WO2010014236A2 - Lieurs clivables à base d'acylhydrazone - Google Patents

Lieurs clivables à base d'acylhydrazone Download PDF

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WO2010014236A2
WO2010014236A2 PCT/US2009/004402 US2009004402W WO2010014236A2 WO 2010014236 A2 WO2010014236 A2 WO 2010014236A2 US 2009004402 W US2009004402 W US 2009004402W WO 2010014236 A2 WO2010014236 A2 WO 2010014236A2
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compound
cleavable linker
support
group
acylhydrazone
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PCT/US2009/004402
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English (en)
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WO2010014236A3 (fr
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Harold L. Kohn
Ki Duk Park
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The University Of North Carolina At Chapel Hill
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems

Definitions

  • biotinylated product release step Several methods have been advanced to address the difficulty of the biotinylated product release step. They include using either biotin analogs (Hirsch et al., 2002; Zeheb et al., 1986) or protein-engineered streptavidin mutants (Howarth et al., 2006; Malmstadt et al., 2003; Morag et al., 1996; Wu et al., 2005), structural modifications that weaken the biotin- (strept)avidin interaction. While these methods improve the release of biotinylated molecules, the reduced association constant for the complex does not allow the application of stringent wash conditions to remove nonspecific protein interactions from the resin and adversely affects the capture efficiency of the biotin-tagged adducts.
  • linkers that contain a proteolytic (Dieterich et al., 2007; Speers et al., 2005) or a chemically labile (e.g., disulfide (Finn et al., 1985; Marie et al., 1990; Shimkus et al., 1985), acid-sensitive (van der Veken et al., 2005), base-sensitive (Ball et al., 1997; Kazmierski et al., 1995), nucleophile-sensitive (Lin et al., 1991), photolytic (Bai et al., 2004; Thiele et al., 1994), reductive (Verhelst et al., 2007)) site.
  • a proteolytic Dieterich et al., 2007; Speers et al., 2005
  • a chemically labile e.g., disulfide (Finn et al., 1985; Marie et al., 1990; Shim
  • a first aspect of the invention is a compound of Formula I:
  • X is a cleavable linker comprising an acylhydrazone
  • Y and R are each independently selected from the group consisting of covalent coupling groups and members of a specific binding pair.
  • a further aspect of the invention is a support (e.g., a solid support) useful for binding a compound of interest from a mixture, wherein the compound of interest is a first member of a binding pair and the support comprises a second member of the binding pair coupled to the support by a cleavable linker, the improvement comprising: employing a compound as herein above and below as the cleavable linker (e.g. where R is the first member of the binding pair).
  • the cleavable linker can be covalently coupled or specifically bound (e.g., by biotin-avidin binding) to the support.
  • Figure 1 General Strategy of Cleavable Linker for Isolation, Identification and Detection of Target Proteins.
  • FIG. 3 Comparison of the Efficiency of Protein Release from BSA Adducts (9 and 11) Modified with either Cleavable Linker 1 or Non-cleavable Linker 10.
  • A Chemical structure of non-cleavable linker 10 and two different BSA samples (9 and 11) modified with either cleavable linker 1 or non-cleavable linker 10.
  • B All reactions were run in aqueous 50 mM HEPES (pH 5.8) using optimized condition (7 (10 mM) and SDS (20 mM) at 50 0 C (1 h)) (data not shown).
  • Lanes marked “s” correspond to the supernatant removed from the bead mixture after the initial treatment, while the lanes marked “b” refer to the sample obtained after the beads were washed and then treated with loading buffer and heated at 95 0 C for either 5 min (Ab) or 15 min (lane Bb).
  • reaction B 5- to 10-fold sample volumes from the recovered Bb were loaded to facilitate comparison. The proteins were visualized by silver staining.
  • Figure 5 Use of 1 for Enolase Capture, Release, and Detection.
  • A Scheme of enolase capture, release, and detection. The single cysteine unit in enolase (19) was treated with 13 to give approximately 30% of alkyne functionalized enolase 20 based on MS (data not shown) and then converted to enolase 21 under Cu(I) -mediated cycloaddtion condition.
  • B Use of fluorescein hydrazide 22 for detection and isolation of streptavidin-bound modified enolase 21.
  • Lane 1 the enolase recovered after incubation of enolase 21 with streptavidin beads in aqueous 50 mM HEPES (pH 5.8), followed by washing of the beads, and then treatment of the beads with 22 (1.5 mM) using an optimized condition (p-anisidine [10 mM], 37 °C, 4 h); lane 2, 24 (reduction of 23 with NaCNBH 3 at pH 3.8); lane 3, control (all reactions were the same as 23 except the cleavable linker was excluded in the cycloaddition step).
