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WO2018165666A1 - Espèce d'aryldiazonium à libération enzymatique - Google Patents

Espèce d'aryldiazonium à libération enzymatique Download PDF

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Publication number
WO2018165666A1
WO2018165666A1 PCT/US2018/022046 US2018022046W WO2018165666A1 WO 2018165666 A1 WO2018165666 A1 WO 2018165666A1 US 2018022046 W US2018022046 W US 2018022046W WO 2018165666 A1 WO2018165666 A1 WO 2018165666A1
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Prior art keywords
triazabutadiene
pro
molecule
compound
triazabutadiene molecule
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Inventor
John C. Jewett
Lindsay E. Guzman
Bereketab T. Mehari
Jie He
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University of Arizona
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University of Arizona
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Priority to US16/566,659 priority Critical patent/US10954195B2/en
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    • 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
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members 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
    • C07D233/88—Nitrogen atoms, e.g. allantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4168—1,3-Diazoles having a nitrogen attached in position 2, e.g. clonidine
    • 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
    • A61K47/545—Heterocyclic compounds
    • 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
    • C07D403/12—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 chain containing hetero atoms as chain links
    • 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

Definitions

  • the present invention relates to protected aryl diazonium species that can be selectively released using enzymes.
  • carboxylation on N1 of triazabutadienes reversibly yields stabilized or protected versions of triazabutadienes, e.g., pro-triazabutadienes that are generally stable in acidic conditions.
  • carboxylation on N1 yielded a pro-triazabutadiene molecule that is stable in concentrated HCI in methanol; treating the pro-triazabutadiene molecule with NaOH in methanol returned the original triazabutadiene molecule (which can then be degraded, e.g., in acidic conditions).
  • the pro- triazabutadiene molecule may function as a means to protect triazabutadienes from degradation, e.g., under acidic conditions.
  • the release of the triazabutadiene molecule from the pro-triazabutadiene molecule can be selectively triggered (under appropriate conditions), e.g., via enzymatic triggers. This can allow for many applications that could benefit from selective triazabutadiene activation.
  • the terms "protected triazabutadiene,” “releasable triazabutadiene” and “pro-triazabutadiene” refer to molecules that comprise an inactive form of a triazabutadiene molecule (e.g., a protected version of a triazabutadiene) but can yield or release an active triazabutadiene molecule (possibly further yielding an aryl diazonium species) upon appropriate conditions.
  • the present invention features triazabutadiene molecules, e.g., water- soluble triazabutadiene molecules.
  • the present invention also features methods of use (applications) of said triazabutadiene molecules, methods of cleavage of said triazabutadiene molecules (e.g., decomposition in water, reductive cleavage, pH- dependent cleavage, light-catalyzed cleavage, etc.), and methods of synthesis of said triazabutadienes.
  • the present invention features methods and compositions for yielding an aryl diazonium species from a triazabutadiene molecule, e.g., a protected aryl diazonium species in the form of a triazabutadiene.
  • an enzyme catalyzes the reaction yielding the aryl diazonium species from the triazabutadiene molecule.
  • the molecules of the present invention (and or the products of triazabutadiene molecule cleavage (e.g., diazonium species) may be used for a variety of applications.
  • the pro-triazabutadiene molecules of the present invention may be used in drug delivery systems.
  • the present invention features pro-triazabutadiene molecules according to Formula B.
  • X 1 is a moiety conferring water solubility
  • Y 1 is a tri-substituted aryl, an alkyl, a carboxylic acid, an alcohol, or an amine
  • Z 1 is an aryl
  • Z 2 is a carboxyl group.
  • the pro-triazabutadiene molecule releases a triazabutadiene molecule and a derivative of Z 2 when subjected to a trigger, e.g., an enzymatic trigger, that results in deprotection of N1 nitrogen.
  • a trigger e.g., an enzymatic trigger
  • X 1 is a mesityl group
  • Y 1 is a mesityl group
  • both X 1 and Y 1 are mesityl groups.
  • Y 1 is a NHS-ester moiety; an oligonucleotide; a peptide; a fluorescence quencher; a pro-fluorophore; an alkyne; a triazene; or a combination thereof.
  • X 1 is a moiety of the formula -R 1 -Q 1 , wherein R 1 is a C 1-6 alkylene, and Q 1 is a sulfate, phosphate, or a quaternary ammonium cation.
  • Y 1 is t-butyl. In certain embodiments, Y 1 is methyl. In certain embodiments, Z 1 is a drug with a phenolic functional group. In certain embodiments, the enzymatic trigger comprises phosphatases, sulfatases, esterases, or nitroreductases. In certain embodiments, the pro-triazabutadiene molecule is incorporated into an amino acid. In certain embodiments, the derivative of Z 2 comprises a drug or a pro-drug. In certain embodiments, the triazabutadiene molecule released from the pro-triazabutadiene molecule yields an aryl diazonium species.
  • the present invention also features a pro-triazabutadiene molecule comprising a molecule according to Formula B, wherein X 1 is a moiety conferring water solubility, Y 1 is a tri-substituted aryl, an alkyl, a carboxylic acid, an alcohol, or an amine; Z 1 is an aryl; and Z 2 is a carboxyl group.
  • X 1 is a mesityl group
  • Y 1 is a mesityl group
  • both X 1 and Y 1 are mesityl groups.
  • Y 1 is a NHS-ester moiety; an oligonucleotide; a peptide; a fluorescence quencher; a pro-fluorophore; an alkyne; a triazene; or a combination thereof.
  • X 1 is a moiety of the formula -R 1 -Q 1 , wherein R 1 is a C 1-6 alkylene, and Q 1 is a sulfate, phosphate, or a quaternary ammonium cation.
  • Y 1 is t-butyl.
  • Y 1 is methyl.
  • Z 1 is a drug with a phenolic functional group.
  • the pro-triazabutadiene molecule is incorporated into an amino acid. In certain embodiments, the pro-triazabutadiene molecule releases a triazabutadiene molecule and a derivative of Z 2 when subjected to a trigger that results in deprotection of N1 nitrogen. In certain embodiments, the derivative of Z 2 comprises a drug or a prodrug. In certain embodiments, the triazabutadiene molecule released from the pro- triazabutadiene molecule yields an aryl diazonium species. In certain embodiments, the trigger is an enzymatic trigger, e.g., phosphatases, sulfatases, esterases, or nitroreductases. In certain embodiments, the trigger is basic conditions. In certain embodiments, the trigger is light.
  • the present invention also features a pro-triazabutadiene molecule comprising a molecule according to Formula B, wherein X 1 is a mesityl group, Y 1 is a mesityl group, or both X 1 and Y 1 are mesityl groups; Z 1 is an aryl; and Z 2 is a carboxyl group.
  • the pro-triazabutadiene molecule releases a triazabutadiene molecule and a derivative of Z 2 when subjected to an enzymatic trigger that results in deprotection of N1 nitrogen.
  • the present invention also features a method of drug release or cargo release.
  • the method comprising introducing an enzymatic trigger to a pro-triazabutadiene molecule according to any the embodiments herein, wherein the enzymatic trigger causes deprotection of N1 nitrogen thereby causing release of a triazabutadiene molecule and a derivative of Z 2 , wherein the derivative of Z 2 comprises a drug or a cargo.
  • the cargo or drug is a pro-drug.
  • the present invention also features a method of selectively activating a triazabutadiene.
  • the method comprises introducing a trigger to a pro-triazabutadiene molecule according to any of the embodiments herein, wherein the trigger causes formation of an active triazabutadiene.
  • the trigger comprises basic conditions.
  • the trigger is an enzyme.
  • the trigger is light.
  • the pro-triazabutadiene molecule is stable in acidic conditions, e.g., a solution having a pH of 8.0 or less, a solution having a pH of 7.0 or less, a solution having a pH of 6.0 or less, etc.
  • the method is used for drug release or cargo release.
  • FIG. 1 shows non-limiting examples of triazabutadiene molecules.
  • FIG. 2A shows triazabutadiene molecules undergoing decomposition to diazonium salts (and cyclic guanidine species). Note the reaction/equilibrium arrows are not to scale.
  • FIG. 2B shows a triazabutadiene molecule breaking down (in low pH conditions) to a diazonium species and a cyclic guanidine species.
  • FIG. 3 shows reductive cleavage of triazabutadiene molecules.
