METHODS AND SYSTEMS FOR SYNTHESIS OF A D-AMINOLUCIFERIN PRECURSOR AND RELATED COMPOUNDS
Methods and systems to generate 6-amino-6-deoxy-D-luciferin precursor, 2-cyano-6-aminobenzothiazole and related compounds and derivatives
This application is related and claims priority to U.S. Provisional Application No. 61/308,317 entitled “Alternate Syntheses of Precursor to D-Aminoluciferin” filed on Feb. 26, 2010, docket IL-12088 to U.S. Provisional Application No. 61/331,072 entitled “Bioluminescent Protease Probe For The Detection Of Disease” filed on, May 4, 2010 with docket number IL-12087, and to U.S. Provisional Application No. 61/331,094 entitled “Carboxylate-Modified Luciferin, Amino Acid/Peptide Probes For Bioluminescent Protease Assays” filed on, May 4, 2010 with docket number IL-11088, each of which is incorporated herein by reference in its entirety.
STATEMENT OF GOVERNMENT INTERESTThe United States Government has rights in this invention pursuant to Contract No. DE-AC52-07NA27344 between the United States Department of Energy and Lawrence Livermore National Security, LLC for the operation of Lawrence Livermore National Laboratory
FIELDThe present disclosure relates to bioluminescence and in particular to methods and systems for the synthesis of
Bioluminescence provides a useful read out for the detection of reactions in a background of optically complex materials such as cells and tissues and the signal-to-noise ratio can be very high.
Luciferases are enzymes that generate visible light through the oxidation of a specific substrate in the presence of oxygen and usually a source of energy (such as Mg2+ and ATP). The reaction between firefly luciferase and the substrate luciferin, yields oxyluciferin, carbon dioxide and visible light with a maximum around 560 nm and with other wavelengths of light output identifiable by a skilled person.
For these reasons, bioluminescence and in particular luciferin and related compounds are routinely used for in vivo imaging applications and for additional techniques for in vitro and/or in vivo detection of targets and/or reactions.
SUMMARYProvided herein are methods and systems to synthesize a precursor of
According to a first aspect a method and system to provide 2-cyano 6-amino-benzothiazole from a monofunctional benzothiazole is described. The method comprises providing a monofunctional benzothiazole attaching in position C2 a functional group of formula (I) (C(═X1)NH2) wherein X1 is O or S, and converting the functional group of formula (I) to a cyanide group, through elimination of H2X1 from the monofunctional benzothiazole. In the method the monofunctional benzothiazole either comprises an amino group in position C6 or is modified to comprise an amino group in position C6. The system comprises one or more monofunctional benzothiazoles attaching in position C2 a functional group of formula (I), together suitable reagents for simultaneous combined or sequential use in the method to provide 2-cyano-6-aminobenzothiazole herein described
According to a second aspect a method and system to provide 2-cyano-6-aminobenzothiazole from a monofunctional benzothiazole is described. The method comprises providing a monofunctional benzothiazole attaching in position C2 a functional group of formula (II) (C(═X1)X2R) wherein R is an alkyl group, or a halogen atom; and X1 and X2 are independently O or S. The method also comprises converting the functional group of formula (II) into an amide of formula (I) (C(═X1)NH2) and converting the functional group of formula (I) to a cyanide group through elimination of a H2X1 from the monofunctional benzothiazole. In the method the monofunctional benzothiazole either comprises an amino group in position C6 or is modified to comprise an amino group in position C6. The system comprises at least two of one or more monofunctional benzothiazole attaching in position C2 a functional group of formula (I), and/or one or more monofunctional benzothiazole attaching in position C2 a functional group of formula (II) together suitable reagents for simultaneous combined or sequential use in the method to provide 2-cyano-6-aminobenzothiazole herein described.
According to a third aspect, a method and system to provide an amino acid labeled with 6-amino-6-deoxy-
According to a fourth aspect an intermediate in the synthesis of an amino acid labeled with 6-amino-6-deoxy-
wherein R1 is an amino acid attached to the remainder of the compound of formula (III) through a peptide bond, a carbamate bond or a urea/thiourea bond.
According to a fifth aspect, a method and system to provide a peptide labeled with 6-amino-6-deoxy-
According to a sixth aspect an intermediate in the synthesis of a peptide labeled with 6-amino-6-deoxy-
wherein R1 is a single amino acid and R2 is a peptide attached to the remainder of the compound of formula (IV) through a peptide bond a carbamate bond or a urea/thiourea bond.
According to a seventh aspect, a labeled peptide is described. The labeled peptide is obtainable by the method to provide a peptide conjugated to 6-amino-6-deoxy-
According to an eighth aspect a carboxylate-modified luciferin amino acid/peptide probe, is described and related method for synthesis and/or use of said carboxylate modified probe. The carboxylate-modified
The methods and systems described allow in several embodiments facilitating the generation of amounts of pure 6-amino-6-deoxy-
The methods and systems described allow in several embodiments facilitating the coupling of 6-amino-6-deoxy-
The methods and systems described allow in several embodiments generation of pure 6-amino-6-deoxy-
The methods and systems described herein can be used in connection with applications wherein production of
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.
The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and the examples, serve to explain the principles and implementations of the disclosure.
Provided herein are methods and systems to synthesize a precursor of 6-amino-6-deoxy-
The reaction between the luciferase enzyme and substrate
Methods and systems are herein described to provide a
In some embodiments, methods and systems are described to provide the precursor with method starting from a monofunctional benzothiazole. The term “benzothiazole” as used herein indicates an organosulfur compound of formula (V)
which can be substituted or unsubstituted in the various positions. Benzothiazole is typically colorless, and takes the form of a slightly viscous liquid while most of its derivatives are solid. Benzothiazoles are commercially available and can be prepared using methods such as treatment of 2-aminobenzenethiol with acid chlorides according to the reaction C6H4(NH2)SH+RC(O)Cl→C6H4(NH)SCR+HCl+H2O and to additional procedures identifiable by a skilled person.