  • the proteins were visualized by fluorescent detection by excitation at 488 nm and detection at 520 nm.
  • FIG. Proteomic Target Search in Mouse Liver Proteome with the Cleavable Linker 1 Using 25 and Comparison with Non-cleavable Linker 10.
  • A Chemical structures of 25 and 26.
  • B After labeling with by 25 (5 ⁇ M or 25 ⁇ M) followed by Cu(I)-mediated cycloaddition with 26, the signal was detected by in-gel fluorescence scanning. Using 5 ⁇ M 25, ALDH-I (arrow) was selectively labeled. At 25 ⁇ M 25, ALDH-I (arrow) and an abundant protein(s) (asterisk) in liver lysate were labeled.
  • probe-labeled proteins were captured by streptavidin beads after Cu(I)-mediated cycloaddition to either cleavable linker 1 or non-cleavable linker 10.
  • the beads were washed and treated using a mild cleavage condition (7 (100 mM), /7-anisidine (10 mM) in aqueous 50 mM HEPES (pH 5.8) (37 °C, 4 h)).
  • Lanes marked "s” correspond to the supernatant removed from the bead mixture after the initial treatment, while the lanes marked "b” refer to the sample obtained after treatment of the remaining beads with loading buffer (95 0 C, 5 min). The proteins were visualized by silver staining.
  • Alkyl refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms.
  • Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.
  • Loweralkyl as used herein, is a subset of alkyl, in some embodiments preferred, and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms.
  • Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like.
  • Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups
  • Cycloalkyl refers to a saturated or partially unsaturated cyclic hydrocarbon group containing from 3, 4 or 5 to 6, 7 or 8 carbons (which carbons may be replaced in a heterocyclic group as discussed below).
  • Representative examples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. These rings may be optionally substituted with additional substituents as described herein such as halo or loweralkyl.
  • the term “cycloalkyl” is generic and intended to include heterocyclic groups as discussed below unless specified otherwise. Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups.
  • alkenyl refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms (or in loweralkenyl 1 to 4 carbon atoms) which include 1 to 4 double bonds in the normal chain.
  • alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4- pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like.
  • Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups.
  • Alkynyl refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms (or in loweralkynyl 1 to 4 carbon atoms) which include 1 triple bond in the normal chain.
  • Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2- butynyl, 4-pentynyl, 3- pentynyl, and the like.
  • alkynyl or “loweralkynyl” is intended to include both substituted and unsubstituted alkynyl or loweralkynyl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above. Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups.
  • Heterocyclo refers to an aliphatic (e.g., fully or partially saturated heterocyclo) or aromatic (e.g., heteroaryl) monocyclic- or a bicyclic-ring system.
  • Monocyclic ring systems are exemplified by any 3 to 8 membered ring containing 1 , 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur.
  • the 5 membered ring has from 0-2 double bonds and the 6 membered ring has from 0-3 double bonds.
  • monocyclic ring systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine,
  • Bicyclic ring systems are exemplified by any of the above monocyclic and heterocyclic ring systems fused to an aryl group as defined herein, a cycloalkyl group as defined herein, or another monocyclic or heterocyclic ring system as defined herein.
  • bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like.
  • Such groups can be unsubstituted or substituted with one or more (e.g
  • Aryl refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings.
  • Representative examples of aryl include, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.
  • aryl is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above. Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups.
  • Heteroaryl as used herein is as described in connection with heterocyclo above. Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups.
  • Arylalkyl refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Representative examples of arylalkyl include, but are not limited to, benzyl, 2- phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.
  • Heteroarylalkyl refers to a heteroaryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Heterocycloalkyl refers to a heterocyclo group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Electrode-withdrawing and “electron donating” refer to the ability of a substituent to withdraw or donate electrons relative to that of hydrogen if the hydrogen atom occupied the same position in the molecule.
  • Examples of such electron withdrawing and electron donating groups or substituents include, but are not limited to halo, nitro, cyano, carboxy, loweralkenyl, loweralkynyl, loweralkanoyl (e.g., formyl), carboxyamido, aryl, quaternary ammonium, aryl (loweralkanoyl), carbalkoxy and the like; acyl, carboxy, alkanoyloxy, aryloxy, alkoxysulfonyl, aryloxysulfonyl, and the like; hydroxy, alkoxy or loweralkoxy (including methoxy, ethoxy and the like); loweralkyl; amino, lower alkylamino, di(loweralkyl) amino, aryloxy (such as phenoxy), mercapto, loweralkylthio, lower alkylmercapto, disulfide (loweralkyldithio) and the like; 1-piperidino, 1-pipe
  • Acyl as used herein alone or as part of another group refers to a -C(O)R radical, where R is any suitable substituent such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl or other suitable substituent as described herein.