  • FIG. 4A shows light catalyzed cleavage of triazabutadiene molecules.
  • FIG. 4B shows photochemically generated bases, (i) A masked base may decompose to reveal a basic nitrogen atom upon exposure to light; (ii) The basic nitrogen atom of a molecule obscured by a steric wall may be reversibly swung away in a photochemically triggered fashion; (Hi) The intrinsic basicity of a nitrogen- containing functional group may be altered by a photochemical event.
  • FIG. 5A shows time-dependent photo-induced degradation of triazabutadienes.
  • the reaction was monitored by comparing starting materials (A, C, D, and E) with product (B);
  • Peak absorption and extinction coefficients for all of the compounds were excitable by the UV source used;
  • Time-dependent conversion of compounds was measured by NMR integration.
  • FIG. 5B shows (i) Compound A is rendered more basic upon exposure to light; that basicity recovers (to some extent) in the absence of light; (ii) Oscillating UV irradiation provides a saw-tooth pH trend over time.
  • FIG. 5C shows the lone-pair of electrons on the N1 nitrogen atom becomes more electron-rich upon isomerization from E to Z
  • FIG. 5D shows the use of the photobase as a catalyst, (i) Structures of water-soluble Compound A versus organic soluble Compound F; (ii) The Henry reaction between Compound G and Compound H was carried out at room temperature and varying amounts of catalyst; (iii) The reactions were monitored by ReactIRTM, following consumption of aldehyde Compound H.
  • FIG. 6A - FIG. 6H show non-limiting examples of triazabutadienes or reaction schemes involving triazabutadienes.
  • FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D show non-limiting examples of reaction schemes involving triazabutadienes.
  • FIG. 8A shows formulas for releasable triazabutadienes of the present invention.
  • FIG. 8B shows synthesis of a pro-triazabutadiene (Compound 57) from a triazabutadiene (Compound 58) and ethylchloroformate.
  • FIG. 8C shows a triggered release of a pro-triazabutadiene (Compound 58b).
  • FIG. 8D shows a proposed substrate (a pro-triazabutadiene, Compound 59) for beta-lactamase. Upon cleavage of the beta-lactam, the compound will decompose to release carbon dioxide and return the triazabutadiene (Compound 58).
  • FIG. 8E shows synthesis of the pro-triazabutadiene (Compound 59) via known beta-lactam (Compound 61 ).
  • FIG. 8F shows a tetrahedral intermediate.
  • FIG. 8G shows synthesis of several protected triazabutadienes that have varying electronic properties on the aryl ring.
  • FIG. 8H shows synthesis of a protected triazabutadiene bearing a t-butyl ester.
  • FIG. 8I shows removal of the protection of the t-butyl ester triazabutadiene of FIG. 8H under basic conditions.
  • FIG. 9A shows synthesis of protected triazabutadienes and their prospective yields using various chloroformate reagents.
  • Initial concentration of compound was 31 ⁇ in buffers of various pH ranges. Buffers pH 8, 9, and 10: 25 mM sodium borate; buffer pH 7: 100 mM phosphate; buffer pH 2: 200 mM KCI buffer.
  • FIG. 9E shows a graph of In(abs) vs. time (s) to obtain Kots under pseudo- first order reaction conditions: a plot of Kobs vs. hydroxide concentration (M) to obtain the second order rate constant (k 2 ).
  • the chart shows various protected triazabutadienes with their corresponding second order rate constants (k 3 ).
  • FIG. 9F shows synthesis of a protected triazabutadiene with a photocleavable protecting group.
  • FIG. 9G shows a photocleavable protected triazabutadiene exposed to 365 nm light to yield a regular triazabutadiene.
  • FIG. 9H shows a non-limiting example of a triazabutadiene that is photocleavable but not cleavable using acids or bases.
  • FIG. 10A shows an example of a triazabutadiene molecule adapted to modify a protein.
  • FIG. 10B shows an example of a triazabutadiene molecule conjugated to an antibody, wherein the conjugate is used for labeling a protein of interest.
  • FIG. 11A shows a schematic representation of a protein modified with a fluorogenic probe that becomes fluorescent upon conjugation with another protein.
  • FIG. 1 1 B shows Compound 53 delivering a coumarin diazonium salt to label tyrosine derivative (Compound Tyr 6).
  • FIG. 11 C shows experiments involving BSA fluorescence.
  • FIG. 12A Compound 56 (diazonium) becoming a fluorescent dye (Compound 55) upon reaction with tyrosine.
  • FIG. 12B shows Compound 57 delivering Compound 56 upon protonation.
  • FIG. 12C shows probes 58 and 59, which are proposed to fluorescently report on protein-protein interactions.
  • FIG. 13A shows diazonium Compound 56 can be bound to a resin (e.g., 56- bead) and reacts with an electron rich aryl ring to generate a fluorophore.
  • a resin e.g., 56- bead
  • FIG. 13B shows an assortment of commercially available aryl rings, which can be evaluated in a plate format.
  • FIG. 13C shows the fluorophore from Compound 60 and Compound 61 will target lysosomes and mitochondria (respectively).
  • FIG. 13D shows the fluorophores from Compound 62 and Compound 63 can be further derivatized with copper catalyzed click chemistry to produce large libraries of fluorophores.
  • FIG. 14A shows synthesis of a triazabutadiene containing an N- hydroxysuccinimide ester.
  • FIG. 14B shows Compound 68.
  • FIG. 14C shows derivatives related to Compound 68.
  • FIG. 15 shows lysine-reactive triazabutadiene Compound 80 is designed to self-immolate to return the starting lysine residue if the resulting diazonium ion,
  • Compound 81 fails to undergo a reaction with tyrosine.
  • FIG. 16A shows an example of cargo release from a triazabutadiene molecule.
  • FIG. 16B shows an example of how a prodrug is released.
  • FIG. 16C shows an example of a prodrug comprising a phenolic functional group masked as a triazylidine moiety.
  • FIG. 16D shows an azide functional group reacted with a carbine to produce an acid labile prodrug comprising a triazylidine moiety.
  • FIG. 17A shows an example of triazabutadiene (left molecule) and a reaction to create a protected the triazabutadiene (middle molecule), e.g., a triazabutadiene with N1 nitrogen protection to help prevent the rapid release of the aryl diazonium that may typically occur if the triazabutadiene is not protected.
  • the protected triazabutadiene may, upon an enzymatic trigger (right molecule), yield an aryl diazonium species.
  • FIG. 17B shows Formula B (left) and non-limiting examples of Y 1 .
  • the present invention features triazabutadiene molecules (e.g., water- soluble triazabutadienes).
  • triazabutadienes are according to Formula A.
  • Examples of Formula A are shown as Formula I, II, III, and IV.
  • X 1 is a moiety conferring water solubility.
  • Y 1 is a tri-substituted aryl, an alkyl, a carboxylic acid, an alcohol, or an amine (see also FIG. 17B).
  • Y 1 is a tri-substituted aryl group, the tri-substituted aryl group comprising a NHS-ester moiety (e.g., for protein linkage); an oligonucleotide; a peptide; a fluorescence quencher; a pro-fluorophore; an alkyne (e.g., for click chemistry); a triazene (e.g., from click reaction); the like, or a combination thereof.
  • Y 1 comprises an aldehyde; an amine (e.g., Fmoc protected), aminooxy, halogen (e.g., radio isotope); the like, or a combination thereof.
  • Z 1 is an aryl, e.g., a substituted aryl.
  • Z 1 comprises a NHS-ester moiety; an oligonucleotide; a peptide; a fluorescence quencher; a pro-fluorophore; a biologically active acid labile compound; a prodrug comprising a phenolic functional group; releasable cargo; an alkyne (e.g., for click chemistry); a triazene (e.g., from click reaction); the like, or a combination thereof.
  • Z 1 comprises an aldehyde; an amine (e.g., Fmoc protected), aminooxy, halogen (e.g., radio isotope); the like, or a combination thereof.
  • X 1 may comprise a functional group that confers water solubility.
  • X 1 comprise a moiety of the formula -R 1 -Q 1 , wherein R 1 is C 1-6 alkylene, and Q 1 is sulfate, phosphate, or a quaternary ammonium cation.