The term “monofunctional benzothiazole” indicates a benzothiazole including one functional group in the C2 position wherein the term “functional group” as used herein indicates a specific group of atoms within a molecular structure that are responsible for the characteristic chemical interactions or reactions of that structure. In general, exemplary functional groups include hydrocarbons, groups containing halogen, groups containing oxygen, groups containing nitrogen and groups containing sulfur all identifiable by a skilled person. In particular, exemplary functional groups in the sense of the present disclosure comprise alkoxy or alkyl groups, secondary amines, amides and additional functional groups can be identified by a skilled person upon reading of the present disclosure. In some embodiments, the monofunctional benzothiazole can be substituted in any positions, wherein the terms “substituted” or “substitution” herein indicates replacement of one or more hydrogens with chemical groups that do not substantially interfere with the characteristic chemical interactions or reactions of the functional group as described herein.
In some embodiments, methods and systems are described to provide the precursor with methods starting from a monofunctional benzothiazole attaching in position C2 a functional group of formula (I) (C(═X1)NH2) wherein X1 is O or S, wherein the amide in the functional group of formula (I) is converted to a cyanide group by elimination of a H2X1 compound.
The term “amide” as used herein indicates an organic compound that contains the carbonyl group (R4—C═O ) linked to a nitrogen atom (N) (herein also carboxamide) or a thiamide group R4—CS—NR′R5, where R4, R′, and R5 are same or different and are organic groups with R′ and R5 possibly formed by H. The term refers both to a class of compounds and a functional group within those compounds.
In particular, the term “carboxamide” as used herein indicates an organic compound that contains a carbonyl group (R4-C═O) linked to a nitrogen atom (N). Typically, carboxamides of the general structure R6-CO—NR7R2R8 can be synthesized by replacement of the hydroxyl group (OH) of a carboxylic acid by an amino group (NR1R2), where R6, R7, and R8 are same or different and are organic substituents with R7 and R8 possibly formed by H. Exemplary compounds containing carboxamide groups comprise asparagine and glutamine.
The term thioamides indicates sulfur analogues to amides, where the oxygen atom (O) in amide is replaced by a sulfur atom (S). The general structure of thioamide is R4—CS—NR′R5, where R4, R′, and R5 are same or different and are organic groups with R′ and R5 possibly formed by H. Typically thioamides are analogous to carboxamides but they exhibit greater multiple bond character along the C—N bond, resulting in a larger rotational barrier. Thioamides are typically prepared by treating amides with phosphorus sulfides such as phosphorus pentasulfide and, in more specialized applications, Lawesson's reagent. An alternative route entails the reaction of nitriles with hydrogen sulfide. The Willgerodt-Kindler reaction also affords benzylthioamides.
The terms “eliminate” and “elimination reaction” as used herein with reference to a reaction indicate an elimination reaction. An elimination reaction is a type of organic reaction in which two substituents are removed from a molecule in either a one or two-step mechanism (E2 and E1 mechanisms, respectively). Either the unsaturation of the molecule increases or the valence of an atom in the molecule decreases by two. E1 mechanism: generally follows the following form: the first step requires the loss of the leaving group, forming a carbocation intermediate. A nucleophilic species, usually a base, then attacks a neighboring hydrogen forming the double bond. E2 mechanism generally follows the following form: a nucleophilic species or base attacks a hydrogen neighboring the leaving group, pushing the electrons into the double bond as the leaving group leaves. In elimination reactions herein described does not necessarily result in an actual production of an H2X1 product, as long as the H2X1 compound is removed from the starting compound. In this connection for example, in embodiments where X1 is oxygen and a POCl3 reagent is used the oxygen atom is eventually attached to phosphorus. Exemplary elimination reactions comprise dehydration and dehydrosulfurization. A dehydration reaction is usually defined as a chemical reaction that involves the loss of water from the reacting molecule. Dehydration reactions are a subset of elimination reactions where the leaving group is water (H2O). Dehydrosulfurization reactions or H2S elimination reactions are another subset of elimination reaction where the leaving group is (H2S).
In some embodiments, in the functional group of formula (I) X1 is S, the conversion from thioamide to CN group can be performed by dehydrosulfurization directed to elimination the H2S compound. The dehydrosulfurization can be performed with suitable reagents (e.g. tellurium chloride) and under suitable conditions, such as the ones described for example in ref. 17. A skilled person will be able to identify additional reactions and related procedures that are functional to the dehydrosulfurization of the monofunctional benzothiazole herein described.
In some embodiments, in the functional group of formula (I) X1 is O the reduction can be performed by dehydration of the corresponding ester group with production of H2O according to procedures such as the ones exemplified in the Examples section and additional procedures identifiable by a skilled person.
In some embodiments, the benzothiazole presenting the functional group of formula (I) in position C2 and/or the benzothiazole presenting the functional group CN comprises an amino group in position C6. In some embodiments, the benzothiazole presenting the functional group of formula (I) in position C2 is modified to comprise an amino group in position C6. In particular in some of those embodiments, a nitrile group is first introduced in position C6 before or after converting the functional group of formula (I) to a CN group. The nitrile group in C6 of the benzothiazole is then converted to an amino group. A skilled person will be able to identify suitable methods to introduce an NO2 group in monofunctional benzothiazoles herein described at any stage of the procedures. For example, typical nitrations use nitric acid and sulfuric acid to produces the nitronium ion (NO2), which is the active species of nitration reactions and attacks the electron-rich reactant, such as a benzene ring, to initiate electrophilic substitution reaction.
In some embodiments, an NH2 is introduced directly into the C6 position of the benzothiazole ring. A skilled reader will be able to identify suitable reaction for introducing an NH2 group directly in monofunctional benzothiazoles herein described at any stage of the procedures herein described.
