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WO2008145609A1 - Procédé de fabrication de conjugués covalents avec des protéines étiquetées his - Google Patents

Procédé de fabrication de conjugués covalents avec des protéines étiquetées his Download PDF

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WO2008145609A1
WO2008145609A1 PCT/EP2008/056361 EP2008056361W WO2008145609A1 WO 2008145609 A1 WO2008145609 A1 WO 2008145609A1 EP 2008056361 W EP2008056361 W EP 2008056361W WO 2008145609 A1 WO2008145609 A1 WO 2008145609A1
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compound
nta
group
formula
protein
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PCT/EP2008/056361
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English (en)
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Manfred Auer
Martin Hintersteiner
Thierry Kimmerlin
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Novartis Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0042—Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/04—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D233/20—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D233/26—Radicals substituted by carbon atoms having three bonds to hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes

Definitions

  • the present invention refers to novel agents for covalently conjugating His- tagged proteins, to covalent conjugates of His-tagged proteins and to methods of making such conjugates.
  • Fluorescent probes allow detection of molecular interactions, mobility and conformational changes of proteins with high temporal and spatial resolution by using fluorescence spectroscopy such as steady-state, time-resolved, anisotropy, correlation spectroscopy (FCS), fluorescence resonance energy transfer (FRET), single-molecule detection, lifetime imaging microscopy and surface-sensitive fluorescence detection (for review of spectroscopic techniques see (Hovius, et al 2000)).
  • acyl carrier protein ACP
  • DNA repairing enzyme (f- alkylguanine-DNA alkyltransferase, hAGT) (Keppler, et al 2003), known as SNAPtag.
  • a third strategy, the affinity labeling makes use of the strong interactions between a ligand and its receptor, for instance the biotin- streptavidin system (Cronan 1990) or the haloalkane dehalogenase protein and its ligand known as Halo-tag (Zhang, et al 2006).
  • metal-ligand interactions are also used for selective labeling of proteins.
  • FIAsH-tag biasenical fluorescein dye
  • FIAsH-tag biasenical fluorescein dye
  • IMAC immobilized metal affinity chromatography
  • NTA nitrilo triacetate
  • WO 01/72458 describes a heterofunctional crosslinking agent which may be covalently conjugated to proteins, including to His-tag proteins.
  • the method of making such crosslinking agents described therein suffers from drawbacks such as an incomplete description of synthetic procedures to synthesize the claimed tagging molecules.
  • no protocol for an application of named tagging molecules for in-vitro labeling of proteins and for labeling of proteins on living cells is provided.
  • the present invention describes the synthesis of new agents for irreversible site-specific labeling of His-tagged proteins using the directing effect of the nitrilo triacetate (NTA) moiety towards histidine, in combination with covalent linkage groups, e.g. photoreactive functionalities such azides or electrophilic groups such as acryloyl groups.
  • NTA nitrilo triacetate
  • covalent linkage groups e.g. photoreactive functionalities such azides or electrophilic groups such as acryloyl groups.
  • the new prototype compounds were shown to represent irreversible labeling reagents for His-tagged proteins in-vitro and on living cells. Furthermore, a convenient synthesis strategy and a simple labeling protocol is provided for these agents.
  • a first aspect of the present invention refers to a compound of formula (I)
  • L 1 is a linker
  • L 2 and L 3 are independently chemical bonds or linkers
  • F is a reporter moiety
  • NTA is a nitrilo triacetic moiety and n is from 1 -3, or a salt, chelate or derivative thereof.
  • a salt of the compound (I) may be formed by replacing acidic protons, e.g. of the NTA moiety with organic or inorganic cations and/or by protonating basic groups in the presence of organic or inorganic anions.
  • a chelate of the compound (I) is formed by the NTA moiety and a chelating cation, e.g. a transition metal cation such as Ni 2+ .
  • a derivative of the compound (I) may be a protected derivative which comprises at least one protection group at a reactive moiety. Acid protection groups may be present, e.g. at the NTA moiety and/or base protection groups at amino moieties. Acid protection groups such as t-butyl (tBu) are well known in the art.
  • Base protection groups e.g. amino protection groups such as butoxycarbonyl (Boc) or benzoyl are well known in the art.
  • a derivative may also be a synthesis intermediate of the compound (I) which comprises amino groups or protected amino groups instead of the groups -NHF and/or -NHC.