  • X 1 is a moiety of the formula -R 1 -Q 1 , wherein R 1 is C 1-6 alkylene, and Q 1 is sulfate (e.g., -(0) n S03R a , where n is 0 or 1 , and R a is C1-6 alkyl or typically H), phosphate (e.g., -(0) n P0 3 R a , where n is 0 or 1 , and R a is C1-6 alkyl or typically H), or a quaternary ammonium cation (e.g., - [NR a R b R°l + , where each of R a , R b , and R c is independently H or C 1-6 alkyl).
  • R 1 is C 1-6 alkylene
  • Q 1 is sulfate (e.g., -(0) n S03R a , where n is 0 or 1 , and R a is C1-6 alkyl or typically H
  • alkyl refers to a saturated linear monovalent hydrocarbon moiety of one to twelve, typically one to six, carbon atoms or a saturated branched monovalent hydrocarbon moiety of three to twelve, typically three to six, carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 2-propyl, tert- butyl, pentyl, and the like.
  • alkene refers to a saturated linear divalent hydrocarbon moiety of one to twelve, typically one to six, carbon atoms or a branched saturated divalent hydrocarbon moiety of three to twelve, typically three to six, carbon atoms.
  • alkene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, and the like.
  • Triazabutadiene molecules of the present invention may be readily soluble in water.
  • the solubility of the triazabutadiene molecule in water is at least 23 g/L of water (50 mM).
  • the triazabutadiene molecule is stable in pH 7.4 phosphate buffer.
  • the phosphate buffer solutions are commercially available or can be prepared, for example, as described in http://cshprotocols.cshlp.Org/content/2006/1/pdb.rec8247.
  • the half-life of the triazabutadiene molecules of the present invention in pH 7.4 phosphate buffer solution is at least 24 hours.
  • Stability of the triazabutadiene molecule may be measured in various ways. In some embodiments, stability is measured by the half-life of the molecule (or the half-life of the molecule in a particular buffer at a particular pH). In some embodiments, the molecule has a half-life of at least 12 hours in a pH 7.4 buffer. In some embodiments, the molecule has half-life of at least 24 hours in a pH 7.4 buffer. In some embodiments, the molecule has half-life of at least 36 hours in a pH 7.4 buffer.
  • the triazabutadiene molecule has a half-life of at least 8 hours, at least 10 hours, at least 12 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 36 hours, etc.
  • the present invention is not limited to the aforementioned examples of stability measurements.
  • the triazabutadiene molecules of the present invention are advantageous because the triazabutadiene molecules can be easily modified (e.g., various different functional groups can be easily used as X 1 , Y 1 , or Z 1 (see FIG. 1). And, the release of the diazonium species following triazabutadiene molecule breakdown (via certain mechanisms, as described below) may provide a new functional group that can be taken advantage of in various applications. Also, it may be considered advantageous that the breakdown of the triazabutadiene molecule is irreversible.
  • the present invention shows that triazabutadiene molecules may break down in the presence of water to generate reactive aryl diazonium compounds.
  • FIG. 2A shows that triazabutadiene molecules of the present invention can undergo decomposition to diazonium salts (reactive aryl diazonium compounds) and cyclic guanidine species.
  • Aryl diazonium compounds may react with electron-rich aryl rings (e.g., aryl species wherein the bond of interest is a nitrogen-carbon bond; indoles, anilines, phenol-containing compounds such as resorcinol or tyrosine, etc.) to form stable azobenzene linkages (e.g., an aryl azo dye, e.g., Sudan Orange).
  • electron-rich aryl rings e.g., aryl species wherein the bond of interest is a nitrogen-carbon bond; indoles, anilines, phenol-containing compounds such as resorcinol or tyrosine, etc.
  • stable azobenzene linkages e.g., an aryl azo dye, e.g., Sudan Orange.
  • an aryl azo dye e.g., Sudan Orange
  • imidazole compounds may be used in lieu of a phenol-containing compound.
  • the diazonium species may not necessarily react with an electron-rich aryl rings compound (e.g., phenol species), for example if a phenol species is not present.
  • the diazonium species may irreversibly extrude nitrogen gas to generate an aryl cation, which will rapidly be quenched by solvating water, thus synthesizing a new phenolic compound (e.g., HO-Ph, wherein Ph refers to the phenyl ring); thus, the diazonium portion of the triazabutadiene molecule may function as a masked hydroxyl group.
  • the triazabutadiene molecules are acid labile, e.g., unstable at particular pH levels (see FIG. 2B). For example, decreases in pH may increase the rate at which the triazabutadiene molecules break down (the half life of the molecule decreases).
  • the triazabutadiene molecules are unstable at low (lowered) pH levels (e.g., lowered pH as compared to a particular pH that the molecule may be stored at, e.g., a pH wherein the molecule has a particular desired half life).
  • Low pH levels in some example, may be a sub-physiological pH (7.4 or less).
  • the triazabutadiene molecules are (more) unstable at pH 7.0 or less, pH 6.8 or less, pH 6.5 or less, pH 6.2 or less, pH 6.0 or less, pH 5.8 or less, pH 5.6 or less, pH 5.5 or less, pH 5.2 or less, pH 5.0 or less, etc.
  • the term 'low pH may refer to several different pH levels. Since the functional groups attached to the molecule (e.g., see X 1 , Y 1 , Z 1 of Formula I) affect the stability of the molecule (as well as water solubility), the pH that is necessary to increase the rate of breakdown of the triazabutadiene molecule (e.g., the "lowered pH") may be different for different molecules.
  • the low pH is a pH of 7.4 or less, 7.2 or less, 7.0 or less, etc. In some embodiments, the low pH is a pH of 6.8 or less, 6.6 or less, 6.5 or less, 6.4 or less, 6.2 or less, 6.0 or less, etc. In some embodiments, the low pH is a pH of 5.8, 5.5 or less, 5.0 or less, etc.
  • the triazabutadiene molecules can break down without the presence of the low pH (the molecules have half lives); however, in some embodiments, a lowered pH enhances the reaction (e.g., increases the rate of reaction). As such, a low pH may or may not be used with the molecules and/or methods of the present invention.
  • the triazabutadiene molecule has a half-life of no more than 1 hour in a pH 7.4 aqueous solution. In some embodiments, the triazabutadiene molecule has a half-life of no more than 30 minutes in a pH 7.4 aqueous solution. In some embodiments, the triazabutadiene molecule has a half-life of no more than 15 minutes in a pH 7.4 aqueous solution.
  • the present invention also features methods of breaking down triazabutadiene molecules.
  • the method comprises subjecting the molecule to water.
  • the method comprises subjecting the molecule to a low pH (e.g., a low pH that is appropriate for the molecule, e.g., a lowered pH that increases the rate at which the triazabutadiene molecule breaks down).
  • the reaction of the triazabutadiene molecule to the diazonium species occurs in water within 10-30 seconds. In some embodiments, the reaction of the triazabutadiene molecule to the diazonium species occurs in water within 30 seconds to 1 minute. In some embodiments, the reaction of the triazabutadiene molecule to the diazonium species occurs in water within 1 to 5 minutes. In some embodiments, the reaction of the triazabutadiene molecule to the diazonium species occurs in water within 5-10 minutes. In some embodiments, the reaction of the triazabutadiene molecule to the diazonium species occurs in water within 10-15 minutes.
  • the reaction of the triazabutadiene molecule to the diazonium species occurs in water within 15-20 minutes. In some embodiments, the reaction of the triazabutadiene molecule to the diazonium species occurs in water within 25 minutes, within 30 minutes, within 45 minutes, or within 60 minutes.
  • the diazonium species may be visually differentiated from the triazabutadiene species, e.g., the diazonium species is visually distinct (e.g., a different color) from the triazabutadiene molecule.
  • the aryl azo dye may be visually differentiated from the triazabutadiene species and the diazonium species, e.g., the aryl azo dye is visually distinct (e.g., a different color) from the triazabutadiene species and the diazonium species.
  • the present invention also features methods of producing a visually detectable molecule.
  • the method comprises providing a triazabutadiene molecule according to the present invention and subjecting the triazabutadiene molecule to water and/or a low pH (or light as discussed below, or light and low pH, etc.).
  • the low pH or light, or light and low pH, etc.
  • initiates e.g., increases the rate of the irreversible reaction to produce the diazonium species and the cyclic guanidine species.