  • alkanoyl refers to the group -C(O)R', wherein R' is lower alkyl. Hence “alkanoyl” groups are particular examples of “acyl” groups, as described above.
  • Halo or "halogen,” as used herein refers to -Cl, -Br, -I or -F.
  • Oxy refers to an -O- group.
  • Sulfonyl refers to an -SO 2 - group.
  • “Support” as used herein may be any suitable support, including but not limited to controlled pore glass, oxalyl-controlled pore glass, silica-containing particles, polymers of polystyrene, copolymers of polystyrene, copolymers of styrene and divinylbenzene, copolymers of dimethylacrylamide and N,N'-bisacryloylethylenediamine, soluble support medium such as dendrimers, PEPS, lipid particles such as liposomes, etc. See, e.g., US Patent No. 6,653,468; see also US Patent Nos. 7,202,264 and 6,664,372.
  • Solid support as used herein is meant to comprise any solid (flexible or rigid) substrate (e.g., a particulate or non-particulate substrate) onto which it is desired to apply an array of one or more binding agents.
  • the substrate may be biological, non-biological, organic, inorganic or a combination thereof, and may be in the form of particles, strands, precipitates, gels, sheets, tubings, spheres, containers, capillaries, pads, slices, films, plates, slides, etc, having any convenient shape, including disc, sphere, circle, etc.
  • the substrate surface supporting the array may have any two-dimensional configuration and may include, for example steps, ridges, kinks, terraces and the like and may be the surface of a layer of material different from that of the rest of the substrate. See, e.g., US Patent No. 7,563,587.
  • Covalent coupling group includes, but is not limited to, electrophilic affinity bait groups, photochemical affinity bait groups, and reactive groups.
  • Electrophilic affinity bait group includes, but is not limited to, NCS, C(O)H, and acyl halide, haloketones, alkyl halide, and alkyl sulphonate bait groups.
  • Photochemical affinity bait group includes, but is not limited to, aryl azide, diazirine, and benzophenone bait groups.
  • Reactive groups as used herein includes, but is not limited to, halo, sulfonate, aldehyde, ketone, ester, and activated ester reactive groups.
  • Specific binding pair (abbreviated “sbp”) as used herein describes a pair of molecules (each being a member of a specific binding pair) which are naturally derived or synthetically produced.
  • One of the pair of molecules has a structure (such as an area or cavity) on its surface that specifically binds to (and is therefore defined as complementary with) a particular structure (such as a spatial and polar organization) of the other molecule, so that the molecules of the pair have the property of binding specifically to each other.
  • Examples of types of specific binding pairs are antigen- antibody, antibody-hapten, biotin-avidin, ligand-receptor (e.g., hormone receptor, peptide- receptor, enzyme-receptor), carbohydrate-protein, carbohydrate-lipid, lectin-carbohydrate, nucleic acid-nucleic acid (such as oligonucleotide-oligonucleotide). See, e.g., US Patent No. 7,563,587; 7,506,556.
  • Nucleic acid refers to a deoxyribonucleotide polymer (DNA) or ribonucleotide polymer (RNA) in either single- or double-stranded form, and also encompasses synthetically produced analogs that can function in a similar manner as naturally occurring nucleic acids. While natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, nucleotides or bases. These include, for instance, peptide nucleic acids (PNAs) as described in, e.g., U.S. Pat. No.
  • PNAs peptide nucleic acids
  • pyranosyl nucleic acids as described in, e.g., WO 99/15540 (p-RNAs), WO 99/15539 (p-RNAs), and WO 00/1 101 1 (p-DNAs); locked nucleic acids (LNAs), as described in, e.g., U.S. Pat. No. 6,316,198; and phosphothionates and other variants of the phosphate backbone of native nucleic acids. See, e.g., US Patent No. 7,563,587.
  • Oligonucleotide refers to single stranded nucleotide multimers of from about 5 to about 100 nucleotides. See, e.g., US Patent No. 7,563,587.
  • Antibody as used herein means an immunoglobulin which may be naturally, or partly or wholly synthetically, produced, and also includes active fragments thereof, including Fab antigen-binding fragments, univalent fragments and bivalent fragments. The term also covers any protein having a binding domain which is homologous to an immunoglobulin binding domain. Such proteins can be derived from natural sources, or partly or wholly synthetically produced. Exemplary antibodies are the immunoglobulin isotypes and the Fab, Fab', F(ab') 2 , scFv, Fv, dAb, and Fd fragments. See, e.g., US Patent No. 7,563,587.
