 g, 2.13 mmol) in 40 mL dry dichloromethane. The reaction mixture is heated to reflux for 5 h. Additional catalyst (200 mg) is added and heating continued for 15 h. The solvent is removed in vacuo, and the product purified by column chromatography using a solvent gradient petrol ether/ethyl acetate 1 :1 to 1 :5, to yield 530 mg (0.58 mmol, 54%) of dimer 85.
- TBDMS protected dimer 85 (200 mg, 0.22 mmol) is dissolved in 3 mL dry THF, and tetrabutylammonium fluoride (2.5 mmol) and acetic acid (2.5 mmol) are added. The reaction mixture is stirred at room temperature for 15 h. After evaporation of the solvent, the product is purified by column chromatography using a solvent gradient petrol ether / ethyl acetate 1:1 to ethyl acetate 100% to yield 80 mg (0.11 mmol, 50%) of a colourless solid.
- dimer 86 as a colourless solid (35 mg, 0.036 mmol, 41%).
- Example 54 Demonstration of reactivity of compounds of formula 1 with human O 6 - alkylguanine-DNA alkyltransferase (hAGT) fusion proteins
- Lane “C” of Figure 1A shows the reaction product of PGEA hAGT-GST with the biotinylated oligonucleotide. If PGEA hAGT-GST is first reacted with 11, no further reaction with biotinylated oligonucleotide is observed (lanes 1 and 2, Figure 1 A). This proves that O 6 -triazolylmethylguanines are efficient substrates for hAGT.
- Example 55 Immobilization of a human O 6 -alkylguanine-DNA alkyltransferase (hAGT) fusion protein on a solid phase
- a solution of PGEA hAGT-GST fusion protein (200 ⁇ g/mL in HBS buffer) are incubated with 20 ⁇ L of a 0.4 mM solution of O 6 -benzylguanine-PEG-amino- ⁇ -N-(biotinyl-6-amino- caproyl)- ⁇ -N-([digoxigenin-3-oxymethylcarbonyl]-6-aminocaproyl)-lysine 54 for 10 min at room temperature.
- This solution is passed over a Neutravidin Biacore chip for 35 min at a flow rate of 1 ⁇ L/min. The chip is then purged with buffer (HBST) for 40 min at the same flow rate.
- ⁇ GST-antibody (1980 ⁇ g/mL) is injected to further elucidate the previous binding of the fusion protein, followed again by washing with HBST.
- Immobilization experiments are measured by surface plasmon resonance.
- Surface plasmon resonance analysis is performed using a Biacore 1000 optical biosensor equipped with research grade SA sensor chips (Biacore).
- EDTA-free HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% Tween 20) is freshly prepared and filtered through a 22 ⁇ m membrane. The resulting sensogram is shown in Figure 2.
- Example 56 Dimerization of a human O 6 -alkylguanine-DNA alkyltransferase (hAGT)
- HA- w160 hAGT is expressed in E. coli BL21 in LB medium at 24°C for 2 h after addition of IPTG (1 mM) and substrate 61 in different concentrations (0, 10, 20, and 50 ⁇ M) to an exponentially growing culture. Cells are harvested by centrifugation. For each labelling experiment the total volume of extract is about 1 ml (volume adjusted to a constant OD 600 ).
- the pellets are resuspended in 50 ⁇ l 1xSDS followed by addition of O 6 -benzylguanine to a concentration of 1 mM. After heating for 5 min to 95°C, samples are loaded to a 12% SDS-page.
- Western Blot is obtained using an goat anti-MGMT (1:100 in TBST, Chemicon) and an anti-goat IgG (1:1000 in TBST, Santa Cruz Biotechnology) as s second antibody. The result is shown in Fig. 3B.
- 3HY hAGT is expressed as fusion protein with glutathione S-transferase (GST- 3HY hAGT) in E. coli, the fusion protein purified and its activity determined in an in vitro assay.
- GST- 3HY hAGT is incubated with an excess of 78 or 79 and the reaction is followed by Western Blotting using an anti-methotrexate antibody.
- GST- 3HY hAGT shows second-order rate constants of about 1500 sec "1 M "1 for the reaction with 78 and 900 sec "1 M "1 for the reaction with 79. Accordingly, at concentrations of 78 of 1 ⁇ M and at an excess of 78 over 3HY hAGT (pseudo first-order conditions), 50% of the protein is labelled in less than 10 min.