  • the diazonium species may be visually distinct from the triazabutadiene molecule; therefore the reaction produces a visually detectable molecule.
  • triazabutadiene molecules e.g., triazabutadiene scaffolds
  • reducing agents such as but not limited to sodium dithionite (sodium hydrosulfite) (Na 2 S204) (see FIG. 3).
  • the reducing agent comprises lithium aluminum hydride, sodium borohydride, or the like.
  • electrochemical reduction may be used in accordance with the present invention.
  • Reductive cleavage of the triazabutadiene molecules provides a urea functionality and a terminal aryl triazene.
  • the aryl triazene is further reduced in the presence of excess reducing agent (e.g., sodium dithionite).
  • excess reducing agent e.g., sodium dithionite
  • the reduction can be observed visually by the change in color of a solution. For example, there may be a subtle change of yellows that results from a loss of a shoulder in UV vis spectrum.
  • the ratio of the concentration of the triazabutadiene to the reducing agent is about 1 :1. In some embodiments, the ratio of the concentration of the triazabutadiene to the reducing agent is about 1 :2.
  • the present invention is not limited to the aforementioned ratios. For example, in some embodiments, the ratio of the concentration of the triazabutadiene to the reducing agent is about 2:3, 4:5, etc. The present invention is not limited to the aforementioned ratio of concentrations.
  • the reduction can occur within about 10 minutes, within about 15 minutes, within about 20 minutes, within about 25 min, within about 30 min, etc., at room temperature.
  • reductive cleavage of the triazabutadiene molecules is advantageous because it can occur rapidly (e.g., within 10 minutes, within 15 minutes).
  • the triazabutadiene molecules that are highly stable in acid e.g., a p-CN derived triazabutadiene
  • reductive cleavage of triazabutadiene molecules may also be used to cleave unreacted triazabutadienes that did not undergo diazonium formation/reaction chemistry that is associated with a drop in pH (or other mechanism) as described above (a sort of quench for the pH chemistry).
  • light increases the rate at which the triazabutadiene molecule breaks down (into the cyclic guanidine species and the diazonium species) (see FIG. 4A).
  • the present invention also features water-soluble triazabutadienes that, upon photo-irradiation, may be rendered more basic in a reversible fashion.
  • a protecting group of a masked base may decompose to reveal a basic nitrogen atom upon exposure to light.
  • a basic nitrogen atom of a molecule obscured by a steric wall may be reversibly swung away in a photochemically-triggered manner.
  • the present invention shows the intrinsic basicity of a nitrogen-containing functional group may be altered by a photochemical event.
  • triazabutadiene molecules of the present invention may readily photoisomerize to a more reactive Z-form.
  • an aqueous solution of Compound A was irradiated with a simple handheld UV lamp ("365 nm," measured at 350 nm). Consumption of Compound A was observed after only a few hours. The non-irradiated reaction under similar conditions was stable for days as partial degradation rapidly rendered the solution mildly basic. Without wishing to limit the present invention to any theory or mechanism, it was hypothesized that if a two-electron process were happening, then Compound A-Z would be more basic than Compound A-E. A 1.0 N NaOH solution of Compound A was treated with light.
  • Resorcinol was chosen because it can serve a dual role as a radical scavenger and a trap benzene diazonium species that could be formed.
  • An excess of resorcinol was added to a pH 9 borate-buffered solution of Compound A and the mixture was irradiated with light.
  • the known azobenzene, Sudan Orange G was formed in a 65% yield (versus 4% for the non-irradiated reaction).
  • Derivatives of Compound A were made to examine the effects of electronic perturbations on the light-induced degradation. Electron deficient aryl rings are more stable at lower pH, and this trend generally holds true for the photochemical reactions as well.
  • a buffered borate solution was chosen due to its alkaline nature and lack of complicating signals in the NMR experiment.
  • Compounds C-E all have absorption spectra that are well within the range of the UV lamp (see FIG. 5A(ii)).
  • Both /77-NO2 (Compound C) and p-CN (Compound D) had similar rates of reaction, both slower than Compound A.
  • -NO2 derivative Compound E was irradiated because of its significantly red-shifted spectrum. Compound E absorbed in a range that was not irradiated with the UV lamp and as such was recalcitrant to degradation (see FIG. 5A(iii)).
  • Compound A may be useful as a photo-catalytic base in the context of organic reactions. With limited solubility in all but DMSO, the stability of Compound A was tested. As noted previously, Compound A is quite stable to an excess of acetic acid in DMSO, showing only 12% degradation over 14 hours at room temperature. Upon irradiation with light, Compound A in presence of acetic acid completely fell apart over the same time frame. To confirm that this was due to the acid, a solution of Compound A (in pure DMSO) was irradiated. After four hours of constant irradiation in acid-free DMSO, an E.Z ratio of nearly 50:50 was observed.
  • FIG. 5D due to the limited organic solubility of Compound A, Compound F (FIG. 5D(i)) was synthesized. With Compound F, a similar light- induced acid sensitivity was observed in DMSO (and slow thermal isomerization). Based on the apparent p b of Compound F, pK a were matched to condensation substrates. A Henry reaction between nitroethane (Compound G) and p- nitrobenzaldehyde (H) was chosen to demonstrate the virtues of Compound F (FIG. 5D(ii)). The reaction between Compound G and Compound H occurred rapidly at room temperature in a light and catalyst dependent manner (FIG. 5D(iii)).
  • the present invention features methods of breaking down triazabutadiene molecules by subjecting the molecule to light.
  • the light may, for example, include wavelengths of about 400 nm.
  • the present invention is not limited to wavelengths of 400 nm or about 400 nm.
  • the wavelength is from 350 nm to 400 nm (e.g., 370 nm).
  • the wavelength is from 360 nm to 410 nm.
  • the wavelength is from 330 nm to 420 nm.
  • the wavelength is from 340 nm to 430 nm.
  • the method comprises subjecting the molecule to a low pH and to light.
  • a system such as a UV- LED pen may be used for these reactions, however the present invention is not limited to a UV-LED pen and may utilize any appropriate system.
  • the UV-LED pens may allow for relatively narrow bandwidth irradiation of these compounds (but are not limited to these bandwidths).
  • the color of the bulk material shifts as a result of electronic perturbations to the aryl azide starting material. For example, nitro derivative Compound 6e of FIG. 6G (described below) is rust-red, versus an orange phenyl Compound 6c of FIG.
  • buffers were made to the appropriate pH in a 9:1 mix of H20: D20. These solutions were added to the compound being assayed such that the buffer capacity was at least 10 fold the concentration of the compound.
  • Some experiments used 5 mg compound in 0.5 ml_ of buffer (e.g., pH 5 acetate buffer at 25 degrees C). These were immediately inserted into an NMR instrument and scans were taken at even time intervals to calculate the half-life of the compound based on integration.
  • an azide e.g., NHS-azide
  • N- heterocyclic carbene (NHC) route may be used to synthesize triazabutadiene molecules (e.g., see FIG. 6C).
  • a triazabutadiene molecule was synthesized from dimethyl imidazole derived NHC and phenyl azide.
  • methanolic HCL a rapid color change occurred. This change was confirmed to coincide with diazonium formation by trapping the reactive species with resorcinol to provide known diazo dye Sudan Orange G.
  • the triazabutadiene molecule Compound 4 was treated with the much less acidic acetic acid, the same product was obtained. Compound 4 was not water-soluble.
  • methyl imidazole was alkylated with propane sultone to provide the Zwitterionic NHC precursor Compound 5a (see FIG. 6E).
  • Formation of the NHC under basic conditions in the presence of phenyl azide yielded the highly water soluble Compound 6a (see FIG. 6E).
  • Compound 6a was highly colored, so its pH dependence was studied using UV Vis. The reactions were not only pH-, but also scan-frequency dependent. Upon finding this, the stability of Compound 6a was studied in D20 in the dark using NMR. Even in the dark it was unstable, but not in the diazonium-forming way.
  • the decomposition to diazonium salts and Compound 7 was measured as a function of pH in phosphate/citrate buffers from pH 4-7 and in a phosphate buffer from pH 6-8. All runs provided linear correlations of concentration and time, indicating a pseudo-zero order reaction (first order with respect to hydronium ion with a large excess of hydronium ions). While the peaks for Compound 7 remained constant, the peaks associated with Compound 6c drifted downfield as the reaction progressed. This drifting was highly reproducible across samples and buffers, but the underlying cause is not understood at this time. A sigmoidal correlation between rate and buffer pH centered at pH 6 was obtained.