  • the compound (I) is based on a trifunctional core which has two amino functions and one carboxy function.
  • the amino functions may be linked via chemical bonds and/or linker groups to the core.
  • a detectable moiety F is bound.
  • a covalent coupling moiety C is bound.
  • NTA nitrilo thacetic
  • the carboxy moiety of the core is bound via an amide or an ester group to l_i, more preferably via an amide group.
  • the linker Li preferably has a length of at least two atoms, more preferably of at least 4 atoms and preferably up to 20, more preferably up to 14 atoms, wherein the atoms are selected from carbon atoms and optionally heteroatoms such as nitrogen and/or oxygen atoms.
  • the linker L 1 has a structure -l_i'-CO-NH-l_r, wherein Li 1 and l_r are a chemical bond or a linker group with a length of at least 1 atom, more preferably of at least 2 atoms and preferably up to 6 atoms, wherein at least one of Li 1 and l_r is a linker group. More preferably, the linker L 1 has the structure -(CH 2 ) n1 -CO-NH- (CH 2 )n2-, wherein ni and n 2 are independently from 1 -6.
  • L 2 and L 3 are independently chemical bonds or linkers which may have a length of at least one atom, preferably at least two atoms and preferably up to 12 atoms, more preferably up to 8 atoms, wherein the atoms are selected from carbon atoms and optionally heteroatoms such as nitrogen and/or oxygen atoms.
  • L 2 is a chemical bond and L 3 is a linker.
  • L 3 is a chemical bond and L 2 is a linker.
  • the linkers L 2 and L 3 may comprise the structures -(CH 2 ) n3 - and -(CH 2 ) n4 - respectively wherein n 3 and n 4 are independently 0-6. More preferably, one of n 3 and n 4 is 0 and the other from 2-4.
  • the compound of formula (I) may comprise 1 , 2 or 3 nitrilo triacetic groups, i.e. n may be 1 , 2 or 3. If n is 2 or 3, the NTA groups are preferably coupled to a cyclic group, e.g. a azamacrocycle such as cyclam which is bound to the linker L 1 . Preferably, n is 1.
  • the reporter moiety F is a moiety the presence of which can be detected.
  • the reporter moiety may be an optically detectable label such as a fluorescence group.
  • Preferred fluorescent groups are rhodamine groups such as TMR or rhodamine B.
  • Further preferred optically detectable moieties are direct labels such as gold or latex particles or enzyme groups.
  • F may be a quencher group, i.e. a group the presence of which quenches the fluorescence of adjacent fluorescent groups.
  • An example of a quencher group is QSY7.
  • the covalent coupling moiety C is a group, which is capable of forming covalent groups with a protein, more particularly with reactive groups of a protein such as carboxy, amino, thio and/or hydroxy groups.
  • C is a photoactivatable group, i.e. a group which upon illumination forms a covalent linkage to a complementary group present in a protein.
  • Photoactivatable groups are known in the art and comprise e.g. arylketones, diazo groups, diazirene groups or azide groups.
  • C is an arylazide group, more preferably a hydroxy arylazide group, such as azido salicylic acid, which reacts upon illumination (preferably at a wavelength ⁇ 380 nm) with a complementary group via an azacycloheptatetraene intermediate formed by a rapid intramolecular rearrangement of a singlet nitrene (Brunner 1993).
  • Hydroxyarylazides are highly electrophilic, but less reactive than other nitrene groups and do not insert into non-activated C-H bonds.
  • C may also be a non-photoactivatable group.
  • This non-photoactivatable group is a reactive group which may form a covalent bond with a complementary reactive group, wherein the reaction is accelerated by the proximity of a receptor ligand complex.
  • High local concentrations of the reactive group of compound (I) and complementary reactive groups in the protein due to the immobilization via the receptor- ligand-complex allow the reaction to take place.
  • the immobilization is effected by the Ni 2+ /NTA-poly-His complex.
  • the reactive group of compound (I) may be an electrophilic moiety such as an acryloyl moiety, which may react with nucleophilic moieties present in the protein, e.g. NH, OH, SH groups, when this reaction is accelerated by high local concentration of the respective groups.
  • inventive compound is of formula (Ia):
  • ni and n 2 are independently from 1 -6, n 3 and n 4 are independently from 0-6, and F and C are as defined above, or a salt, or derivative thereof.