In some embodiments, the functional group of formula (I) can be provided in the monofunctional benzothiazole herein described starting from a monofunctional benzothiazole attaching in position C2 a functional group of formula (II) (C(═X1)X2R) wherein R is an alkyl group, or a halogen atom and X1 and X2 are independently O or S.
The term “alkyl” as used herein refers to a linear, branched, or cyclic saturated hydrocarbon group typically although not necessarily containing 1 to about 10 carbon atoms, preferably 1 to about 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Generally, although again not necessarily, alkyl groups herein contain 1 to about 6 carbon atoms. The term “cycloalkyl” intends a cyclic alkyl group, typically having 4 to 8, preferably 5 to 7, carbon atoms. The term “substituted alkyl” refers to alkyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkyl” and “heteroalkyl” refer to alkyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms “alkyl” and “lower alkyl” include linear, branched, cyclic, unsubstituted, and/or substituted. In some embodiments, in the functional group of formula (I), R can be a substituted alkyl which can comprise heteroatoms such as N, O, S, P, Si, F, Cl, Br, and I, as well as additional groups and heteroatoms identifiable by a skilled person.
In embodiments where X2 is O, the resulting X2—R group can be an alkoxy group or an acid halide. The term “alkoxy” as used herein intends an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group may be represented as —O-alkyl where alkyl is as defined above. A “lower alkoxy” group intends an alkoxy group containing 1 to 6 carbon atoms. Exemplary alkoxy group comprised in the functional group of formula (I) comprise methoxy, ethoxy, propoxy and additional alkoxy group comprising a lower alkyl. The term “acid halide” indicates a chemical compound that can be typically derived from an oxoacid by replacing a hydroxyl group with a halide group (i.e. a binary compound, of which one part is a halogen atom and the other part is an element or radical that is less electronegative (or more electropositive) than the halogen). Exemplary acid halides comprise (C(═O)Cl) and additional halides identifiable by a skilled person. In some embodiments, the acyl halide can be treated with ammonia, and the reaction is expected to proceed faster than the reaction performed with the ester group.
In embodiments where X2 is S the resulting X2—R group is an alkyl group and in particular a lower alkyl group, or a halogen such as Cl. Exemplary functional groups of formula (II) comprise (C(═S)OR), which can be made for example by treating the ester with P2S5 as described in ref. 19; (C(═O)SR) which can be made for example by reacting the ester with a mercaptan under basic conditions as identifiable by a skilled person; and (C(═S)SR), which can be made for example by treating the chloromethylbenzothiazole with sulfur and methyl iodide, according to the procedure described in ref. 20. A skilled reader will be able to identify additional functional groups of formula (II) and suitable procedure to provide monofunctional benzothiazole comprising those functional groups.
In some embodiments, wherein the functional group of formula (I) and (II) X1 is S, the conversion from ester to thioamide can be performed using reagents and procedures identifiable by a skilled person. For the conversion from ester to thioamide can be performed by using NH3, then P2S5 as described in ref 18. Additional reactions will be identifiable by a skilled person upon reading of the present disclosure.
In some embodiments, wherein the functional group of formula (I) and (II) X1 is O, the conversion from ester to carboxamide can be performed by using amines alone or in presence of reagents (such as dicyclohexylcarbodiimide (DCC)) which are suitable to convert the hydroxyl oxygen in a more efficient leaving group. Examples of those conversions are reported in the Example section of the present disclosure. A skilled reader will be able to identify additional reactions using amines or other reagents that are suitable to convert an ester according to formula (I) wherein X1 is oxygen into a carboxamide of formula (II) wherein X1 is oxygen.
In some embodiments of methods and systems wherein the
In an embodiment, wherein X1 is O the method can be performed as schematically illustrated in
In the exemplary illustration of
In the exemplary illustration of
In the exemplary illustration of
In some embodiments according to the illustration of
A skilled person will be able to identify variants of the procedure schematically illustrated in
For example additional exemplary reaction schemes starting from a different monofunctional benzothiazole herein described, are schematically illustrated in
Some specific routes are further illustrated in
In the exemplary illustration of
In the exemplary illustration of
In some embodiments, converting the 2-carboxamide-benzothiazole to a corresponding nitrile in C2 position can be performed elimination reactions and additional reactions identifiable by a skilled person. In the methods herein described for example conversion of the C2 carboxamide can be performed by dehydration to the nitrile using suitable reagents such as phosphoryl chloride in pyridine, proceeding with quantitative conversion. In some of those embodiments, benzothiazole-2-carboxamide can be converted to the nitrile to yield 2-cyano-benzothiazole with 100% conversion (analytical HPLC, mass spect) (see Example 8). Without purification, the nitrile was reacted with H2SO4/HNO3 and the reaction was monitored over time (over a period of ˜2 h) via analytical HPLC. (see Example 11).
A skilled person will be able to identify alternative reactions schemes according to the illustration of
In an embodiment of methods and systems provide the
In some embodiments, the 6-amino-6-deoxy-
As used herein the term “amino acid”, “amino acid monomer”, or “amino acid residue” refers to any of the twenty naturally occurring amino acids including synthetic amino acids with unnatural side chains and including both D and L optical isomers. The term “polypeptide” or “peptide” as used herein indicates an organic polymer composed of two or more amino acid monomers and/or analogs thereof. The term “polypeptide” includes amino acid polymers of any length including full length proteins and peptides, as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called a protein oligomer or oligopeptide. The term “amino acid analog” refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, isotope, or with a different functional group but is otherwise identical to its natural amino acid analog.
The term “couple” or “conjugate” as used herein indicates formation of a covalent bond between two compounds. Typically, in some embodiments, coupling or conjugation of the amino acid or to the amino group in 2-cyano-6-aminobenzothiazole can be performed with an efficient peptide coupling method such as DCC, or N-methyl morpholine and isobutylchloroformate, since the aniline amino group is relatively unreactive. In other embodiments, coupling or conjugation can be performed reactions suitable to form a urea/thiourea (NH(C═X)NH) or a carbamate/thiocarbamate (X(C═X)NH) linkage, where X═O or S.