  • Compound 8 When resorcinol was not added to consume the diazonium species, 4- phenylazophenol (Compound 8) was observed (see FIG. 6F). Compound 8 came from the decomposition of one diazonium ion to phenol followed by reaction with a second diazonium ion. The instability of Compound 6c in a pH 7 phosphate buffer was surprising given the stability in D20. Compound 6c was tested in a non-buffered 90:10 H20:D20 solution and observed only >7% after 6 hours.
  • the imidazole core will be alkylated (Compound 9) with either butane sultone to provide imidazolium (Compound 10) and triazabutadiene (Compound 1 1), or a dialkyl aziridinium salt to provide the analogous Compound 12 and Compound 13 which invert the expected charge on the side-chain.
  • the extra methylene in Compound 11 as compared with Compound 6 may alter the way that the side-chain bites back on the triazabutadiene.
  • the tertiary amine will be protonated at physiological pH and as serve to invert the charge of the side arm.
  • a potential bonus of Compound 13 is that the basic nitrogen may help localize this compound in the most acidic subcellular compartments much like LysoTrackerTM dyes.
  • synthesis may be performed with known p-azido dimethyl aniline (Compound 14) because it may lead to a wide range of substituted compounds. From imidazole (Compound 15) one can alkylate with 1 ,3-propanesultone to provide NHC precursor Compound 16, or prior to that one can treat with an NHC to access the wealth of diazonium chemistry to provide Compound 17 in all of its forms.
  • Solvolysis in water or alcoholic solvent may provide a phenol or aryl ether, and copper mediated Sandmeyer-type chemistry may afford cyano, nitro or halogenated aryl species.
  • copper mediated Sandmeyer-type chemistry may afford cyano, nitro or halogenated aryl species.
  • From imidazolium Compound 16 Staudinger chemistry followed by aniline alkylation may provide Compound 18, or traceless Staudinger-Bertozzi ligation may yield Compound 19.
  • These substrates cover a range of Hammett values while also providing an additional site of attachment to proteins, fluorophores, surfaces, etc.
  • Compound 33 contains an aryl ring, positioned ortho to the masked diazonium.
  • the synthesis may start from a diazo transfer reaction to convert aniline Compound 34 to an aryl azide. Coupling with Compound 5c (see FIG. 6E) may complete the synthesis. It is possible that following diazonium unmasking an aromatic substitution reaction will occur to provide benzocinnoline Compound 35. Because this reaction is intramolecular one might be able to use a non-activated ring, rendering the ring electron rich.
  • the methyl ether may serve as a site of attachment to chemical cargos.
  • a second type of intramolecular diazonium trap that could be employed is a beta keto ester that is also ortho to the diazonium produced. Beta keto esters are known to react with diazonium species through enol form, and can generate oxo-cinnolines, which are biologically active cores.
  • the present invention also features triazabutadienes that may be released from pro-triazabutadienes (triazabutadiene precursors), e.g., under appropriate conditions.
  • the present invention also features triazabutadienes that are used to synthesize said pro-triazabutadienes.
  • carboxylation on N1 of triazabutadienes reversibly yields stabilized or protected versions of triazabutadienes, e.g., pro-triazabutadienes that are generally stable in acidic conditions.
  • carboxylation on N1 yielded a pro-triazabutadiene molecule that is stable in concentrated HCI in methanol; treating the pro-triazabutadiene molecule with NaOH in methanol returned the original triazabutadiene molecule (which can then be degraded, e.g., in acidic conditions).
  • the pro- triazabutadiene molecule may function as a means to protect triazabutadienes from degradation, e.g., under acidic conditions.
  • the present invention features pro-triazabutadiene molecules that under appropriate conditions (e.g., chemical conditions, enzymatic conditions, light, etc.) yield or release a triazabutadiene molecule.
  • appropriate conditions e.g., chemical conditions, enzymatic conditions, light, etc.
  • the terms "releasable triazabutadiene” and "pro-triazabutadiene” refer to molecules that comprise an inactive form of a triazabutadiene molecule (e.g., a protected version of a triazabutadiene) but can yield or release an active triazabutadiene molecule upon appropriate conditions.
  • the active triazabutadiene molecule could then go on to release a diazonium species. That diazonium species could then react with a phenol (e.g., a tyrosine molecule), e.g., in a coupling reaction, or the diazonium species could self-immolate to release a phenol.
  • FIG. 8A shows a non-limiting example of a formula (Formula B) for releasable triazabutadiene molecules (pro-triazabutadiene molecules) of the present invention.
  • pro-triazabutadienes may comprise a formula according to Formula B.
  • Z 2 comprises a carboxyl group (N1 nitrogen is carboxylated).
  • X 1 and Y 1 have been previously described.
  • X 1 comprises a mesityl group.
  • Y 1 comprises a mesityl group.
  • Z 2 (the carboxyl group) comprises COaEt.
  • the present invention is not limited to the aforementioned examples and formulas.
  • Z 2 may comprise a carboxyl group different from Co2Et.
  • the pro-triazabutadienes comprise a triazabutadiene, e.g., according to Formula B, wherein the N1 nitrogen of the triazabutadiene is modified such that the modified triazabutadiene is more stabile at a particular pH as compared to the unmodified triazabutadiene.
  • the modified triazabutadiene (pro-triazabutadiene) is more stabile at pH 5.5 as compared to the unmodified triazabutadiene.
  • the modification comprises carboxylation (on the N1 nitrogen). The present invention is not limited to modifications comprising carboxylation on the N1 nitrogen.
  • the modification comprises an ortho-quinone methide linked on the N1 nitrogen.
  • the present invention is not limited to the N1 carboxylations described herein.
  • other carbonyl variations or derivatives may be used, e.g., the ethyl formate side group shown in Compound 58 of FIG. 8B may be different, e.g., the carbonyl oxygen may be replaced with sulfur, one or both oxygen atoms may be replaced with nitrogen, an oxygen atom may be removed, etc.
  • the triazabutadiene molecules that form the pro- triazabutadiene molecules are water-soluble. In some embodiments, the triazabutadiene molecules that form the pro-triazabutadiene molecules are not water-soluble.
  • a variety of triggers may be employed to release the triazabutadiene from the pro-triazabutadiene.
  • the release of the triazabutadiene molecule may in some cases be selectively triggered.
  • the trigger is an enzymatic trigger that selectively reacts with the protected triazabutadiene to deprotect the protected triazabutadiene, possibly allowing for aryl diazonium release.
  • a triazabutadiene is shown on the left.
  • the triazabutadiene may be converted to a protected triazabutadiene (middle molecule), e.g., pro- triazabutadiene.
  • the protected triazabutadiene may feature protection at the N1 nitrogen to help prevent the rapid release of the aryl diazonium that may typically occur if the triazabutadiene is not protected.
  • the protected triazabutadiene may, upon an enzymatic trigger (right molecule), yield an aryl diazonium species.
  • Enzymatic triggers may include but are not limited to phosphatases, sulfatases, esterases, nitroreductases, etc., or any enzyme that cleaves a bond. Such enzymes are well known to one of ordinary skill in the art. A triazabutadiene may be created or tailored to be cleavable by a particular enzyme of choice.
  • the compound used to protect the triazabutadiene may be termed a protecting group.
  • the protecting group comprises a drug or pro-drug.
  • the drug or pro-drug upon an enzymatic trigger, the drug or pro-drug is released.
  • the protecting groups are not limited to drugs; any appropriate molecule may be considered.
  • the present invention features a range of moieties that can be utilized to trigger triazabutadiene release (FIG. 8C shows one example).
  • ortho- nitro derivatives e.g., Compound 58b
  • the light used to un-cage may also speed up diazonium release.
  • the present invention also features enzymatically- triggered derivatives.
  • FIG. 8D shows ⁇ -lactamase substrate
  • FIG. 8F shows a tetrahedral intermediate (see Key Intermediate). This tetrahedral intermediate may be important for release strategies. For example, the loss of carbon dioxide from the intermediate yields the original triazabutadiene (e.g., see Compound A in FIG. 8F).
  • FIG. 8G shows synthesis of several protected triazabutadienes with varying electronic properties on the aryl ring.
