WO2017079538A1 - Synthetic receptor compounds for detection of modified amino acids in peptides and proteins - Google Patents

Abstract

The present invention concerns compounds useful as probes for detecting modified amino acids in peptides or proteins.

Description

Synthetic Receptor Compounds for Detection of Modified

Amino Acids in Peptides and Proteins

Statement of Priority

[0001] This application claims the benefit of U.S. Provisional Application Serial No.

62/251,235, filed November 5, 2015, the entire contents of which are incorporated by reference herein.

Statement of Government Support

[0002] This invention was made with government support under Grant No. CHE- 1306977 awarded by the National Science Foundation. The government has certain rights in the invention.

Field of the Invention

[0003] The present invention concerns compounds useful as probes for detecting modified amino acids in peptides or proteins.

Background of the Invention

[0004] The post-translational modification (PTM) of histone proteins is involved in many important biological processes that regulate the dynamic structure of chromatin and alter the interactions of DNA with protein complexes. Currently, there is great interest in mapping out the epigenetic role of PTMs in development and disease, including methylated lysine (Lys) and arginine (Arg). Established methods to detect PTMs of proteins and peptides include the use of antibodies and mass spectrometry-based proteomics. Even though these methods have been used successfully to identify PTMs, they both face challenges and limitations. While antibodies have been widely used with Western blotting for the identification of PTMs, they are sequence specific and generally require the protein or peptide sequence of interest to be known (Blow, Nature 447:741 (2007); Kazanecki et al, J. Cell Biochem. 102:925 (2007)). This makes the detection of unknown PTMs difficult. Their sequence specificity also hinders the detection of the patterns of PTMs that make up the histone code. Additionally, antibodies suffer from high cost of production and irreproducibility of quality from batch to batch. Mass spectrometry is a another powerful tool that is well-suited for this kind of analysis as it allows for site mapping and quantitative detection of PTMs. MS-based proteomics efforts have been used to explore a number of PTMs including melhylalion of lysine and arginine, aeelylalion of lysine, and phosphorylation of serine and threonine. However, MS analysis can be complicated by analytical challenges including low natural abundance of PTMs, the presence of complex mixtures, and difficulties in analyzing large ionized proteins (Witze et al, Nat. Methods 4:79ft (2007); Sidoli et al., Proteomics 75:3419 (2012)). Generally, enrichment methods are required, and antibodies are currently the primary tool for enrichment.

[0005] A method for chemically labeling proteins containing PTMs would represent a new technology that would be of broad interest to those in the fields of chemical proteomics, chemical biology, and molecular biology. For example, selective fluorophore labeling would facilitate the quantitative detection of the target PTM-containing protein or peptide while installation of a biotin label would allow for the protein or peptide target to be directly removed from a complex mixture.

Summary of the Invention

[0006] A first aspect of the present invention is a compound useful for detecting a modified amino acid in a protein or peptide. Such a compound generally comprises: a cyclic oligomer synthetic receptor {e.g., a calixarene, a disulfanyl-cyclophane, etc.) that binds to the modified amino acid; optionally, but in some embodiments preferably, an electrophilic cross-linking group covalently coupled to said cyclic oligomer, and optionally, but in some embodiments preferably, a detectable group covalently coupled to either the cyclic oligomer or said cross- linking group.

[0007] A second embodiment of the invention is a method of detecting a modified amino acid in a protein or peptide, comprising: (a) contacting a protein or peptide to a compound of the invention under conditions in which said cyclic oligomer binds to said modified amino acid, and then (b) detecting the (transient or covalent) coupling of the detectable group to the protein or peptide to thereby detect a modified amino acid in that protein or peptide.

[0008] Particular features and aspects of the invention are shown in the drawings herein and the specification set forth below.

Brief Description of the Drawings

[0009] Figure 1 A: Schematic illustration of labeling methods using synthetic receptors as affinity probes (case 1). The circle represents a modified amino acid; X represents an electrophilic cross-linking group; nuc represents a nucleophilic amino acid; and the wavy line represents a linking group. [0010] Figure IB: Schematic illustration of labeling methods using synthetic receptors as affinity probes (case 2a). The circle represents a modified amino acid; X represents an electrophilic cross-linking group; nuc represents a nucleophilic amino acid; and the wavy line represents a linking group.

[0011] Figure 1C: Schematic illustration of labeling methods using synthetic receptors as affinity probes (case 2b). The circle represents a modified amino acid; X represents an electrophilic cross-linking group; nuc represents a nucleophilic amino acid; and the wavy line represents a linking group.