  • X is a cleavable linker comprising an acylhydrazone
  • Y and R are each independently selected from the group consisting of covalent coupling groups and members of a specific binding pair.
  • Y is a covalent coupling group ⁇ e.g., N 3
  • R is a member of a specific binding pair ⁇ e.g., avidin).
  • the cleavable linker further comprising a polyalkylene oxide group ⁇ e.g., poly(ethylene glycol)).
  • the compound has the structure of Formula II:
  • A, B, C, and X are each independently selected from the group consisting of O, S, N(H), N(R), N(R')(R") + , CH 2 , C(R')(R"), single covalent bond, and double covalent bond;
  • F' is selected from the group consisting of hydrogen, lower alkyl, and aryl (which can be unsubstituted or substituted with one or more electron-donating or electron-withdrawing groups);
  • W is selected from the group consisting of a single covalent bond, O, S, N(H), and N(R');
  • R and R" are each independently selected loweralkyl; n is 0 or 1-4; and m is 0 or 1-4.
  • R is selected from the group consisting of ssDNA, RNA, antigenic peptides, N-terminal polyhistidines, covalent coupling groups, and a group of the Formula:
  • Y is selected from the group consisting of N 3 , CCH, electrophilic affinity bait groups, photochemical affinity bait groups, reactive groups, peptides, proteins, nucleic acid, carbohydrates, lipids, modified drugs, inhibitors, enzyme substrates or mimics, and organic groups.
  • a further aspect of the invention is a support (e.g., a solid support) useful for binding a compound of interest from a mixture, wherein the compound of interest is a first member of a binding pair and the support comprises a second member of the binding pair coupled to the support by a cleavable linker, the improvement comprising employing a compound as described herein as the cleavable linker (e.g. where R is the first member of the binding pair).
  • the cleavable linker can be covalently coupled or specifically bound (e.g., by biotin-avidin binding) to the support.
  • the present invention provides a method of releasing a compound of interest bound to a support from that support, wherein the compound of interest is bound to the support through a cleavable linker.
  • the method employs a cleavable linker comprising an acylhydrazone group therein (e.g., a cleavable linker as described herein and above), and is carried out by cleaving the acylhydrazone by contacting an acylhydrazide and/or an anionic detergent (e.g., sodium dodecyl sulfate; SDS) thereto.
  • an acylhydrazone group e.g., a cleavable linker as described herein and above
  • the contacting step is carried out under near neutral pH conditions (e.g., pH 4, 5 or 6 up to pH 8 or 9).
  • near neutral pH conditions e.g., pH 4, 5 or 6 up to pH 8 or 9.
  • the acylhydrazide is contacted to the acylhydrazone linker in solution in an amount of at least 1, 10 or 50 mM.
  • the acylhydrazide is contacted to the acylhydrazone in solution in an amount up to 500 or 1000 mM.
  • the anionic detergent is contacted to the acylhydrazone in solution in an amount of at least 0.5, 1, 5 or 10 mM. In some embodiments of the foregoing, the anionic detergent is contacted to the acylhydrazone in solution in an amount up to 50 or 100 mM.
  • the contacting step is carried out at a temperature of at least 4, 10 or 20 0 C.
  • the contacting step is carried out at a temperature of up to 40 or 50 °C.
  • the contacting step is carried out for a time of at least 10, 20 or 30 minutes.
  • the contacting step is carried out for a time of up to 2 or 4 hours.
  • At least 50, 60, 70 or 80 percent by weight of the compound of interest is released following the contacting step.
  • the acylhydrazide comprises a detectable group that reacts with the cleavable linker in the cleaving step, whereby the compound of interest can be detected after the cleaving step by detecting the detectable group coupled thereto.
  • Cleavable linkers of the present invention can be implemented in a variety of additional ways, such as in the methods, compounds and products described in US Patent Application Publication No. 20090181860 (Applied Biosystems) in place of the disulfide bond cleavable linkers described therein.
  • the present invention provides, among other things, a collection of at least two distinguishably labeled oligonucleotide probe families (optionally wherein probes in each probe family comprise a constrained portion and an unconstrained portion, optionally wherein each position in the constrained portion is at least 2-fold degenerate, and and optionally wherein probes in each family comprise a scissile internucleoside linkage).
  • each probe comprises a terminus that is not extendable by ligase. In some embodiments, each probe comprises a detectable moiety at a position between the scissile linkage and the terminus that is not extendable by ligase. In some embodiments, the scissile linkage is a phosphorothiolate linkage. In some embodiments, the collection comprises 2, 3, or 4 or more probe families.