The term “carbamate” as used herein indicates organic compounds derived from carbamic acid (NH2COOH). Specifically, the term refers to any salt or ester of carbamic acid with the general structure R8O—CO—NR9R10, where R8, R9, and R10 are same or different and are organic substituents with one or two possibly formed by H. The term “thiocarbamate” as used herein indicates sulfur analogues of carbamates, where one of the oxygen atoms (O) in a carbamate is replaced by a sulfur atom (S). Specifically, there are two structurally isomeric types of thiocarbamates: O-thiocarbamates, ROC(═S)NR2, where the carbonyl group (C═O) is replaced with a thiocarbonyl group (C═S) and S-thiocarbamates, RSC(═O)NR2, where the R—O—group is replaced with an R—S— group. Further, O- and S-thiocarbamates can interconvert, for example in the Newman-Kwart rearrangement.
In particular, in some embodiments coupling of the peptide with the precursor can be performed according to a two step process schematically illustrated in
In some embodiments, conjugation of the amino group of the 2-cyano-6-aminobenzothiazole to single amino acids or peptides can be achieved using isobutylchloroformate activation of the free acid of the amino acid in the presence of N-methyl morphiline in THF as previously reported.11, 13 (see Example 10). In some embodiments, conjugation of the amino group of the 2-cyano-6-aminobenzothiazole to single amino acids can be achieved using carbodiimide activation. In particular carbodiimide activation involves attack of the carboxylate oxygen on the central carbodiimide carbon, creating a leaving group with that oxygen atom according to reactions identifiable by a skilled person. The amine then attacks the carboxyl carbonyl, displacing the now urea leaving group. The insolubility of some ureas can in some cases help drive the reaction.
In some embodiments, suitable amino acids to be conjugated with the
In some embodiments, the amino acids comprise a side chain, which have been surprisingly found to be labelable with the precursor and then converted in to functional amino acid labeled with
In some embodiments, an intermediate in the synthesis of peptide labeled at the carboxy terminus with 6-amino-6-deoxy-
In some embodiments, methods and systems to provide a peptide labeled at the carboxy terminus with 6-amino-6-deoxy-
In an embodiment, a method and system to provide a peptide labeled with 6-amino-6-deoxy-
In some embodiments, modification of
In some embodiments, methods and systems herein described provide an alternate route for the synthesis of the precursor 2-cyano-6-aminobenzothiazole to
In some embodiments, methods and systems herein described provide convenient synthetic route to
In some embodiments a carboxylate-modified Luciferin, amino acid or peptide probe, is described, that comprises a
The carboxylate-modified luciferin, amino acid or peptide probe can be provided using methods and systems based on reactions performed on a resin base. In particular the amino acid or peptide can be provided on a resin base (e.g. by synthesizing the peptide on the resin base). A
The carboxylate-modified Luciferin, amino acid or peptide probe can be used to perform various assays and procedure aimed at detection of targets and/or reactions. For example in some embodiments the carboxylate modified luciferin amino acid or peptide can be used in connection with luciferase for releasing the
In particular, in some embodiments, carboxylate-modified luciferin probes are provided, where
The term “derivative” as used herein with reference to a first compound (e.g.,
In some embodiments, derivatives of
The term “detect” or “detection” as used herein indicates the determination of the existence, presence or fact of a target or signal in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate including a platform and an array. In particular,
In some embodiments, the
Exemplary suitable assays are provided by in vitro assays for certain protease developed with different reporting techniques. The most common techniques are the proteolytic release of p-nitro aniline for UV detection, the proteolytic release of fluorescence dyes for fluorescence detection and mass spectrometry detection of proteolytic cleavage products. However, there are various problems associated with the detection techniques currently available when developing an assay for detecting specific reactions in a complex background (e.g.in vivo—the low sensitivity of UV absorption, auto-fluorescence of cells or fluorescent interference from chemical and natural products, and the vacuum and other expensive and cumbersome parts required for mass detection).
In methods and systems herein described, any of the above compounds can be synthesized or added according to techniques identifiable by a skilled person.
As disclosed herein, the compounds herein described can be provided as a part of systems to detect targets according to any of the methods described herein. The systems can be provided in the form of kits of parts.
In a kit of parts, the monofunctionalized benzothiazoles, reducing agents, and other reagents to perform the methods can be comprised in the kit independently. One or more compounds and reagents can be included in one or more compositions alone or in mixtures identifiable by a skilled person. Each of the one or more of compounds and reagents can be in a composition together with a suitable vehicle.
Additional reagents can include salts (such as Mg2+) and reagents (e.g. ATP) and/or molecules suitable to enhance or favor the reaction according to any embodiments herein described and/or molecules, standards and/or equipment to allow detection of pressure temperature and possibly other suitable conditions. For example luciferin/luciferase as control reagents can be comprised in a kit to serve as the “parent compounds” that will provide the baseline for comparison to the modified bioluminescence substrates.
In particular, the components of the kit can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here described. The kit will normally contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes or C
Further advantages and characteristics of the present disclosure will become more apparent hereinafter from the following detailed disclosure by way of illustration only with reference to an experimental section.
EXAMPLESThe methods system herein described and related intermediates and derivatives are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.
In particular, the following examples illustrate exemplary synthesis and uses of 2-cyano-6-aminobenzothiazole starting from 6-nitrobenzothiazole, ethyl 6-nitrobenzothiazole-2-carboxylate, and other compounds performed according to the reaction schemes summarized in
The following experimental procedures and characterization data were used for all compounds and their precursors exemplified herein.