  • the inductively electron donating p-methyl substituent, 8, slowed the rate of deprotection moderately. Without wishing to limit the present invention to any theory or mechanism, it is believed that this can be rationalized by a model whereby the carbonyl is stabilized by being more electron rich and thus less prone to nucleophilic attack.
  • the p-methoxy substituted compound, 9, also slowed hydrolysis, so much so that the rate could not be determined due to the error of the analysis.
  • the acid-stabilization may make the triazabutadiene compatible with most traditional Boc-strategies of solid-phase peptide synthesis.
  • a protected triazabutadiene bearing a f-butyl ester was synthesized (see FIG. 8H, FIG. 81).
  • the acid-labile ester was removed using trifluoroacetic acid (TFA) in dichloromethane. Following removal of the ester the carbamate protection was removed under basic conditions and finally the resulting triazabutadiene was treated with acid and resorcinol to provide an azo-benzene product.
  • TFA trifluoroacetic acid
  • FIG. 9 shows analyses of various protected triazabutadienes.
  • FIG. 9A shows synthesis of protected triazabutadienes and their prospective yields using various chloroformate reagents.
  • FIG. 9A shows synthesis of protected triazabutadienes and their prospective yields using various chloroformate reagents.
  • FIG. 9E shows a graph of In(abs) vs. time (s) to obtain K 0 bs under pseudo-first order reaction conditions: a plot of K obs vs. hydroxide concentration (M) to obtain the second order rate constant (k 2 ).
  • the chart shows various protected triazabutadienes with their corresponding second order rate constants (k 3 ).
  • the protected triazabutadiene is protected with a photocleavable protecting group.
  • FIG. 9F shows synthesis of a protected triazabutadiene with a photocleavable protecting group.
  • FIG. 9G shows a photocleavable protected triazabutadiene exposed to 365 nm light to yield a regular triazabutadiene.
  • the protected triazabutadiene is not susceptible to cleavage using acids or bases.
  • the protected triazabutadiene is photocleavable (see FIG. 9H as an example of a photocleavable protected triazabutadiene that is not susceptible to cleavage using acids or bases).
  • the present invention also features releasable triazabutadienes that can be appended to proteins, e.g., an azide-containing compound.
  • the releasable triazabutadiene molecules may provide opportunities for selective diazonium delivery as biochemical probes.
  • the releasable triazabutadiene molecules may provide for spatially selective release of aryl diazonium ions.
  • the reactivity may be generalized to be broadly applicable and triggered. For example, by parlaying the release chemistry into amide bonds it may be possible to target proteases. And, endosomal proteases that viruses and other pathogens encounter upon entry should facilitate cleavage. If the caged triazabutadiene compounds are not substrates for the enzyme then a quinone-methide strategy may be implemented.
  • a variety of triggers may be employed to release the triazabutadiene from the pro- triazabutadiene.
  • the release of the triazabutadiene molecule may in some cases be selectively triggered.
  • the pro-triazabutadiene molecules may allow for use in two-step drug release systems or other cargo release systems.
  • the pro- triazabutadiene molecules may provide for an easier means of protein modification, e.g., because the molecules can be worked with in a wider range of pHs.
  • the increased stability of the pro-triazabutadiene molecules in acidic environments may provide for enhanced processes of self-assembling monolayers for the production of biosensors.
  • the pro-triazabutadiene may be protected until it is in the presence of any given enzyme, and upon that trigger, the triazabutadiene molecule can be released, which can then go on to form a diazonium species, which can couple with particles such as nanoparticles, polymers, and biomacromolecules.
  • the molecules of the present invention can be used to help couple target compounds to a sensor (e.g., see U.S. Pat. No. 8,668,978, the disclosure of which is incorporated herein in its entirety).
  • the triazabutadiene molecules of the present invention may be utilized for a variety of purposes.
  • the triazabutadiene molecules of the present invention are utilized for a chemoselectively-cleavable linkage for use in biological/complex settings where rapid, clean cleavage is of interest.
  • the triazabutadiene molecules are used for systems including but not limited to drug delivery systems, protein-protein interaction systems, pH environment detection systems, etc. Applications of these triazabutadienes may fall under one (or more) categories of reactivity. a. Diazonium Coupling Applications
  • the triazabutadiene molecules may be used for applications involving pH-dependent protein coupling.
  • General examples involve methods for detecting protein-protein proximity or protein-protein interactions (in a sample).
  • the method comprises providing a first protein, wherein the first protein is conjugated with a triazabutadiene molecule according to the present invention.
  • the first protein may be introduced to a sample.
  • the triazabutadiene molecule encounters a low pH in the sample; in some embodiments, acid is added to the sample to lower the pH appropriately.
  • the triazabutadiene molecule undergoes the irreversible reaction yielding the diazonium species and the cyclic guanidine species.
  • the diazonium species is adapted to react with a phenol group; thus if there is a nearby protein with a tyrosine residue, the diazonium species may react with it yielding an azobenzene product (often colored, e.g., the dye Sudan Orange G is an azobenzene-containing dye) that is visually distinct from the triazabutadiene molecule and the diazonium species.
  • detection of the azo dye e.g., Sudan Orange
  • the method comprises adding a second protein to the sample, wherein a tyrosine of the second protein may react with the diazonium species.
  • the second protein is already in the sample.
  • a tyrosine or phenol species conjugated to the second protein is added to the sample.
  • the method comprises introducing to the sample a first antibody specific for a first protein, wherein the first antibody is conjugated with a triazabutadiene molecule according to the present invention.
  • the method comprises introducing to the sample a second antibody specific for a second protein.
  • the second antibody comprises a tyrosine.
  • the second antibody is conjugated with a phenol species.
  • the method comprises introducing an acid to the sample to appropriately lower the pH of the sample. As previously discussed, in the low pH environment, the triazabutadiene molecule undergoes the irreversible reaction yielding the diazonium species and the cyclic guanidine species.
  • the diazonium species is adapted to react with a phenol group; thus if the phenol species is nearby, the diazonium species may react with it yielding an azo dye that is visually distinct from the triazabutadiene molecule and the diazonium species. As such, detection of the azo dyemay be indicative of proximity or interaction of the first protein and the second protein.
  • the acid-labile reactivity of triazabutadienes may be used to assist in work deducing interaction partners between a virus and endosomally localized host proteins.
  • a viral-bound diazonium species may be unmasked and this may go on to react with Tyr- containing proteins that are associating with the virus. It is possible that this system could be used to trap an interaction that is relevant at a key point of viral entry, e.g., the fusion of membranes.
  • synthesis of compounds that may be used in such systems, e.g., for modifying the viral surface. Lysine-reactive probes may be used to modify the surface of viral proteins. Referring to FIG.
  • triazabutadiene Compound 36 bearing an N- hydroxysuccinimide (NHS) ester it may be possible to couple the compound to one of many reactive Lys on the surface of the virus.
  • a triazabutadiene molecule may be attached to a viral protein (e.g., a purified viral protein).
  • a system such as a cell line (e.g., mosquito cell line, human cell line, or even mosquitos themselves) may be infected with the viral protein.
  • the infected system can be treated appropriately.
  • the azo dye may "label" any proteins that interact with or are nearby the viral protein (in the low pH environment). The present invention is not limited to this example.
  • Lys-NHS conjugation chemistry may work well on the basic side of neutral, which may be beneficial for pH sensitive probes.
  • Compound 36 may be made in a straightforward fashion from NHC precursor Compound 5c (see FIG. 6E) and an aryl azide. It is possible that the steric congestion about the NHC may favor the unencumbered azide over the potentially reactive NHS ester. If the NHS ester presents a problem during the synthesis it is possible to go into the reaction with a carboxylate instead and follow that by a coupling with N-hydroxysuccinimide.
  • a monoclonal antibody e.g., mouse anti-biotin
  • Compound 36 may be modified with Compound 36.
  • the extent of labeling may be quantified by coupling to resorcinol (or other appropriate alternative) in a low pH solution and the extent of modification may be analyzed by mass spectrometry. This may show the number of reactive triazabutadienes.
  • a fluorescent goat anti-mouse secondary antibody may be added, and then a gel-shift assay may be used to show that the two antibodies are covalently linked in a pH dependent manner.