  • the detectable moiety is preferably attached by a cleavable linker, and optionally is photobleachable.
  • the cleavable linker preferably comprises an acylhydrazone as described herein, and in use is cleaved by an acylhydrazide as described herein. The present invention is explained in greater detail in the following non-limiting Examples.
  • cleavage reaction would allow the incorporation of a traceable tag (i.e., isotopic, radioactive, fluorescent) that permits the detection and identification of the captured (bio)molecule by chromatographic and spectroscopic methods when it is released.
  • a traceable tag i.e., isotopic, radioactive, fluorescent
  • acylhydrazones are stable under near neutral-to-basic pH conditions (pH ⁇ 8- 10), but they undergo both hydrolysis (Flinn et al., 2004; Smith et al., 2007) and hydrazone exchange with hydrazides (King et al., 1986) in moderately acidic solutions (pH -4-5).
  • biotinylated probes with an imbedded acylhydrazone unit are readily cleaved in high yields at near neutral pH values with acylhydrazides that contain a traceable tag providing (bio)molecules that can be identified in proteomic searches by conventional analytical techniques.
  • incorporation of an acylhydrazone unit within a linker will be generalized, allowing for the selective release of tagged molecules from non- covalently or covalently bound supports.
  • Compound 1 was selected and synthesized as our test linker ( Figure 2). It contained an acylhydrazone cleavage site, a biotin unit, a terminal azide on 1 to permit capture with an alkyne-modified protein using a copper(I)-mediated cycloaddition reaction ("click chemistry") (Agard et al., 2006; Dieterich et al., 2007; MacKinnon et al., 2007; Rostovtsev et al., 2002; Speers et al., 2003; Tornoe et al., 2002), and a polyethylene glycol linker to increase its water solubility and to minimize adverse steric interactions with the immobilized streptavidin during protein capture (Marie et al., 1990).
  • Click chemistry Copper(I)-mediated cycloaddition reaction
  • BSA sample 9 (Scheme 2, proposed general structure of the photoadduct; the structure of the azide photoadduct has not been determined) ( Figure 3A) after photocross-linking with photoactivable lacosamide probe followed by click chemistry with 1 (data not shown).
  • the BSA sample 9 was captured by streptavidin-agarose beads and divided into equal aliquots. Each portion was either treated with or without 7 (50, 200 mM) at different pH values (5.8, 7.4) to release the captured proteins.
  • mouse soluble liver lysate (pH 8.0) was incubated with 25 (5, 25 ⁇ M) at room temperature (1 h) and then treated with rhodamine azide (RhN 3 , 26), TCEP, TBTA and CuSO 4 .
  • the lysate was separated by SDS-PAGE and the labeled proteins visualized by in-gel fluorescence (Figure 6B).
  • Figure 6A In agreement with earlier reports (Speers et al., 2004), only a single protein band was observed at ⁇ 50 kDa when 5 ⁇ M of 25 was used ( Figure 3A, lane 1). Increasing the concentration of 25 to 25 ⁇ M led to increased intensity of this band and the appearance of lower molecular weight bands ( Figure 6B, lane 3).
  • the in-gel detection of the -50 kDa band was consistent with the selective tagging of ALDH-I by activity probe 25 as detailed in the literature (Speers et al., 2004).
  • SDS serves as either a specific or general base in the hydrolysis step, or SDS-mediated acylhydrazone loss is due to direct attack of the hydrazone linkage by SDS, or if SDS-mediated loss is due, in part, to the formation of micelles that facilitates hydrazone modification (Camilo et al., 2004; Gogoi et al., 2005; Wang et al., 2005). Most important for our study was our demonstrating that 7 induced hydrazone exchange in 1. Acethydrazide exchange of acylhydrazones has been described by King et al. (1986).
  • linker 1 contained a terminal azide unit we installed an alkyne moiety (moieties) within the test proteins to permit Cu(I)- mediated cycloaddition reaction. Accordingly, BSA was randomly modified with the photoaffinity lacosamide probe to give an alkyne functionalized BSA (data not shown). Correspondingly, the single cysteine unit in S.
  • cleavable linker 1 The utility of cleavable linker 1 was tested in a proteomic search. Cravatt and coworkers have elegantly demonstrated that ALDH-I was selectively labeled in the mouse soluble liver lysate by phenyl sulfonate ester 25 at pH 8.0. The specificity of this activity probe for ALDH-I provided a stringent test for our linker. We asked if linker 1 selectively reacted with 25-modified ALDH-I by Cu(I)-mediated cycloaddition and whether, upon streptavidin bead capture and treatment of the resin with 7 under mild conditions, the supernatant would be enriched in ALDH-I and be devoid of background proteins.