  • n-i and n 2 are preferably 2-4. Further, it is preferred that one of n 3 and n 4 is O and the other from 2-4.
  • the present invention refers to a covalent conjugate of a compound of formula (I) or (Ia) as described above with a protein.
  • the protein may be any protein which has a group capable of forming a receptor-ligand-complex with a nitrilo triacetic acid moiety, preferably in the presence of a heavy metal ion such as Ni 2+ .
  • the protein comprises polyhistidine sequence, wherein the number of histidine residues is at least 2, preferably at least 3 and more preferably at least 4.
  • the protein comprises a hexahistidine sequence.
  • the polyhistidine sequence is preferably present at the N- and/or C-terminus of the protein.
  • the protein may be any prokaryotic or eukaryotic protein.
  • the protein comprises a domain which is localized at the surface of a cell, preferably incorporated into a cell membrane.
  • examples of such molecules are receptors, e.g. cytokine receptors, such as interleukin receptors, or growth-factor receptors.
  • the protein comprises a domain which is capable of interacting with a complementary protein present on the surface of a cell. Examples thereof are ligands capable of binding to a receptor present on the surface of a cell, e.g. cytokines.
  • the protein is a fusion protein comprising a biologically functional domain, e.g. a receptor or ligand as indicated above and a fluorescent domain such as GFP or a variant thereof.
  • Preferred applications for the compounds and conjugates of the invention are the labeling of biological components, e.g. cell components, cells, viruses, etc. Especially preferred is the labeling of living cells.
  • the labelled biological components may be detected via the reporter group F. Due to the covalent association of the reporter group to the protein, a very sensitive detection is possible, e.g., single-molecule detection.
  • the detection preferably involves optical methods, more preferably a fluorescence detection, e.g. by Fluorescence Correlation Spectroscopy (FCS) or by other fluorescence detection methods as described above.
  • FCS Fluorescence Correlation Spectroscopy
  • the compounds (I) are particularly suitable in screening assays, e.g. in assays wherein the effect of a candidate agent on a biological system, e.g. a living cell, is tested.
  • the compounds and conjugates of the invention allow a fast and reliable detection of interactions between protein components of the conjugates and biological systems and may thus be used in High Throughput Screening assays, e.g. for the detection of an effect of a candidate agent on a biological system, particularly on a cell surface protein.
  • the compounds of the invention are preferably synthesized by a procedure which comprises the steps:
  • pNTA is a protected nitrilo triacetic group
  • n is 1 -3
  • Li is a linker, with a compound of formula (III):
  • Still a further aspect of the invention refers to a method of irreversibly conjugating a compound of formula (I) to a protein, comprising the steps: (i) providing a chelate of a compound of formula (I), (ii) reversibly binding said chelate to a protein,
  • Figure 1 shows a Ni 2+ -NTA chelate.
  • Figure 1 b shows a non-covalent complex of a Ni 2+ -NTA chelate and a hexahistidine sequence present in a protein.
  • Figure 2 shows a non-covalent complex of a tridentate chelator, e.g. a Ni 2+ - NTA 3 chelate and a hexahistidine sequence in a protein.
  • a tridentate chelator e.g. a Ni 2+ - NTA 3 chelate and a hexahistidine sequence in a protein.
  • Figure 3 shows the structure of a tris-Ni 2+ -NTA chelate having a functional amino group to which e.g. a dye R is bound.
  • Figure 4 shows a scheme for the synthesis of a tris-Ni 2+ -NTA chelate linked to a dye.
  • Figure 5 shows a scheme for the synthesis of a mono-Ni 2+ -NTA chelate linked to a dye and a covalent coupling moiety.
  • Figure 6 shows structures of the quencher compound QSY7 and the fluorescent dyes TMR and RhB.
  • the indicated absorption wavelength and ⁇ - values are as determined in MeOH.
  • Figure 7 shows a schematic representation of the irreversible covalent coupling of an inventive compound to a protein.
  • the NTA moiety directs the probe to the labeling site, e.g. a hexahistidine sequence.
  • the reactive group covalent coupling moiety
  • Fiqure 8 shows the structure of the preferred photo-crossl inker moiety N- hydroxysuccinimide (NHS)-ASA, an example of a hydroxyarylazide moiety.
  • Figure 9 shows a scheme for the proximity-accelerated nucleophile- electrophile reaction triggered by the interaction between an NTA group and a polyhistidine sequence.