General. Commercially available reagents and solvents were used as received without further purification. Anhydrous pyridine and phosphoryl chloride were purchased from Aldrich. 6-nitrobenzothiazole was purchased from Alfa Aesar. Anhydrous ammonia was purchased from Mattheson Tri-Gas, ethyl pyruvate from Fluka, ferrous sulfate from Mallinckrodt Chemicals, sodium bicarbonate from EMD Biosciences, and tin(II) chloride from Sigma. The amino acid compounds were purchased from NovaBioChem and the biotin-conjugated peptides were specifically designed and purchased from Peptides International.
Characterization/Instrumentation. Analytical thin layer chromatography (TLC) was carried out using aluminum sheets coated with silica gel 60 F254. Reaction conversions were followed by analytical HPLC at 1 mL/min on an Agilent 1100 machine (Waters Symmetry C18, 5 μm, 4.2×150 mm column, diode array detector) with a linear gradient from 95% H2O (0.1% TFA) to 80% MeCN (0.1% TFA) over 15 mi. For analytical characterization, small portions were purified by semi-preparative HPLC at 10 mL/min on a Waters preparative machine (Waters Symmetry prep C18, 7 μm, 19×300 mm column, photodiode array detection) with a linear gradient from 90-95% H2O (0.1% TFA) to 50-90% MeCN (0.1% TFA) for 30-35 min. Semi-preparative HPLC fractions were collected and lyophilized using Kinetics Flexi-Dry freeze-dryer. Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker 500 MHz machine in d6-DMSO. Splitting patterns are denoted s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet; br s, broad singlet. Mass spectra were acquired on a Micromass Quattro Micro API mass spectrometer operating in positive ion mode. The samples were dissolved in MeCN/H2O (1:1), 0.1% formic acid for mass spectrometry analysis.
EXAMPLE 1 Synthesis of ethyl 6-nitrobenzothiazole-2-carboxylate (2)Ethyl 6-nitrobenzothiazole-2-carboxylate was synthesized from 6-nitrobenzothiazole according to the following reaction scheme.
In particular, 6-nitrobenzothiazole 1 (16.7 mmol, 3.0 g) was suspended in 6.6 ml deionized H2O. 2.7 ml conc. H2SO4 was added dropwise to the reaction flask. Separately, 30% H2O2 (167.6 mmol, 5.7 g) was added dropwise to a solution of ethyl pyruvate (75.6 mmol, 8.4 ml) at 0° C. The resultant oxyhydroperoxide solution and a solution of FeSO4.7H2O (48.9 mmol, 13.6 g) in 13.2 ml deionized H2O were simultaneously added dropwise to the 6-nitrobenzothiazole reaction flask at 0° C. After 30 min, the reaction was poured onto ice and basified with NaHCO3 (pH from 2 to ˜pH 6). The organic product 2 was washed with a saturated NaCl solution, extracted with EtOAc, dried over Na2SO4, and concentrated in vacuo. The material was additionally filtered with 200 proof EtOH yielding a pale yellow solid (˜61% yield crude material). Agilent HPLC displayed partial conversion to the crude product at 10.4 min with a peak at 8.7 min corresponding to the starting material.
Compound 2 was used in the subsequent step without purification. For analytical characterization, a small portion was purified by semi-preparative HPLC at 10 mL/min on a Waters preparative machine (Waters Symmetry prep C18, 7 μm, 19×300 mm column, photodiode array detection) with a linear gradient from 95% H2O (0.1% TFA) to 90% MeCN (0.1% TFA) for 35 min yielding a large product peak at 27 min. The fractions were collected and lyophilized using Kinetics Flexi-Dry freeze-dryer. ESI-MS: m/z calcd for C10H8N2O4S (M+H)+ 253.26, found 252.85 (100%). 1H (d6-DMSO) δ (ppm)=1.39 (t, J=7.0 Hz, 3H), 4.48 (q, J=7.0 Hz, 2H), 8.44 (m, 2H), 9.32 (s, 1H). 13C NMR (d6-DMSO) δ (ppm)=13.8, 63.0, 120.1, 122.1, 125.5, 136.5, 145.9, 155.9, 159.3, 164.1.
Bernadi et al reported almost quantatative conversion of benzothiazole to ethyl 6-nitrobenzothiazole-2-carboxylate.14 In this case, the low solubility of starting material 1 in aqueous H2SO4 limited the conversion to the desired product (˜40% by TLC). The product 2 was also impossible to separate from the starting material 1 either by flash or gravity silica column chromatography.
EXAMPLE 2 Synthesis of 6-nitrobenzothiazole-2-carboxamide (3)6-Nitrobenzothiazole-2-carboxamide was synthesized from ethyl 6-nitrobenzothiazole-2-carboxylate according to the following reaction scheme.
In particular, crude ethyl 6-nitrobenzothiazole-2-carboxylate 2 (7.9 mmol, 2.0 g) was dissolved in 140 ml MeOH and purged with NH3 gas. Conversion to the product was monitored by analytical TLC (3 Hexane: 2 EtOAc). Following complete conversion to the product (20 min) the solvent was removed in vacuo. The crude material was filtered with chloroform (30 ml) to yield a pale tan-colored solid (80% yield crude material). Agilent HPLC displayed partial conversion to the crude product 3 at 8.1 min. The compound 3 was used in the subsequent step without purification.
For analytical characterization, a small portion was purified by semi-preparative HPLC at 10 mL/min on a Waters preparative machine (Waters Symmetry prep C18, 7 μm, 19×300 mm column, photodiode array detection) with a linear gradient from 90% H2O (0.1% TFA) to 50% MeCN (0.1% TFA) for 30 min yielding a large product peak at 22.5 minutes. The fractions were collected and lyophilized using Kinetics Flexi-Dry freeze-dryer. ESI-MS: m/z calcd for C8H5N3O3S (M+H)+ 224.22, found 223.88 (100%). 1H (d6-DMSO) δ (ppm)=8.30 (br s, NH), 8.31 (dd, J=9.0 Hz, J=0.5 Hz), 8.41 (dd, J=9.0 Hz, J=2.5 Hz), 8.65 (br s, NH), 9.28 (d, J=2.0 Hz). 13C NMR (d6-DMSO) δ (ppm)=120.3, 122.1, 124.8, 137.1, 145.6, 156.6, 160.8, 171.1.