  • Some advantages associated with this method include: (1) the ease of linker synthesis; (2) stability of the linker to bioorthogonal coupling transformations (e.g., Cu(I) -mediated cycloaddition reactions); (3) linker stability at near neutral and moderately basic pH values; (4) chemoselective cleavage of the linker with acylhydrazides with minimal release of background proteins non-specifically bound to (streptavidin) supports; (5) high recovery yields of the cleaved proteins; and (6) ability to incorporate a traceable tag (e.g., isotopic, fluorescent) in the captured protein to facilitate detection and identification.
  • a traceable tag e.g., isotopic, fluorescent
  • the acylhydrazone linker compares favorably with previously introduced cleavable linkers. Like 1, each linker has advantages and disadvantages to consider prior to selection and use. For instance, photolytic-based cleavable linkers typically undergo efficient release, but upon cleavage, they release reactive carbonyl compounds (aldehydes, ketones) that can react with the protein (Bai et al., 2004; Thiele et al., 1994). Acid-sensitive linkers that require trifluoroacetic acid (TFA) can lead to non-specific cleavage and TFA use requires its removal prior to MS analysis (van der Veken et al., 2005).
  • TFA trifluoroacetic acid
  • Linkers that rely on proteolytic enzymes provide a selective cleavage method but require that a designed peptide sequence be installed within the linker that is efficiently cleaved (Dieterich et al., 2007; Fonovic et al., 2007; Speers et al., 2005).
  • proteolytic enzymes for cleavage may prevent the isolation of the intact protein.
  • Proteomic experiments utilizing disulfide linkers require the use of buffers that are devoid of reducing reagents (Finn et al., 1985; Fonovic et al., 2007; Marie et al., 1990; Shimkus et al., 1985).
  • these disulfide linkers can be cleaved under cellular conditions. This concern for premature cleavage of the linker is not an issue for the diazobenzene linker advanced by Bogyo and coworkers and where Na 2 S 2 O 4 serves as the chemoselective reductant (Fonovic et al., 2007; Verhelst et al., 2007).
  • Ci 8 H 32 N 7 O 5 S 458.2186 Anal. (C 18 H 3 iN 7 O 5 S, 0.25 H 2 O) Calcd.: C, 46.79%; H, 6.87%; N, 21.22%; S, 6.94%. Found: C, 46.67%; H, 6.82%; N, 20.88%; S, 6.88%.
  • Enolase (19) Treatment of Enolase (19) with Maleimide 13 to Afford Enolase 20.
  • a 200 ⁇ M solution (1 mL) of S. cerevisiae enolase (19) (Sigma, E6126) in aqueous 50 mM HEPES (pH 7.4) was added a 200 mM solution (0.1 mL) of 13 (2.7 mg, 20.0 //mol) in 5% CH 3 CN/aqueous 50 mM HEPES (pH 7.4).
  • the solution was incubated at room temperature (2 h) and then the reaction solution was diluted with aqueous 50 mM HEPES buffer (pH 7.4) to 5 mL and passed through NAP-5 columns pre-equilibrated with 50 mM HEPES buffer (pH 7.4).
  • the eluents ( ⁇ 10 mL) were combined and stored at 4 °C.
  • reaction mixture was rotated using Roto-shake (8 rpm, Scientific Industries Inc., Model No. SI-1100, Bohemia, NY) at room temperature (1 h), then divided in two equal portions and passed through separate NAP- 5 columns pre-equilibrated with HEPES buffer (pH 7.4) to give an aqueous solution of enolase 21.
  • the eluents were combined ( ⁇ 2 mL) and stored at 4 °C.
  • Use of Fluorescent Hydrazide 22 for Detection and Isolation of Streptavidin- bound Modified Enolase 21 and Reduction of Imine 23 to 24 (Method B).
  • the supernatant (50 ⁇ L) was treated with a 10 mM solution Of NaCNBH 3 (50 ⁇ L) in 50 mM NaOAc buffer (pH 3.8) and rotated using Roto-shake (8 rpm) at room temperature (2 h) to give a mixture containing 24. Both the supernatant containing 23 and the reaction mixture containing 24 were loaded on a 10% SDS-PAGE gel. Labeled proteins were visualized using a typhoon 9400 scanner (Amersham Bioscience) with excitation at 488 nm and detection at 520 nm.