[0012] Figure ID: Schematic illustration of labeling methods using synthetic receptors as affinity probes (case 3). Y represents any amino acid. The circle again represents a modified amino acid, nuc again represents a nucleophilic amino acid, and the wavy line represents a linking group.

[0013] Figure IE: Schematic illustration of labeling methods using synthetic receptors as affinity probes (case 4). Cat represents a catalytic group, RO₂C represents a reactive ester, and Lys represents lysine. The circle again represents a modified amino acid, and the wavy line again represents a linking group.

[0014] Figure 2: Turn-on fluorescence affinity labeling of a histone Kme3 peptide using a CX4-ONBD probe (example of case 1).

[0015] Figure 3: Analysis of distance dependence in the labeling of histone peptides by probe 5. Fluorescence intensity is relative to probe 5 alone. Conditions: 40 uM 5 and 20 uM peptide in 25 mM bicine, 137 mM NaCl, 2.7 mM KC1, 1 mM MgCl₂, pH 8.02, 24 h.

[0016] Figure 4: Turn-on Fluorescence Labeling of K9me3Kl 4 and K9K14 Peptides with Probe 5.

[0017] Figure 5: Turn-on fluorescence HDAC1 activity assay. Fluorescence monitoring of the deacetylation of histone H3 peptides K4me3 K9ac and K27ac K36me3 with

probe 5 after reaction with HDAC1 for 1 h at 37 °C. Enzymatic conditions are described in Example 1. Fluorescence response is shown after labeling for 24 h. The fluorescence intensity for each reaction was normalized to the reaction containing HDAC1 and 5.

[0018] Figure 6: Turn-on fluorescence labeling SAHA control experiment. Labeling of 40 uM 5 alone or in the presence of 5 uM SAHA in 25 mM Bicine buffer, 137 mM NaCl, 2.7 mM KC1, 1 mM MgCl₂ (pH 8.02). Each data point represents an average of three runs. Error bars are standard deviations.

[0019] Figure 7: Turn-on fluorescence HDAC1 activity assay: fluorescence increase of deacetylated K4me3 K9ac after labeling for 6, 12, or 24 hours. Fold increase in fluorescence intensity was normalized to the negative control containing HDAC1 and 5 alone after 6, 12, or 24 hours. Each data point represents an average of three runs. Error bars are standard deviations.

[0020] Figure 8: Fluorescence monitoring of the deacetylation of histone H3 peptide K4me3K9ac with probe 5 after reaction with HDAC1 or HDAC3/NCOR1 in the absence or presence of S AHA.

[0021] Figure 9: Dose-response curve of the inhibition of the HDAC3/NCOR1 catalyzed deacetylation of K4me3K9ac by SAHA.

[0022] Figure 10: Photo-affinity labeling of labeling of histone H3 peptide (example of case 3).

[0023] Figure 11 : Probe 2 before (high peak) and after (low peak) irradiation with U VA light.

Detailed Description of Illustrative Embodiments

[0024] The present invention is now described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art

[0025] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and claims and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well- known functions or constructions may not be described in detail for brevity and/or clarity.

A. Definitions.

[0026] "Modified amino acid" as used herein refers to a methylated lysine or methylated arginine.

[0027] "Nucleophilic amino acid" as used herein includes lysine, methyllysine, glutamic acid, aspartic acid, tyrosine, histidine, and cysteine. [0028] "Reactive group" and "electrophilic cross-linking group" as used herein includes any suitable group reactive with a nucleophilic group, examples of which reactive groups v include, but are not limited to, aryl ethers, esters, alky] sulfonates, aldehydes, ketones, alpha,beta unsaturated carbonyls, disulfides, alkyl halides, ketenes, isocyanates,

isothiocyanates, acyl imidazoles, alkynes, alkenes, azides, etc.

[0029] "Nucleophilic group" as used herein includes any suitable group reactive with a reactive group as described above, examples of which nucleophilic groups include, but are not limited to, amines, thiols, alcohols, carboxylates, oxamines, substituted hydrazines, hydrazides, alkynes, azides, etc.

[0030] "Catalytic group", or "catalytic detection-inducing group" as used herein includes any suitable group that catalyzes the reaction between a reactive group and a nucleophilic group as described above, examples of which catalytic groups include, but are not limited to, aniline catalysts (e.g., aniline, m-phenylenedi amine, etc.), phosphine catalysts (e.g., triphenylphosphine and substituted triphenylphosphine), pyrrolidine catalysts (e.g., proline and proline derivatives), chelated transition metal catalysts, such as rhodium, palladium, ruthenium, rhenium, and iron catalysts, (e.g., dirhodium tetracarboxylate), etc.