EXAMPLE 3 Synthesis of 2-cyano-6-nitrobenzothiazole (4)2-Cyano-6-nitrobenzothiazole was synthesized from 6-nitrobenzothiazole-2-carboxamide according to the following reaction scheme.
In particular, crude 6-nitrobenzothiazole-2-carboxamide 3 (4.5 mmol, 1.0 g), was dissolved in anhydrous pyridine (0.74 mol, 60 ml) and stirred at RT under N2. The temperature of the reaction was dropped to 0° C. and POCl3 (0.14 mol, 12.5 ml) was added dropwise to the reaction flask. After 20 min, the acetone/ice bath was removed and the reaction was stirred at RT for an additional 2 h. The contents were then transferred to a larger reaction flask containing 150 ml EtOAc at 0° C. While stifling, the reaction was quenched with the dropwise addition of water (150 ml).
The organic layer was separated, dried over Na2SO4, and concentrated in vacuo yielding an orange/brown solid material. TLC analysis (3 Hexane: 7 CH2Cl2) displayed 100% conversion to the product 4. Agilent HPLC displayed conversion to the crude product at 10.1 min. The product 4 was purified by gravity silica column chromatography (3 Hexane: 7 CH2Cl2) to yield a pale white solid (˜24% pure product). ESI-MS: m/z calcd for C8H3N3O2S (M+H)+ 206.20, found 205.97 (100%). 1H (d6-DMSO): δ (ppm)=8.47 (s, 2H, 1H), 9.39 (s, 1H). 13C NMR (d6-DMSO): δ (ppm)=112.7, 120.3, 122.7, 125.3, 136.0, 143.2, 146.4, 154.6.
EXAMPLE 4 Synthesis of 2-cyano-6-aminobenzothiazole (5)2-Cyano-6-aminobenzothiazole was synthesized from 2-cyano-6-nitrobenzothiazole according to the following reaction scheme.
In particular, purified 2-cyano-6-nitrobenzothiazole 4 (0.49 mmol, 100 mg) was dissolved in 5 ml EtOH (200 Proof). 2.5 equivalents of SnCl2 (1.3 mmol, 322 mg) was added to the flask. The reaction was heated to 60° C. under N2, and stirred for 2 h. Over time the mixture changed from a bright fluorescent yellow to an orange/yellow coloring. After cooling to room temperature (RT), the reaction was poured into ice water (˜5 ml) and the pH was adjusted using NaHCO3 (pH 7).
The product 5 was extracted using EtOAc, dried over MgSO4 and removed in vacuo (˜65% yield). Agilent HPLC displayed conversion to the crude product at 7.4 min. The product 5 was purified by gravity silica column chromatography (3 Hexane: 2 EtOAc) to yield an orange/yellow solid. ESI-MS: m/z calcd for C8H5N3S (M +H)+ 176.22, found 175.85 (100%). 1H NMR (d6-DMSO, 500 MHz) δ (ppm)=6.15 and 6.55 (br s, NH2), 6.97 (dd, J=9.0 Hz, J=2.0 Hz), 7.15 (d, J=2.5 Hz), 7.86 (d, J=9.0 Hz). 13C NMR (d6-DMSO) δ (ppm)=102.2, 114.1, 117.3, 125.0, 127.7, 138.2, 143.2, 150.4.
EXAMPLE 5 Synthesis of ethyl 6-nitrobenzothiazole-2-carboxylate (7)Ethyl 6-nitrobenzothiazole-2-carboxylate was synthesized from ethyl benzothiazole-2-carboxylate according to the following reaction scheme.
In particular, commercially available ethyl benzothiazole-2-carboxylate 6 (24.1 mmol, 5.0 g) was suspended in 22 ml conc. H2SO4 at 0° C. At 10° C. KNO3 (26.4 mmol, 26 g) was added portionwise over 30 min to the stifling solution, not to exceed 15° C. Color of reaction changed from bright green/yellow to yellow over time. The reaction warmed from 25° C. to 40° C. for an additional 30 min. When the temperature of the reaction was decreased, the solution was poured over ice/water). The precipitate forming in solution was collected via glass frit vacuum filtration to yield a light yellow solid 7.
Resultant solid 7 was additionally washed with water, extracted into EtOAc, and concentrated in vacuo. Agilent HPLC displayed partial conversion to the crude product at ˜10.0 min with a peak at ˜9.9 min corresponding to the starting material. Attempts to purify the product were made using gravity silica column chromatography (4 Hexane: 1 EtOAc) to yield a yellow solid (˜10% yield). Compound 7 was identified with LC/MS spectroscopic analysis as illustrated in
Benzothiazole-2-carboxamide was synthesized from ethyl benzothiazole-2-carboxylate according to the following reaction scheme.
In particular, commercially available ethyl benzothiazole-2-carboxylate 6 (2.4 mmol, 0.5 g) was dissolved in 34 mL MeOH and purged with NH3 gas. Conversion to the product was monitored by TLC (3 Hexane: 2 EtOAc). Following complete conversion to the product 8 (˜30 min), the solvent was removed in vacuo. Agilent HPLC displayed 100% conversion to the desired product 8 with a single peak at ˜8.0 min. The solid white compound was used in the subsequent step without purification. ESI-MS: m/z calcd for C8H6N2OS 179.02 (M+H)+, 179.88 found (100%) (see LC/MS spectra of
6-nitrobenzothiazole-2-carboxamide was synthesized from benzothiazole-2-carboxamide according to the following reaction scheme.