  • Figure 10 shows fluorescence spectra before and after addition of non- fluorescent ths-Ni 2+ -NTA quencher compound H to a GFP-C-His protein.
  • Figure 11 shows a fluorescence resonance energy transfer (FRET) between a tris-Ni 2+ -NTA TMR compound 10 non-covalently coupled to a GFP-C-his protein.
  • FRET fluorescence resonance energy transfer
  • Figure 12 shows visualization of cell surface receptors with a ths-Ni 2+ -THR compound 10, targeted to an IL-4R alpha chain-GFP-C-His fusion protein.
  • Figure 13 shows a covalent cross-linking of NTA-ASA derivatives in solution, a) The emitted GFP-C-His fluorescence (15 nM, solid dark line) is quenched by addition of non-fluorescent NTA-ASA-QSY7 35 (10 ⁇ M, line annotated as "2") due to FRET.
  • Figure 15 shows autocorrelation curves for e-GFP-C-His and NTA-ASA-TMR 34.
  • Figure 16 shows calibration curves for e-GFP-C-His and NTA-ASA-TMR 34.
  • Figure 17 shows Kd values of NTA-I and NTA-II vs eGFP-C-His.
  • concentration dependent fluorescence quenching of eGFP-C-His by NTA-I and NTA-II was fitted with the Hill-equation, as previously published.
  • Kd values of 0.25 ⁇ M and 3.5 ⁇ M were obtained for NTA-I and NTA-II, respectively.
  • Figure 18 shows the binding of His-tagged receptors by NTA-ASA-TMR.
  • HCLH-GIU(OBZI)-OBU was purchased from Senn Chemicals.
  • succinimidyl ester of TMR was purchased from Fluka Biochemika, the succinimidyl esters of QSY 7 and of Rhodamine Red X from Invitrogen.
  • Boc-Lys(Z)-OH was purchased from Novabiochem.
  • Sulfo-SFAD Sulfosuccinimidyl- [perfluoroazidobenzamido]ethyl-1 ,3 ' -dithiopropionate
  • SANPAH N- Succinimidyl-6-[4 ' -azido-2 ' -nitrophenylamino]hexanoate
  • 6-(Boc-amino)caproic acid 6-(Boc-amino)caproic acid, benzyl N-(2-aminoethyl) carbamate hydrochloride, benzylethane-1 ,2-diamine and 1 ,4,8,11 -tetraaza- cyclotetradodecane were purchased from either Fluka or Sigma Aldrich.
  • Analytical reversed phase (RP) HPLC Agilent 1100 Series System (Quat pump G1311A, degasser G1322A, multiwavelengths and fluorescence detector DAD G1315B and FLD G1321A).
  • N ;i2+ complexed compounds were analyzed using the previous gradient but with A: ammonium acetate (AcONH 4 ) in water (400 mg/l) and B: MeCN.
  • Retention times (Rt) are given in minutes.
  • Preparative reversed phase (RP) HPLC Agilent 1100 Series preparative system (Prep pump G1361A, multiwavelengths detector MWD G1365B). Column: Agilent prep Ci 8 (21.2 x 150 mm, 10 ⁇ m).
  • Crude products were purified using the following linear gradient of A (95% H 2 O, 5% MeCN, 1 % TFA) and B (95% MeCN, 5% H 2 O, 1 % TFA) at a flow rate of 20 ml/min with UV detection at the corresponding wavelengths.
  • N ;i2+ complexed compounds were purified using the same gradient, flow, and UV detection with A: ammonium acetate (AcONH 4 ) in H 2 O (400 mg/l) and B: MeCN.
  • Retention times (R t ) are given in minutes.
  • TFA/triisopropyl-silane (TIS)/H 2 O 95:2.5:2.5
  • the compound containing the free amine was dissolved in NaHCO 3 buffer (pH 8.4) to a final concentration of 5-10 mg/ml.
  • the dye NHS ester dissolved in dry DMF (5-10 mg/200 ⁇ l) was then added and the reaction mixture was stirred for 2 h at RT.
  • the labeled compound was purified by preparative HPLC and analyzed by MS.
  • the compound foreseen to be complexed was dissolved in H 2 O to a final concentration of 5-10 mg/ml and treated with a 0.1 M solution of NiCI 2 (1 eq). The mixture was stirred for 2 h at RT. Finally, the Ni 2+ complex was purified by preparative HPLC and analyzed by MS.