  • triazabutadiene molecules may be used in applications involving fluorogenic molecules (e.g., see FIG. 11 , FIG. 12, and FIG. 13).
  • triazabutadiene molecules may be configured to generate a fluorescent compound when combined with a second molecule, e.g., upon reaction with a tyrosine molecule or other appropriate molecule (see FIG. 11 A).
  • Triazabutadienes may be a way to provide stable diazoniums that can form azobenzenes, which can be fluorophores.
  • the wavelengths (e.g., absorption/emission) of the fluorophores are (or can be can be tuned) within the UV to visible range.
  • the emission wavelength is from 350 nm to 450 nm.
  • the emission wavelength is from 400 nm to 500 nm.
  • the emission wavelength is from 450 nm to 550 nm.
  • the emission wavelength is from 500 nm to 600 nm.
  • the Stokes shift of the fluorophore is between 30 to 60 nm. In some embodiments, the Stokes shift of the fluorophore is between 40 to 70 nm.
  • the Stokes shift of the fluorophore is between 70 to 90 nm. In some embodiments, the Stokes shift of the fluorophore is between 80 to 100 nm. In some embodiments, the Stokes shift of the fluorophore more than 100 nm.
  • the fluorescence is in the blue spectrum, green spectrum, yellow spectrum, and/or red spectrum.
  • the present invention is not limited to the aforementioned wavelength ranges or spectrums.
  • Bovine serum albumin was used as a model protein to help assess the ability of Compound 53 to label proteins.
  • BSA is known to bind to small molecules.
  • Compound 53 was subjected to acid for varying times and then added to a solution of BSA at a pH sufficient to minimize background release of the diazonium ion (FIG. 1 1C). It was observed that BSA was labeled in a pH dependent manner and this labeling was gone upon reduction of the sample with sodium dithionite. To assess whether the standard azobenzene reduction is operative with these compounds, the resulting amino phenol may be oxidized and the orffto-iminoquinone may be trapped via established Diels-Alder chemistry.
  • molecules of the present invention can help distinguish between tyrosine and histidine modifications.
  • a complementary fluorophore based on a scaffold was synthesized, wherein a highly electron deficient boron atom coordinates the nitrogen of an azo-benzene to form a push-pull system. Recognizing that tyrosine could serve as the push half of the dye (Compound 55, Figure 1 1 A) aryl diazonium Compound 56 was synthesized, which contains an o/f jo-borane. When this non-fluorescent salt was mixed with resorcinol, a highly fluorescent compound was obtained.
  • derivative Tyr 6 Tyr 6 shown in FIG. 11 B
  • compound 55 the resulting azobenzene adduct
  • Probes that can be used to modify proteins can be synthesized.
  • coumarin-containing 58 (see FIG. 12C) may be synthesized via classic von Pechmann chemistry.
  • the NHS derivatives may be used to label reactive lysine side chains.
  • An alternative attachment point to the coumarin scaffold may be to use a 3- carboxylic acid derivative.
  • Analogous to Compound 58, Compound 59 may be synthesized. The synthesis of this and other Lewis acidic borane analogs may be challenging (but using a sulfonated NHS may simplify synthesis through keeping compounds in the organic phase until the final step).
  • the schemes, mechanisms, and molecules of the present invention can be used to generate a variety of fluorophores (e.g., the present invention features a library of fluorophores).
  • the aryl diazonium Compound 57 (in FIG. 12) is not fluorescent; because the boron needs an electron-rich push-pull system, that electron rich group can be replaced with an azobenzene part of a molecule, rendering it a fluorophore. Any electron rich aryl ring can react with Compound 57, forming a fluorophore.
  • This allows for the production of a wide range of fluorophores e.g., Compound 60, Compound 61 , see FIG. 13C). For example, as shown in FIG.
  • Compound 56 can be bound to a resin to form 56-bead. This can react with an electron rich aryl ring (e.g., see additional aryl ring examples in FIG. 13B) to generate a fluorophore. Note that Compound 60 can track the lysosome and Compound 61 is targeted to the mitochondria. Further, the fluorophores from Compound 62 and Compound 63 (see FIG. 13D) can be further derivatized with copper catalyzed click chemistry to produce large libraries of fluorophores. With these mechanisms, the number of fluorogenic compounds that can be synthesized expands greatly. c. Triazabutadiene Probes for Protein Modification
  • the present invention also features a lysine-reactive A/-hydroxysuccinimide (NHS) modified triazabutadiene, e.g., Compound 68 (see FIG. 14A).
  • a series of derivatives is shown in FIG. 14C.
  • Compound 70 with a sulfonate-containing NHS ester may provide a protein modified identically to using Compound 8, but it is may be soluble at higher concentrations, which may enable more rapid labeling of dilute protein samples (such as viral samples).
  • Another sulfonate-containing derivative, Compound 71 may have the effect of adding a negative charge to the surface of the protein that it modifies.
  • the AMinked amide probe, Compound 73 may be used to look at those electronic effects in the context of a complex biological sample.
  • the triazabutadiene molecules of the present invention may be used in applications involving diazonium degradation to release cargo or drugs.
  • a group of applications takes advantage of the solvolysis of diazonium salts to produce phenolic byproducts.
  • the degradation of diazonium salts to phenols, via aryl cations, is a first-order process that is not pH dependent in the physiological range of pHs. The half-life of this first order process depends on substitution on the aryl ring; the rate for benzenediazonium is ⁇ 4 hours. Indeed, the product of this degradation and subsequent azo-dye formation was observed if resorcinol is not put into the buffered NMR experiments.
  • the acid-dependent instability of the triazabutadiene molecule may allow for a drug or cargo molecule to be deposited at a desired location and time (e.g., the reaction can be controlled and initiated at a desired time and location).
  • the present invention also features methods of delivering a drug (or a cargo compound) to a subject.
  • the method comprises providing a triazabutadiene molecule according to the present invention, conjugating a drug (or cargo compound) to the triazabutadiene molecule; and administering the conjugate (the drug/cargo-triazabutadiene conjugate) to the subject.
  • the method comprises providing a triazabutadiene molecule according to the present invention wherein the triazabutadiene molecule comprises the drug (or cargo compound); and administering the triazabutadiene molecule to the subject.
  • the diazonium species of the triazabutadiene molecule is part of the drug (or cargo compound).
  • the drug (or cargo compound) is formed when the diazonium species reacts to a phenol species.
  • the drug is an anti-cancer drug.
  • the drug (or cargo compound) is not limited to an anti-cancer drug. Any appropriate drug for any appropriate condition may be considered.
  • the triazabutadiene molecules may be incorporated into drug/cargo-delivery systems for conditions including but not limited to cancer or other conditions associated with low pH states (e.g., gastrointestinal conditions, sepsis, ketoacidosis, etc.).
  • conditions including but not limited to cancer or other conditions associated with low pH states (e.g., gastrointestinal conditions, sepsis, ketoacidosis, etc.).
  • Non-limiting examples of drugs include: Abarelix, Alvimopan, Amoxicillin, Acetaminophen, Arformoterol, Cefadroxil, Cefpiramide, Cefprozil, Clomocycline, Daunorubicin, Dezocine, Epinephrine, Cetrolrelix, Etoposide, Crofelemer, Ezetimibe, Idarubicin, Ivacaftor, Hexachlorophene, Labetalol, Lanreotide, Levodopa, Caspofungin, Butorphanol, Buprenorphine, Dextrothyroxine, Doxorubicin, Dopamine, Dobutamine, Demeclocycline, Diflunisal, Dienestrol, Diethylsti Ibestrol , Doxycycline, Entacapone, Arbutamine, Apomorphine,
  • drug delivery systems featuring triazabutadiene molecules may be enhanced with other reactions, e.g., enzymatic reactions. Such additional reactions may help provide appropriate specificity of the drug delivery system or appropriate timing to the drug delivery system.
  • the triazabutadiene molecules of the present invention may be used for applications involving benzoquinone methides, e.g., it may be possible to synthesize derivatives that can undergo elimination via para-quinone methide chemistry (see FIG. 16A).
  • triazabutadiene Compound 41 may decompose to diazonium salt (Compound 42).
  • This reactive species may decompose to a phenol (Compound 43), which itself decomposes to a quinone methide and may liberate the cargo molecule (Compound 44). It may be possible to modify the electronic properties of the central ring in order to influence the rates at each step.