In particular, 2-carboxamide-benzothiazole 8 (0.88 mmol, 0.156 g) was suspended in 0.655 ml conc. H2SO4 at 0° C. 1.3 equivalents of concentrated HNO3 (1.1 mmol, 0.069 g) was added portionwise over 10 min to the stirring solution; not to exceed 0° C. The reaction remained at 0° C. for ˜6 h and continued overnight; warming to 25° C. overnight. The following day the reaction was poured over ice/water forming a white precipitate in solution. The reaction was washed with water, extracted into EtOAc, and concentrated in vacuo to yield crude product. Attempts to purify the product were made using gravity silica column chromatography (Initially 3 Hexane: 7 CH2Cl2 followed by 3 Hexane: 2 EtOAc) to yield a pure white solid (˜30% yield). (see LC/MS spectra of
2-Cyanobenzothiazole was synthesized from benzothiazole-2-carboxamide according to the following reaction scheme.
Benzothiazole-2-carboxamide 8 (0.61 mmol, 0.1 g) was dissolved in anhydrous pyridine (0.080 mol, 6.5 ml) and stirred under N2 at 0° C. The temperature of the reaction was dropped to 0° C. during dropwise addition of POCl3 (0.015 mol, 1.4 ml). The solution immediately changed from clear to a pale pink coloring with addition of POCl3. After 20 min, the acetone/ice bath was removed and the reaction was stirred at RT for 2 h further. The tan/brown-colored reaction was then transferred to a larger reaction flask containing 10 ml EtOAc at 0° C. The reaction was quenched by the dropwise addition of water. The organic product 10 was washed with water, extracted with EtOAc, and concentrated in vacuo to yield a light yellow/orange product. TLC analysis (3 Hexane: 2 EtOAc) and Agilent HPLC displayed 100% conversion to the desired product (single peak at 10.5 min). The compound was used in the subsequent step without purification (95% yield). ESI-MS: m/z calcd for C8H4N2S 161.01 (M +H)+, 160.86 found (100%). (see LC/MS spectra of
2-Cyano-6-nitrobenzothiazole was synthesized from 2-cyanobenzothiazole according to the following reaction scheme.
In particular 2-cyano-benzothiazole 10 (0.84 mmol, 0.134 g) was suspended in 0.563 ml conc. H2SO4 at 0° C. 1.3 equivalents of concentrated HNO3 (1.1 mmol, 0.069 g) was added portionwise over 10 min to the stirring solution; not to exceed 0° C. over a total of ˜45 min. The reaction proceeded further for 2 h and at 5 h the product was evident. The reaction was then poured over ice/water, extracted into EtOAc, and concentrated in vacuo to yield a crude yellow residue 11. For analytical characterization, a small portion was purified by semi-preparative HPLC at 10 mL/min on a Waters preparative machine (Waters Symmetry prep C18, 7 μm, 19×300 mm column, photodiode array detection) with a linear gradient from 90% H2O (0.1% TFA) to 50% MeCN (0.1% TFA) for 30 min yielding a large product peak at ˜27 minutes. The fractions were collected and lyophilized using Kinetics Flexi-Dry freeze-dryer.
Further NMR can be performed to detect the monofunctional benzothiazole comprising the NO2 at position C6 versus thiazole substituted in the C4, C5, or C7 positions.
EXAMPLE 10 Synthesis of Tyrosine-Aminocyano Derivative of 2-cyano-6-aminobenzothiazoleThe tyrosine-aminocyano derivative of 2-cyano-6-aminobenzothiazole was synthesized according to the following reaction scheme.
Tyrosine protected amino acid (0.32 mmoles, 0.11 g) was dissolved in 4 mL of anhydrous THF and stirred under N2 at 0° C. in the dark. N-methylmorpholine (2 eq, 0.64 mmol, 0.070 mL) and isobutyl chloroformate (1.3 eq, 0.42 mmol, 0.055 mL) were added dropwise to the reaction at 0° C. and stirred for 30 min in the dark. Separately, purified 2-cyano-6-aminobenzothiazole 5 (0.32 mmol, 0.056 g) was dissolved in 1 mL of anhydrous THF and then added dropwise to the reaction flask over a period of 30 min. The reaction was stirred under N2 in the dark at 0° C. for 2 h, followed by an overnight stir at room temperature in the dark. 24 h later the anhydrous THF was removed in vacuo. The product 12 was dissolved in EtOAc and washed with saturated NaHCO3 and NaCl to quench any remaining isobutyl chloroformate. The organic layer was collected, dried with MgSO4, and evaporated to dryness. The protected group on the tyrosine portion of the product was removed using 20% TFA in anhydrous CH2Cl2; stirred at room temperature in the dark for 2 h. Following deprotection, the product 12 was evaporated to dryness and placed on vacuum.
The residue was redissolved in 50/50 0.1% TFA in MeCN & H2O (+drops of DMF to solubilize) and any solid residue removed by filtration through a 0.45 μm filter. Analytical HPLC with a linear gradient from 95% H2O (0.1% TFA) to 80% MeCN (0.1% TFA) over 15 min yielded a peak at 7.3 min. The material was purified using the semi-preparative HPLC at 10 mL/min on a Waters preparative machine (Waters Symmetry prep C18, 7 μm, 19×300 mm column, photodiode array detection) with a linear gradient from 90% H2O (0.1% TFA) to 50% MeCN (0.1% TFA) for 30 min. Semi-preparative HPLC fractions at 17 min were collected and lyophilized using Kinetics Flexi-Dry freeze-dryer. Compound 12 was identified with LC/MS spectroscopic analysis as illustrated in
This same procedure can be applied for various other amino acids (e.g. the 20 amino acids) as a means to develop new conjugates as bioluminescence probes that may vary in their kinetic activity (pharmacodynamics/pharmacokinetics) in vitro and in vivo
EXAMPLE 11 Synthesis of tyrosine-The tyrosine-d-aminoluciferin compound was synthesized according to the following reaction scheme
The deprotected and purified amino acid-conjugated 2-cyano-6-aminobenzothiazole (e.g. tyrosine conjugated; 0.048 mmoles, 0.024 g) was dissolved in anhydrous THF (0.5 mL), followed by dropwise addition of
The peptide-conjugated 2-cyano-6-aminoacid-aminobenzothiazole was synthesized according to the following reaction scheme.