  • GIu-NTA(OtBu) 3 -OH (3) (1 eq, 1.43 mmol, 617 mg) was dissolved in dry dichloromethane (DCM) (55 ml), followed by addition of 1 ,4,8,11 tetraaza- cyclotetradecane (0.33 eq, 0.472 mmol, 94.5 mg) at 0° C.
  • the quencher QSY7-NHS ester was coupled to the amino caproic acid tris- NTA derivative (6) following the general method described in chapter 1.2.3.
  • Preparative HPLC using the general method described in 1.1.2.3) yielded (8), a mauve solid (3.5 mg, 33%).
  • f? t 22 (analytical HPLC as described in 1.1.2.1 ), MS (ESI, C 82 HiO 2 NIiO 26 S): m/z 563.77 [M+3H] 3+ /3; 845.21 [M+2H] 2+ /2.
  • Rhodamine Red X NHS ester was coupled to the amino caproic acid tris- NTA derivative (6) following the general method described in chapter 1.2.3.
  • Preparative HPLC using the general method described in 1.1.2.3) yielded (9), a purple solid (2.9 mg, 57%).
  • f? t 23.2 (analytical HPLC as described in 1.1.2.1 ).
  • UV irradiation was applied with a Strata/inker 1800 (Stratagene, USA) by placing the sample in 10 cm distance to 365 nm UV tubes.
  • An automatic routine deposits a dose of 12O mJ within 40 seconds. The dosage is calibrated internally by a photodiode.
  • Crosslinking of solutions was performed in a droplet placed on aluminium foil from which even ⁇ l volumes could be recovered with a pipette. This treatment increased the x-link efficiency 4-fold compared to crosslinking in 1.5 ml polyethylene vials.
  • Crosslinking of cells was performed by placing the NUNC-chambered coverglass slides (Nunc LAB-TEK Cat. No. 155409) in 10 cm distance to the UV bulbs on aluminium foil.
  • GFP-C-His was displayed with SDS-PAGE using 4-12% Bis-Ths Novex precast gels (Invitrogen) run in IxMES buffer for 1 hour at constant 150 V. The gel was imaged either directly with UV excitation (365 nm) or white light after Coomassie staining (Invitrogen) using a BioSpectrumAC imaging system with integrated quantification software (UVP Cambridge UK). 1.8 Cloning
  • Mammalian expression vectors were produced integrating the coding sequence of human IL-4R ⁇ into pEGFP-N1 from Clontech via directional cloning.
  • the hexahistidine-tag was inserted by site directed mutagenesis ⁇ GeneTailor, Invitrogen).
  • the expressed polypeptide NHis-IL-4Rac-GFP comprises the extracellular His-tagged IL-4R ⁇ (residues 33-297 correspond to amino acids 26-292 Swiss-Prot P24394 with 6 histidine residues inserted after the leader sequence at positions 27-32), a short linker GSTGRH at positions 298-303, and eGFP amino acids 304-542 corresponding to GeneBank Accession No.
  • HEK cells were grown in DMEM H21 (13.38 g/l DMEM Powder (Gibco), 44 mM NaHCO 3 ⁇ Gibco), 50,000 IE Penicillin (30 mg) (BC), 50 mg Streptomycin (Gibco), 2 mM glutamine, 10% complement inactivated fetal calf serum).
  • DMEM H21 13.38 g/l DMEM Powder (Gibco), 44 mM NaHCO 3 ⁇ Gibco), 50,000 IE Penicillin (30 mg) (BC), 50 mg Streptomycin (Gibco), 2 mM glutamine, 10% complement inactivated fetal calf serum.
  • PBS 137 mM NaCI, 1.5 mM KH 2 PO 4 , 2.7 mM KCI, 8 mM Na 2 HPO 4 * 2H 2 O
  • CDS Cell Dissociation Solution
  • Confluent HEK 293 cells were transfected into a 12-well (4 cm 2 surface area per well, Falcon) via Lipofectamine 2000 ⁇ Invitrogen, Cat. No. 11668-019) according to the manufacturer's recommendation. After 12 hours, the cells were transferred into Fibronectin-coated chamber slides (Nunc LAB-TEK 8- well, Cat. No. 155409). Coating was performed by covering the glass with 100 ⁇ g/ml Fibronectin (BD, Cat. No. 356008), followed by incubation for 30 min at 37° C. After aspiration, the cells were directly seeded at a density of 5,000 to 10,000 cells per well (1 cm 2 surface area per well). Within the time range of 48 hours the cells were subconfluent and sufficiently adherent to apply the staining and crosslinking procedure.