[0031] "Linking group" or "linker" as used may be any suitable linking group, examples of which include but are not limited to polyethylene glycol linkers, alkyl linkers, polypeptides linkers, triazole linkers, etc., including combinations thereof. Length and flexibility of the linking group may be selected in accordance with the example embodiments of Figures 1A- 1E, where the modified amino acid is typically within 1, or 2 or 5 to 10, 20 or 30 amino acid residues on the protein or peptide backbone of the nucleophilic amino acid, lysine, or amino acid "Y" to which the detectable group is joined.

[0032] "Water soluble group" as used herein may be any suitable water soluble group, examples of which include, but are not limited to, polar groups such as a hydroxy, alkoxy, carboxy or carboxylic acid, nitro, cyano, amino (primary, secondary and tertiary), amido, ureido, sulfonamido, sulfinyl, sulfhydryl, silyl, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, C -amido, N-amido, sulfonyl, phosphono, morpholino, piperazinyl, tetrazolo, etc.; ionic groups such as a carboxylate, sulfonate, phosphate, amine, N-oxide, ammonium, uronic acid, sulfonic acid, amine, and moieties such as guanidinium, phosphoric acid, phosphonic acid, phosphatidyl choline, phosphonium, borate, sulfate, etc.; amino acids (particularly acidic amino acids such as aspartic acid and glutamic acid); water-soluble oligomers and polymers such as polyethylene oxide, amino acids peptides (particularly those containing at least one acidic amino acid), etc. [0033] "Detectable group," "label," or "tag" as used herein includes, but is not limited to, radioactive groups (e.g., ³⁵S, ^l251, ³²P, ³H, ¹⁴C, ^,131I), enzymatic and catalytic groups (e.g., horseradish peroxidase, alkaline phosphatase), gold beads, quantum dots (e.g., cadmium selenide), dyes (e.g., aniline, rhodamine and phycoerythrin dyes, etc.), luminescent groups, specific binding ligands (or a member of a specific binding pair; e.g., biotin, avidin, digoxin, antigen or antibody, etc.) and/or fluorescent groups (e.g., fluorescein, fluorescent proteins including, but not limited to, a green fluorescent protein or one of its many modified forms), nucleic acid segments in accordance with known techniques, energy absorbing and energy emitting agents, etc.

[0034] "Alkyl" as used herein refers to a straight- or branched-chain alkyl group having from 1 to 4, 8 or 12 carbon atoms in the chain. Example alkyl groups include methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like. Alkyl groups may be unsubstituted or substituted unless specifically specified otherwise, and when substituted may be substituted (e.g., from 1 or 2 to 3 or 4 times) with any suitable substituent, including but not limited to halo, mercapto, azido, cyano, formyl, carboxylic acid, hydroxyl, nitro, etc.

[0035] "Aryl" as used herein refers to a monocyclic, or fused or spiro polycyclic, aromatic carbo cycle (ring structure having ring atoms that are all carbon) having from 3 to 12 ring atoms per ring. Illustrative examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracyl, etc. Aryl groups may be unsubstituted or substituted unless specifically specified otherwise, and when substituted may be substituted (e.g., from 1 or 2 to 3 or 4 times) with water soluble groups, and/or with any other suitable substituent, such as those substituents given in connection with "alkyl" above.

[0036] "Heteroaryl" as used herein refers to a monocyclic, or fused or spiro bicyclic or polycyclic, aromatic heterocycle (ring structure having ring atoms selected from carbon atoms as well as nitrogen, oxygen, and sulfur heteroatoms) having from 3 to 12 ring atoms per ring. Illustrative examples of heteroaryl groups include, but are not limited to, pyridyl, pyrazyl, pyrimidyl, pyridazyl, quinolyl, indolyl, etc. Heteroaryl groups may be unsubstituted or substituted unless specifically specified otherwise, and when substituted may be

substituted (e.g., from 1 or 2 to 3 or 4 times) with water soluble groups, and/or with any other suitable substituent, such as those substituents given in connection with "alkyl" above. B. Overview.