The peptide sequence was designed to be recognized by a specific protease in the blood that is indicative of disease (e.g. prostate specific antigen (PSA), which correlates to prostate cancer; a wide variety of proteases are also present and these levels may change in response to biological insults conferred by infection, malignant growth and autoimmune responses16. The protected peptide sequence (0.033 mmoles, 0.067 g) was dissolved in 2.3 mL of anhydrous THF (+small amount of anhydrous DMF), sonicated, and stirred under N2 at 0° C. in the dark (material in suspension). N-methylmorpholine (2 eq, 0.07 mmol, 0.007 mL) and isobutyl chloroformate (1.3 eq, 0.04 mmol, 0.006 mL) were added drop-wise to the reaction at 0° C. and stirred for 30 min in the dark. Separately, purified amino acid-conjugated 2-cyano-6-aminobenzothiazole (e.g. tyrosine conjugated) 12 (0.03 mmol, 0.011 g) was dissolved in 0.2 mL of anhydrous THF and then added dropwise to the reaction flask over a period of 30 min. The reaction was stirred under N2 in the dark at 0° C. for 2 h, followed by a 72 h stir at room temperature in the dark. After 72 h, the anhydrous THF/DMF was removed in vacuo. The product 14 was dissolved in EtOAc and washed with saturated NaHCO3 to quench any remaining isobutyl chloroformate. The organic layer was collected and evaporated to dryness to yield a bright yellow to golden orange/yellow residue. The protected group on the peptide portion of the product was removed using 50% TFA in anhydrous CH2Cl2; stirred at RT in the dark for 3 h. Following deprotection, the product 14 was evaporated to dryness and placed on vacuum. Compound 14 was identified with LC/MS spectroscopic analysis as illustrated in
This same procedure can be applied for various other peptide sequences as a means to develop new conjugates as bioluminescence probes for detection of various other proteases that link to disease.
EXAMPLE 13 Synthesis of Peptide Conjugated tyrosine-The peptide-conjugated tyrosine-
The deprotected and purified peptide-conjugated 2-cyano-6-aminobenzothiazole-[amino acid] (0.006 mmoles, 0.008 g) was dissolved in anhydrous THF (0.01 mL), followed by drop-wise addition of
Chlorotrityl chloride (CTC) resin can be used to synthesize amino acid/peptide derivatives of
Reference is made to the reaction Scheme of
Carboxypeptidase Y was used but other carboxypeptidases are expected to be suitable depending on the nature of the amino acids attached to the
The scheme of
A blocking carboxypeptidase activity using a blocking agent (e.g. CONH2) on the C. terminus of the conjugates was investigated as outlined in
These determinations are expected to confirm the utility of
The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the compounds, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
It is to be understood that the disclosures are not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.
When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible subcombinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified can be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein can be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the disclosure and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the monofunctional benzothiazoles, compositions, methods steps, and systems set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.
It will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.
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Claims
1. A method to provide a peptide conjugated to 6-amino-6-deoxy-D-luciferin, the method comprising
- conjugating an amino acid with a 2-cyano-6-aminobenzothiazole to provide a 2-cyano-6-amino-aminoacid-benzothiazole, the conjugating performed to allow formation of a bond between the amino group of 6-amino-6-deoxy-D-luciferin and a carboxylic group of the amino acid;
- conjugating the peptide with the 2-cyano-6-amino-aminoacid-benzothiazole to provide a 2-cyano-6-amino-peptide-benzothiazole; and
- reacting the 2-cyano-6-amino-peptide-benzothiazole with a D-cysteine to provide a 6-amino-peptide-6-deoxy-D-luciferin,
- wherein the bond is a peptide bond, carbamate bond or a urea/thiourea bond.
2. The method of claim 1, wherein the bond is a peptide bond.
3. The method of claim 1, wherein the amino acid is an amino acid having a side chain hydrophobic, hydrophilic, acid or basic in nature.
4. The method of claim 3, wherein the amino acid is Alanine, Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan, Tyrosine and Valine.
5. The method of claim 3, wherein the amino acid is Serine, Threonine, Asparagine and Glutamine.
6. The method of claim 3, wherein the amino acid is Arginine, histidine and Lysine, Aspartic acid and Glutamic acid.
7. The method of claim 1, wherein the peptide comprises at least 5 amino acid residues.
8. The method of claim 7, wherein the peptide comprises at least 6 or 7 amino acid residues.
9. The method of claim 1, wherein the peptide further comprises a label.
10. A system to provide a peptide conjugated to a 6-amino-6-deoxy-D-luciferin, the system comprising
- one or more peptides, amino acids, 2-cyano-6-aminobenzothiazole, 2-cyano-6-amino-aminoacid-benzothiazoles and/or 2-cyano-6-amino-peptide-benzothiazoles for simultaneous combined or sequential use in the method to provide a peptide labeled with 6-amino-6-deoxy-D-luciferin according to claim 1.
11. An intermediate in the synthesis of an amino acid labeled with 6-amino-6-deoxy-D-luciferin, the intermediate being a 2-cyano-6-aminobenzothiazole of formula (III) wherein R1 is a single amino acid and R2 is a peptide attached to the remainder of the compound of formula (IV) through a peptide bond a carbamate bond or a urea/thiourea bond.
12. A labeled peptide obtainable by the method to provide a peptide conjugated to a 6-amino-6-deoxy-D-luciferin according to claim 1.
Type: Application
Filed: Feb 28, 2011
Publication Date: Sep 1, 2011
Inventors: Amy L. GRYSHUK (Livermore, CA), Julie Perkins (Sunnyvale, CA)
Application Number: 13/037,163
International Classification: C07K 7/06 (20060101); C07K 1/113 (20060101);