  • Mouse soluble liver lysate 400 ⁇ L was treated with 25 (3.08 ⁇ g, 10 nmol, 25 ⁇ M) at room temperature (1 h) and passed through a NAP-5 column to exchange buffer to an aqueous 50 mM HEPES buffer (pH 7.4) and then divided in two 250 ⁇ L aliquots.
  • reaction mixture was rotated at room temperature (2 h) and then an aliquot (50 //L) containing 16/16-d 3 was analyzed by HPLC using a //Bondapak C-18 column (3.9 x 300 mm, Waters Corp. Cat. No. WAT027324) and a photodiode array detector (210-340 nm).
  • a gradient mobile phase (0/100 CH 3 CN/H 2 O-50/50 CH 3 CN/H 2 O) was employed for 30 min using a flow rate of 1 mL/min. The major peaks were collected and analyzed by ESI-MS.
  • reaction solution (0.95 mL) containing 16/16- ⁇ 3 was treated with a 200 mM solution of NaCNBH 3 (0.95 mL) in 50 mM NaOAc buffer (pH 3.8) and shaken at room temperature (1 h).
  • the reaction solution containing 17/17- ⁇ ? 3 was separated by a HPLC and the major peaks collected.
  • the collected solutions were analyzed by ESI-MS.
  • Preparative Reaction iV-(Prop-2-ynyl)maleimide (13) and iV-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)acetaldehyde Biotinylhydrazone (1) to Give 27.
  • Bovine Serum Albumin (BSA, 28) with (R)-JV-(4-Azidobenzyl)-2- acetamido-3-methoxypropanamide (29) to Give 30 (Method C).
  • BSA Bovine Serum Albumin
  • R R-JV-(4-Azidobenzyl)-2- acetamido-3-methoxypropanamide
  • the aliquots were centrifuged (1000 rpm, 1 min), the supernatant removed, and 50 mM HEPES solution (100 //L, pH 5.8-7.4) added and the beads treated with 7 (0.39-1.56 mg, 5-20 //mol) in the presence or absence of a catalyst (aniline, p- anisidine [final concentration 10 mM]; SDS [final concentration 20 mM]).
  • a catalyst aniline, p- anisidine [final concentration 10 mM]; SDS [final concentration 20 mM].
  • the reaction mixture was gently shaken at a specified temperature (22-50 °C) (1-4 h) and then the supernatant collected.
  • streptavidin beads were washed with 10 mM HEPES buffer (pH 7.4) (3 x 0.15 mL), and then boiled (5 min) with SDS-loading buffer (2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bromophenol blue). The samples were loaded on a 10% SDS-PAGE gel and the proteins visualized by silver staining. The relative intensities of the bands were determined by densitometry.
  • Each portion was suspended in 50 mM HEPES solution (50 ⁇ L, pH 5.8), followed by treating with either 7 (0.39 mg, 5 ⁇ mol) and SDS (0.5%, 20 mM) at 50 °C (1 h) (mild conditions) or SDS-loading buffer (2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bromophenol blue) at 95 °C (5 min) (harsh conditions).
  • SDS-loading buffer 2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bromophenol blue
  • an aqueous 50 mM HEPES solution of 9 (1 ⁇ L) and the flowthrough solution (1 ⁇ L) obtained after incubation with the streptavidin beads were used to determine the amount of biotinylated BSA 9 captured by the streptavidin beads using a dot blot assay (data not shown).
  • Solutions (serially diluted by 2-, 4-, 8-, and 16-fold, respectively) (1 ⁇ L) were loaded to a nitrocellulose membrane (RPN203D, Amersham) and the membrane was washed (10 min) with TBST (25 mM Tris buffer, 150 mM NaCl, 0.1% Tween-20 (pH 7.6)).
  • the membrane was incubated in 5% BSA/TBST solution (50 mL) at room temperature (1 h) and then incubated with a HRP-conjugated streptavidin specific for biotin (N-100, Pierce) in 5% BSA/TBST solution (1.5 mL) at room temperature (1 h). After washing (x 4, 5 min each) with TBST, chemiluminescent reagent (RPN2132, GE Healthcare) was added to the blot and the signal developed in the darkroom.
  • HRP-conjugated streptavidin specific for biotin N-100, Pierce
  • the beads were treated with 7 (0.78 mg, 10 ⁇ mol) and SDS (0.5%, 20 mM) at 50 °C (1 h). The supernatant was collected and then the remaining streptavidin beads were boiled (15 min) with loading buffer (2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bromophenol blue [final concentration]). The samples were loaded on a 10% SDS-PAGE gel and the proteins visualized by silver staining.