  • argon ion (488 nm) or helium/neon (543 nm) laser light was fiber-coupled into the microscope (IX70, Olympus).
  • the excitation beam passes the beam splitting plate (reflection 10%) and is directed onto the back aperture of a high-numerical objective (Olympus Uplan 6Ow NA1.2).
  • the generated fluorescence is collected by the same objective, passes the beam splitting plate (transmission 90%) and focused by the tube lens onto the confocal pinhole (40 ⁇ m).
  • a color splitting dichroic mirror (550 DLRP, Omega Optical, Brattleboro, Vt) splits the fluorescence into the GFP- (band pass 515DF30) and TMR-specific (band pass 585DF20) color channels, which are separately detected by single-photon sensitive avalanche photodiodes (SPMC-AQR-13-FC, Perkin Elmer).
  • SPMC-AQR-13-FC Single-photon sensitive avalanche photodiodes
  • the binary image files generated by the MIPS software contain the number of photon counts accumulated within the residence time of the laser focus (pixel time, PT) at a certain position in the sample.
  • the binary files are a proprietary image format and have been converted into TIF images by a script (Acapella, Evotec Technologies).
  • the PTs used for imaging were 0.5 or 1 ms. Thus, dividing the counts for each pixel by PT in ms, converts the absolute number of photons to kHz intensities.
  • the molecular brightness (kHz per particle) for free eGFP and NTA-ASA-TMR (34) was determined by fluorescence correlation spectroscopy (FCS) in medium ( Figure 15 a, b, Figure 16 a).
  • FCS fluorescence correlation spectroscopy
  • the molecular brightness of the eGFP tagged receptors and free eGFP is the same (determined by FCS measurements at the membrane and in the cytoplasm of transfected cells (not shown). Therefore, dividing the kHz intensities of each pixel by the molecular brightnesses of eGFP and NTA-ASA-TMR (34) yielded absolute particle numbers for each pixel.
  • N cor r G(0)*(1 +U/F) "2 , where G(O) is the intercept of the correlation curve, U the background intensity for a certain laser power and F the average background subtracted signal ( Figure 16 b). Dividing the intensity images by the molecular brightness returns particles per pixel.
  • the average concentrations at the surface membrane were determined with a proprietary image calculation script (MatLab Version 7.2.0.232 (R2006a), MathWorks Inc., USA).
  • the region of interest (ROI) was segmented by threshold settings and manual corrections; a global background fluorescence from the medium was subtracted.
  • the synthesis of the ths-NTA consists of coupling 3 carboxy functionalized NTA moieties, prepared by alkylation of the amino function of a protected glutamate derivative, to the amino groups of a cyclam scaffold. To the last amino group, an amino caproic spacer was attached before the coupling of the different dyes (the QSY7, the TMR and the Rhodamine Red
  • the mono-NTA was obtained starting from the te/t-butyl protected NTA (3)
  • QSY7 is a non-fluorescent acceptor dye and TMR and RhB are fluorescent rhodamine derivatives with similar absorption characteristics.
  • Rhodamine B was chosen to test a cellular assay system addressing the conformational change of a cell surface expressed lnterleukin-4 receptor (IL-4R).
  • IL-4R lnterleukin-4 receptor
  • the brightness of Rhodamine B is known to be sensitive to the polarity of the local environment.
  • protein conjugates of the Rhodamine Red-X dye are frequently brighter (than those of Lissamine Rhodamine B) and are less likely to precipitate during storage.
  • NTA metal-ion-chelating moiety
  • a photoreactive functionality e.g. azides
  • a weak electrophile e.g. an acryloyl
  • ASA azido salicyclic acid
  • Figure 8 was chosen as photo-crossl inker.
  • aryl azides Upon illumination ( ⁇ 380 nm), aryl azides generate radicals (very reactive intermediates) that can react with nucleophiles present in the protein sequence (e.g OH, NH, SH) to form a covalent.
  • Hydroxy aryl azides do not react via the short lifetime singlet nitrenes but through an azacycloheptatetrane intermediate formed by very rapid intramolecular rearrangement of the nitrenes (Brunner 1993) ( Figure 8).