  • the azide- coupling chemistry may render this amenable to wide variety of chemical cargos. In a biological context these compounds may be able to release their desired cargo upon entry into the endosome, or upon exposure to non-virally relevant acidic environments such as in proximity to cancerous tumors.
  • This type of attachment chemistry may be utilized as a method for drug or detection delivery, and may have an added level of specificity if the system was delivered to a desired location using an antibody or aptamer.
  • Z 1 is a prodrug comprising a phenolic functional group, wherein the phenolic group is masked as a triazylidene moiety.
  • An example of how a prodrug is released (e.g., in an acidic environment, e.g., in a patient) is illustrated in FIG. 16B.
  • all drugs such as those approved by the U.S. Food and Drug Administration, that have a phenolic functional group may be masked as a triazylidene moiety.
  • Compound C is a stimulant that is produced by Glaxo Smith-Kline Beecham pharmaceutical company.
  • the phenolic group of Compound C can be converted to an azide group, e.g., by displacement of the hydroxy! group with an azide group.
  • the phenolic group is first converted to a suitable leaving group before subjecting to a nucleophilic displacement reaction with an azide group.
  • the resulting azide Compound A is then reacted with 3-(3-mesityl-2- (phenyltriaz-2-en-1-ylidene)-2, 3-dihydro-1 H-imidazol-1-yl) propane-1 -sulfonate to produce triazylidene Compound B.
  • the acidic environment of the patient's gastrointestinal tract (if administered orally) or patient's blood plasma (when administered intravenously) decomposes it to generate a corresponding diazonium compound regenerates the phenolic group as illustrated in FIG. 16B.
  • the phenolic group e.g., the hydroxy! group that is attached to a phenyl ring
  • the triazylidene moiety serves as a masking group for a phenolic functional group.
  • the present invention also features a method for administering a drug comprising a phenolic function group to a subject in need of such a drug administration.
  • the method comprises converting a drug comprising a phenolic-functional group to a prodrug, wherein said prodrug comprises an acid labile triazylidene moiety; and administering said prodrug to a subject in need of such a drug administration.
  • the triazylidene compound may also comprise a water solubility conferring moiety and/or Y 1 functional group defined in FIG. 1.
  • the present invention also features a method of converting a drug comprising a phenolic-function group to an acid labile prodrug.
  • the phenolic- functional group is converted to an azide group.
  • the azide functional group may then be reacted with a carbene to produce an acid labile prodrug comprising a triazylidene moiety (see FIG. 16D). d.
  • a triazabutadiene molecule is conjugated to another molecule (a conjugate molecule), e.g., a protein (e.g., an amino acid such as but not limited to lysine), a lipid, or other appropriate molecule.
  • a conjugate molecule e.g., a protein (e.g., an amino acid such as but not limited to lysine), a lipid, or other appropriate molecule.
  • the diazonium species part of the triazabutadiene molecule is conjugated to the conjugate molecule.
  • the cyclic guanidine species part of the triazabutadiene molecule is conjugated to the conjugate molecule.
  • the triazabutadiene molecule is attached to the conjugate molecule via a linker.
  • Linkers are well known to one of ordinary skill in the art and may include (but are not limited to) a polyether linkers such as polyethylene glycol linkers.
  • the conjugate molecule to which the triazabutadiene molecule is conjugated comprises an antibody or a fragment thereof.
  • the conjugate molecule to which the triazabutadiene molecule is conjugated comprises a viral protein.
  • the triazabutadiene molecules of the present invention are used for pull-down studies wherein a biomolecule or protein of interest is attached to one side and the other side is appended to something such as but not limited to a small molecule (e.g., hapten such as biotin) or compound.
  • a biomolecule or protein of interest can be pulled down using an avidin bead (which binds strongly to the biotin) and thoroughly washed. This may be useful for protein enrichment.
  • the biomolecule or protein of interest may then be cleaved from the avidin bead by means of reductive cleavage of the triazabutadiene that holds them together.
  • the present invention is not limited to these components, for example this application could also feature the use of a probe (e.g., fluorescent or otherwise) attached to an antibody used to interrogate a complex sample.
  • reductive cleavage of triazabutadiene molecules may also be used to cleave unreacted triazabutadienes that did not undergo diazonium formation/reaction chemistry that is associated with a drop in pH (or other mechanism) as described above (a sort of quench for the pH chemistry).
  • the diazonium species can react with a phenol species such as resorcinol or other appropriate phenol species.
  • a phenol species or resorcinol species is conjugated to a protein, e.g., a protein different from the protein to which the triazabutadiene molecule is conjugated, a protein that is the same protein to which the triazabutadiene molecule is conjugated, etc.
  • the resorcinol species or phenol species that the diazonium species reacts with is the phenol functional group of a tyrosine residue.
  • the present invention also features a method of detecting an environment having a low pH.
  • the method comprises providing a sample (e.g., tissue sample, cell sample, any appropriate sample) and introducing a triazabutadiene molecule according to the present invention to the sample.
  • An environment having a low pH a low pH appropriate for the triazabutadiene molecule
  • the method further comprises introducing a resorcinol species or a phenol species to the sample.
  • the resorcinol species or phenol species may react with the diazonium species to form an azo dye. Since the azo dye is visually distinct from the diazonium species and the triazabutadiene species, detection of the diazonium species and/or the azo dye would be indicative of the low pH environment.
  • the triazabutadiene molecules are used as reagents in buffers for various chemical or biochemical assays (e.g., immunohistochemistry assays, in situ hybridization assays, protein assays such as western blots, ELISAs, etc.).
  • chemical or biochemical assays e.g., immunohistochemistry assays, in situ hybridization assays, protein assays such as western blots, ELISAs, etc.
  • the triazabutadiene molecules may function as masked compounds that, when exposed to water, form reaction products that form covalent bonds with surfaces containing phenols.
  • the triazabutadiene molecule (or a diazonium species) is conjugated to a molecule other than a glass or plastic as described above.
  • the triazabutadiene molecule (or a diazonium species) is conjugated to a surface via a linker.
  • Linkers are well known to one of ordinary skill in the art and may include (but are not limited to) a polyether linkers such as polyethylene glycol linkers.
  • a triazabutadiene molecule is bonded to a surface.
  • Surfaces may include but are not limited to glass, plastic, a biomaterial, or any other appropriate surface, Non-limiting examples of materials also include Tufnol materials such as phenolic cotton laminated plastics, phenolic paper laminated plastics, etc., a phenol formaldehyde resin such as bakelite (or baekelite), etc.
  • the present invention is not limited to the methods and uses described herein.
  • the molecules herein are used as reagents in buffers for various chemical or biochemical assays (e.g., immunohistochemistry assays, in situ hybridization assays, protein assays such as western blots, ELISAs, etc.).
  • references to the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting of is met.

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Abstract

La présente invention concerne des molécules de triazabutadiène et des procédés d'utilisation de molécules de triazabutadiène, par exemple des procédés et des compositions pour produire une espèce d'aryldiazonium à partir d'une molécule de triazabutadiène, par exemple, une espèce d'aryldiazonium protégée sous la forme d'un triazabutadiène. Selon certains modes de réalisation de l'invention, une enzyme catalyse la réaction produisant l'espèce d'aryldiazonium à partir de la molécule de triazabutadiène. À titre d'exemple, les procédés et les compositions de l'invention peuvent être utilisés pour l'administration de médicaments.
PCT/US2018/022046 2015-08-11 2018-03-12 Espèce d'aryldiazonium à libération enzymatique Ceased WO2018165666A1 (fr)

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US10954195B2 (en) 2015-08-11 2021-03-23 Arizona Board Of Regents On Behalf Of The University Of Arizona Substituted triazenes protected from degradation by carboxylation of N1
US11339129B2 (en) 2016-07-29 2022-05-24 Arizona Board of Regents on behalf of the University of Arizon Triazabutadienes as cleavable cross-linkers

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Publication number Priority date Publication date Assignee Title
US10954195B2 (en) 2015-08-11 2021-03-23 Arizona Board Of Regents On Behalf Of The University Of Arizona Substituted triazenes protected from degradation by carboxylation of N1
US11339129B2 (en) 2016-07-29 2022-05-24 Arizona Board of Regents on behalf of the University of Arizon Triazabutadienes as cleavable cross-linkers

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