  • loading buffer 2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bromophenol blue [final concentration]
  • Mouse liver harvested from wild-type C57BL/6 mice and immediately Dounce homogenized in 50 mM sodium/potassium phosphate buffer (pH 8.0) (PB). The lysate was centrifuged at slow speed (1200 x g for 12 min at 4 0 C) to remove debris. The supernatant was centrifuged at high speed (100,000 x g for 1 h at 4 0 C). The supernatant was collected and stored at -80 °C until use. The total protein concentration was determined by using the Bradford assay.
  • PB sodium/potassium phosphate buffer

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Abstract

L'invention concerne des composés de lieurs clivables de formule (I) : dans laquelle : X est un lieur clivable comportant une acylhydrazone; et Y et R sont chacun sélectionnés indépendamment dans le groupe composé de groupes et d'éléments de liaison covalents d'une paire de liaison spécifique. L'invention concerne également des supports solides auxquels sont couplés de tels lieurs clivables, ainsi que des procédés d'utilisation associés.
PCT/US2009/004402 2008-07-30 2009-07-30 Lieurs clivables à base d'acylhydrazone WO2010014236A2 (fr)

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US20120232015A1 (en) * 2009-09-23 2012-09-13 Kohn Harold L Novel N-Benzylamide Substituted Derivatives of 2-(Acylamido)acetic Acid and 2-(Acylamido)propionic Acids: Potent Neurological Agents
WO2013019681A3 (fr) * 2011-07-29 2013-07-11 Avelas Biosciences, Inc. Molécules de délivrance sélective et procédés d'utilisation
WO2016176335A1 (fr) 2015-04-27 2016-11-03 Concert Pharmaceuticals, Inc. Otx-015 deutéré
WO2020010133A1 (fr) * 2018-07-03 2020-01-09 Rutgers, The State University Of New Jersey Composition stratifiée luminescente et procédé d'utilisation de la composition
US12037529B2 (en) 2020-12-30 2024-07-16 Rutgers, The State University Of New Jersey Luminescent layered composition and a method for using the composition

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US5122368A (en) * 1988-02-11 1992-06-16 Bristol-Myers Squibb Company Anthracycline conjugates having a novel linker and methods for their production

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Title
AISSAOUI, ABDERRAHIM ET AL.: 'Novel Cationic Lipids Incorporating an Acid- Sensitive Acylhydrazone Linker: Synthesis and Transfection Properties' J. MED. CHEM. vol. 47, no. 21, 2004, pages 5210 - 5223 *
FELIX KARTZ ET AL.: 'Synthesis of New Maleimide Derivatives of Daunorubicin and Biological Activity of Acid Labile Transferrin Conjugates' BIOORG. MED. CHEM. LETT. vol. 7, no. 5, 1997, pages 617 - 622 *
PAULA C. A. RODRIGUES ET AL.: 'Acid-Sensitive Polyethylene Glycol Conjugates of Doxorubicin: Preparation, In Vitro Efficacy and Intracellular Distribution' BIOORG. MED. CHEM. vol. 7, 1999, pages 2517 - 2524 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8933065B2 (en) * 2008-04-01 2015-01-13 The University Of North Carolina At Chapel Hill N-benzylamide substituted derivatives of 2-(acylamido)acetic acid and 2-(acylamido)propionic acids: potent neurological agents
US20120232015A1 (en) * 2009-09-23 2012-09-13 Kohn Harold L Novel N-Benzylamide Substituted Derivatives of 2-(Acylamido)acetic Acid and 2-(Acylamido)propionic Acids: Potent Neurological Agents
US8829033B2 (en) * 2009-09-23 2014-09-09 The University Of North Carolina At Chapel Hill N-benzylamide substituted derivatives of 2-(acylamido)acetic acid and 2-(acylamido)propionic acids: potent neurological agents
WO2013019681A3 (fr) * 2011-07-29 2013-07-11 Avelas Biosciences, Inc. Molécules de délivrance sélective et procédés d'utilisation
EA030795B1 (ru) * 2011-07-29 2018-09-28 Авелас Биосайнсиз, Инк. Молекулы для селективной доставки пары визуализирующих агентов в раковую ткань
WO2016176335A1 (fr) 2015-04-27 2016-11-03 Concert Pharmaceuticals, Inc. Otx-015 deutéré
WO2020010133A1 (fr) * 2018-07-03 2020-01-09 Rutgers, The State University Of New Jersey Composition stratifiée luminescente et procédé d'utilisation de la composition
US12037529B2 (en) 2020-12-30 2024-07-16 Rutgers, The State University Of New Jersey Luminescent layered composition and a method for using the composition

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