  • This cycle is highly electrophilic but less reactive than a typical nitrene and does not insert non-activated C-H bonds.
  • the acryloyl group is a weak electrophile (Michael acceptor), unreactive toward the nucleophiles present in the protein (e.g NH, OH, SH) unless high local concentrations of reagents accelerate this reaction.
  • the NTA moiety permits to localize the covalent bond on the protein terminus where the histidine sequence is located. Especially for the photo-crossl inkers this property is important as otherwise covalent bonds might be formed somewhere on the peptidic chain during illumination. This would lead to a loss in site selectivity of labeling.
  • the labeling reaction required a purification step which is caused by the presence of a large excess of dye and to the fact that labeled compounds are often not detected by MS in the reaction medium. However, all the desired compounds were produced and characterized.
  • Figure 10 shows fluorescence spectra before (black) and after (green) addition of non-fluorescent ths-NTA-Ni 2+ -QSY7 (11). Within a few seconds, the probe quenches the GFP-C-His fluorescence to ⁇ 15%. Binding was reversed by a large excess of imidazole, the functional moiety of histidine (red).
  • Tris-NTA probes bind the His-tag with higher affinities compared to the mono-NTA tags and are characterized by slow dissociation processes in the time range of minutes to hours.
  • Mono-NTA tags bear the advantage of reduced binding confirmation possibilities and "cleaner" binding events. Therefore, in an effort to expand the spectrum of His-tag directed probes, the reversible mono-NTA tracer was used to add a photo-crosslink functionality (illumination at 365 nm). Covalent conjugation extends the application range for His-tag binding probes to long term observations of cellular processes. It might also provide an alternative protein labeling strategy for proteins in vitro. Labeling can be performed in a broad range of buffers followed by purification steps.
  • covalent conjugation is a prerequisite for single molecule spectroscopy applications in very low concentrations (pM to nM).
  • the system ensures a homogeneous 1 :1 labeling stoichiometry of observed proteins which facilitates the calculation of quantitative data.
  • Binding and fluorescence properties of the new labeling reagent were investigated by conventional fluorescence spectroscopy.
  • FRET fluorescence resonance energy transfer
  • a purified recombinant GFP with a C-terminal His-Tag, GFP-C-His served as a FRET donor for NTA- ASA-TMR (34) or NTA-ASA-QSY7 (35) as acceptors.
  • Quenching of the donor due to energy transfer directly reflects the fraction of complexed GFP- C-His ( Figure 13 a).
  • the affinity was determined by measuring the degree of donor quenching under equilibrium conditions for increasing ligand concentrations ( Figure 13 c).
  • Kd values of previously described compounds NTA-I were also determined ( Figure 17). Without photoactivation, binding of probes 34 and 35 to oligohistidine-tagged proteins is reversible and mediated by a d 8 coordinated Ni 2 . Thus, EDTA, added in excess (> 250-fold), competes for free Ni 2+ -ions, and binding is successively reversed. The kinetics of this competition reaction critically depended on the substituents of the NTA.
  • the fractional fluorescence intensity associated with the GFP-band versus free dye is 9% for a dosage of 120 mJ and 15% for 240 mJ. Further increase of the dosage had no effect on increase in complexation. Neglecting the small background of protein impurities running at higher molecular weights, we recalculated these values with respect to the fraction of bound ligand, which resulted in a crosslink efficiency of 45% (120 mJ) and 75% (240 mJ) at the binding site.
  • IL-4R His-tagged lnterleukin-4 receptor
  • the receptor construct (NHis-IL-4Rac-GFP) comprises a hexahistidine stretch at the N-terminus, followed by the extracellular and transmembrane domains of the lnterleukin-4 receptor ⁇ chain. The cytoplasmic tail of the receptor was replaced by eGFP. In this configuration, the His-tag is expressed extra-cellularly, while the GFP is located in the cytosol.
  • the double-tagged receptors allow estimating crosslinking efficiencies, provided that receptor and ligand densities are observed in orthogonal color channels.
  • Figure 14 a-f illustrates confocal images of a transfected cell in a layer of non-transfected neighbors. In the GFP-channel only the transfected cell is visible. The fluorescent receptors are clearly localized in the membrane. Typical for high expression levels, the unprocessed receptors accumulate in intracellular membrane systems.