- hAGT fusion proteins preferably the mutant Asn157Gly Ser159Glu is used.
- This mutant shows increased activity against benzylguanine derivatives of the type 1 by a factor of approximately 20 compared to wild-type hAGT, see A. Juillerat et al, Chem. Biol. 10: 313-317, 2003.
- the following additional mutations in hAGT have been shown to disrupt DNA binding of hAGT but do not significantly interfere with the activity against benzylguanine derivatives: Lys125Ala, Ala127Thr and Arg128Ala, see A. Lim et al., EMBO J. 15: 4050-4060, 1996, and D.S. Daniels etal, EMBO J.
- Pairs of plasmids encoding LexA- 3HY hAGT and B42-DHFR fusion proteins are transformed into the yeast strain L40 in which the reconstitution of functional LexA-B42 transcription factor leads to transcription of the reporter genes HIS3 and lacZ. It is tested if the expression of different combinations of fusion protein in the presence of either 78 or 79 complemented the histidine auxotrophy of the yeast L40 (Table 1). Co-expression of LexA- 3HY hAGT and B42-DHFR allows yeast L40 to grow on plates lacking histidine but containing either 78 or 79, indicating transcription of HIS3. No growth is observed in the absence of 78 or 79.
- Table 1 Growth rates of different yeast L40 strains on plates lacking histidine and in the presence and absence of compounds 78 or 79.
- B42 fusion proteins is examined (Table 2).
- the activity of the product of the lacZ gene, ⁇ -galactosidase is determined by measuring the hydrolysis rate of the chromogenic substrate o-nitrophenyl- ⁇ -D-galactopyranoside (ONPG) in cell extracts of liquid cultures.
- ONPG o-nitrophenyl- ⁇ -D-galactopyranoside
- Table 2 Quantitative ⁇ -galactosidase assay using ONPG.
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US10/529,651 US7799524B2 (en) | 2002-10-03 | 2003-10-01 | Substrates for O6-alkylguanina-DNA alkyltransferase |
AU2003271669A AU2003271669A1 (en) | 2002-10-03 | 2003-10-01 | Substrates for O6 -alkylguanine-DNA alkyltransferase |
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Cited By (16)
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WO2005085470A1 (en) * | 2004-03-02 | 2005-09-15 | Epfl Ecole Polytechnique Federale De Lausanne | Specific substrates for o6- alkylguanine-dna alkyltransferase |
WO2005085431A2 (en) * | 2004-03-02 | 2005-09-15 | Epfl Ecole Polytechnique Federale De Lausanne | Mutants of o6-alkylguanine-dna alkyltransferase |
WO2006021553A1 (en) * | 2004-08-23 | 2006-03-02 | Covalys Biosciences Ag | Method for protein purification and labeling based on a chemoselective reaction |
WO2006114409A1 (en) * | 2005-04-27 | 2006-11-02 | Covalys Biosciences Ag | Pyrimidines reacting with o6-alkylguanine-dna alkyltransferase |
EP1882688A1 (en) * | 2006-07-25 | 2008-01-30 | EPFL Ecole Polytechnique Fédérale de Lausanne | Labelling of fusion proteins with synthetic probes |
US20090203706A1 (en) * | 2006-09-15 | 2009-08-13 | Enzon Pharmaceuticals, Inc. | Lysine-based polymeric linkers |
FR2936245A1 (en) * | 2008-09-23 | 2010-03-26 | Cis Bio Int | NOVEL O6-ALKYLGUANIN-DNA ALKYLTRANSFERASE SUBSTRATES AND MUTANTS THEREOF |
US7825096B2 (en) | 2004-09-08 | 2010-11-02 | The United States Of America As Represented By The Department Of Health And Human Services | O6-alkylguanine-DNA alkyltransferase inactivators and beta-glucuronidase cleavable prodrugs |
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Also Published As
Publication number | Publication date |
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CN1720332A (en) | 2006-01-11 |
CA2501063A1 (en) | 2004-04-15 |
JP2006501289A (en) | 2006-01-12 |
US7799524B2 (en) | 2010-09-21 |
EP1546371A1 (en) | 2005-06-29 |
JP4976651B2 (en) | 2012-07-18 |
AU2003271669A1 (en) | 2004-04-23 |
KR20050062588A (en) | 2005-06-23 |
US20060024775A1 (en) | 2006-02-02 |
ZA200502210B (en) | 2006-02-22 |
NZ538986A (en) | 2006-06-30 |
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