  • TMR color channel nicely shows that staining of transfected cells with NTA- ASA-TMR (34) is restricted to the surface membrane suggesting that the labeling reagent is not cell penetrating.
  • IL-4Rac-eGFP did not accumulate TMR-fluorescence over background ( Figure 14 g-i).
  • Tsien RY (1998) The green fluorescent protein. Annu.Rev.Biochem.; 67:509- 44.

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Abstract

La présente invention porte sur de nouveaux agents pour conjuguer de façon covalente des protéines étiquetées His, à des conjugués covalents de protéines étiquetées His et sur des procédés de fabrication de tels conjugués.
PCT/EP2008/056361 2007-05-25 2008-05-23 Procédé de fabrication de conjugués covalents avec des protéines étiquetées his WO2008145609A1 (fr)

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WO2011101445A1 (fr) * 2010-02-18 2011-08-25 Johann Wolfgang Goethe-Universität Frankfurt am Main Composés chélateurs multivalents (mch) à haute affinité et leur utilisation pour l'analyse structurelle et fonctionnelle de molécules cibles
WO2012113738A1 (fr) 2011-02-22 2012-08-30 Akzo Nobel Chemicals International B.V. Précurseurs d'agents chélateurs, fluides les contenant et leur utilisation
CN102827016A (zh) * 2012-03-20 2012-12-19 石家庄杰克化工有限公司 新型绿色螯合剂谷氨酸二乙酸四乙酸金属盐的制备
WO2015097313A1 (fr) * 2013-12-27 2015-07-02 The University Of Hong Kong Sondes fluorescentes basées sur la chélation de métaux pour le marquage de protéines ou d'autres biomolécules dans des cellules
CN108976160A (zh) * 2017-06-05 2018-12-11 华东理工大学 一种荧光探针及其制备方法和用途
CN108993415A (zh) * 2018-08-02 2018-12-14 西北大学 一种ids螯合型吸附剂及其金属螯合型吸附剂的除磷应用
JP2020506161A (ja) * 2017-12-28 2020-02-27 新発薬業有限公司 ５ｒ−ベンジルオキシアミノピペリジン−２ｓ−ギ酸又はその誘導体の調製方法
CN114315784A (zh) * 2021-11-30 2022-04-12 浙江工业大学 一种组氨酸标签荧光探针及其制备方法和应用

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011101445A1 (fr) * 2010-02-18 2011-08-25 Johann Wolfgang Goethe-Universität Frankfurt am Main Composés chélateurs multivalents (mch) à haute affinité et leur utilisation pour l'analyse structurelle et fonctionnelle de molécules cibles
WO2012113738A1 (fr) 2011-02-22 2012-08-30 Akzo Nobel Chemicals International B.V. Précurseurs d'agents chélateurs, fluides les contenant et leur utilisation
US9556378B2 (en) 2011-02-22 2017-01-31 Akzo Nobel Chemicals International B.V. Chelating agent precursors, fluids containing them, and their use
CN102827016A (zh) * 2012-03-20 2012-12-19 石家庄杰克化工有限公司 新型绿色螯合剂谷氨酸二乙酸四乙酸金属盐的制备
CN102827016B (zh) * 2012-03-20 2015-04-01 石家庄杰克化工有限公司 新型绿色螯合剂谷氨酸二乙酸四乙酸金属盐的制备
WO2015097313A1 (fr) * 2013-12-27 2015-07-02 The University Of Hong Kong Sondes fluorescentes basées sur la chélation de métaux pour le marquage de protéines ou d'autres biomolécules dans des cellules
CN106461649A (zh) * 2013-12-27 2017-02-22 香港大学 用于在细胞中标记蛋白质或其它生物分子的基于金属螯合的荧光探针
CN108976160A (zh) * 2017-06-05 2018-12-11 华东理工大学 一种荧光探针及其制备方法和用途
JP2020506161A (ja) * 2017-12-28 2020-02-27 新発薬業有限公司 ５ｒ−ベンジルオキシアミノピペリジン−２ｓ−ギ酸又はその誘導体の調製方法
CN108993415A (zh) * 2018-08-02 2018-12-14 西北大学 一种ids螯合型吸附剂及其金属螯合型吸附剂的除磷应用
CN114315784A (zh) * 2021-11-30 2022-04-12 浙江工业大学 一种组氨酸标签荧光探针及其制备方法和应用

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