[0037] Illustrative embodiments of our method transforms synthetic receptors for post- translational modifications (PTMs; e.g., methylated Lys and Arg) (Ingerman et al., Chem. Commun. 46:1839 (2010); James et al. J. Am. Chem. Soc. 755:6450 (2013); Pinkin et al.. Org. Biomol. Chem.12:7059 (2014); Daze et al, Org. Lett. 74:1512 (2012)) into chemical probes capable of selective protein labeling. These probes or probe systems generally contain three characteristic components: a synthetic receptor (grey), a reactive group (X), and a tag unit or label (Figs 1A-1E). The labeling scheme is based on proximity-promoted affinity labeling, which has been used primarily for labeling proteins using high affinity ligands. However, a novel aspect of our approach is the use of synthetic receptors for post- translational modifications. In this labeling scheme, receptor binding places the electrophile (or crosslinking group) on the probe in close proximity to a nucleophile (or crosslinking target) on the target protein or peptide allowing the chemical reaction to take place.

[0038] In case 1 (Fig. 1A), the labeling is trace less and covalently attaches a tag unit but not the receptor to the protein.

[0039] For cases 2a and 2b (Figs. 1B-C), both the tag unit and the receptor are attached to the protein. The difference between these cases is the location of the tag unit: attached at the electrophile (case 2a; Fig. IB) or attached at the receptor (case 2b; Fig. 1C).

[0040] Case 3 (Fig. ID) represents a photo-affinity labeling method where UV excitation covalently crosslinks the probe to the protein.

[0041] Finally, case 4 (Fig. IE), represents a three-reagent system, where a catalytic group is coupled to the synthetic receptor as the first reagent. A second reagent comprises a compound of Formula V: RO₂C-L⁴-X, where X is a reactive group, L⁴ is a linking group or covalent bond, and RO₂C is a reactive ester that binds to all lysines in the protein or peptide. A third reagent comprises a compound of Formula VI: B-L⁵-Nuc, where B is the detectable group, L⁵ is a linking group, and Nuc is a nucleophilic group. When the three are contacted to the protein or peptide together (simultaneously or in sequence), the catalytic group on the compound of Formula IV catalyzes the reaction of the reactive group on the compound of Formula V (now coupled to all lysines) with the nucleophilic group of the compound of Formula VI, thereby coupling the detectable group B only to lysines adjacent a modified amino acid. In this case, the catalyst can also be considered a "catalytic detection-inducing group," and the second and third reagents can be considered the detection system. C. Probe compounds.

[0042] Compounds useful for detecting a modified amino acid in a protein or peptide generally comprise: (a) a cyclic oligomer synthetic receptor that binds to the modified amino acid, (b) optionally, an electrophilic cross-linking group covalently coupled to the cyclic oligomer, and (c) optionally a detectable group covalently coupled to either the cyclic oligomer or the cross-linking group. Particular examples include compounds of Formula I-

IV:

where:

A is a cyclic oligomer synthetic receptor;

X is an electrophilic cross-linking group;

X* is a photoactivatable cross-linking group (e.g., a benzophenone or diazirine cross- linking group);

X" is a fluorescent electrophilic cross-linking group or a catalytic detection-inducing group (e.g., aniline catalysts, triphenylphosphine catalysts, etc.);

B is a detectable group; and

L² and L³ are each an independently selected linking group or a covalent bond.

[0043] Particular examples of the foregoing include compounds of Formulas I '-IV:

where:

each R¹ is an independently selected aryl, alkylaryl, alkylarylalkyl, heteroaryl, alkylheteroaryl, or alkylheteroarylalkyl;

each L¹ is an independently an alkyl, alkenyl, disulfide, thioether, ether, ester, amide, dithioacetal, or combination thereof;

n is from 2 or 3 to 5, 10 or 20;

and remaining substituents are as given above.

[0044] In some embodiments of the foregoing, the electrophilic cross-linking group is an aryl ether, ester, alkyl sulfonate, aldehyde, ketone, alpha,beta unsaturated carbonyl, disulfide, alkyl halide, ketene, isocyanate, isothiocyanate, acyl imidazble, alkyne, alkene, or azide.

[0045] In some embodiments of the foregoing, L² and L³ are independently selected alkyl, peptide, or polyalkylene oxide (e.g., poly(ethylene glycol)).

[0046] In some embodiments of the foregoing, each R¹ is independently selected from phenyl, naphthyl, and 9, 10-dihydro-9, 10-ethenoanthracyl (each of which may be

unsubstituted or optionally substituted, such as with 1, 2, or 3 water soluble groups).

[0047] In some embodiments of the foregoing, each L¹ is independently selected from— S- S-, -CHR-, -OCHR-, or -SCHR-, where R is H, alkyl, or aryl.

[0048] In some embodiments of the foregoing, the cyclic oligomer is a disulfanyl- cyclophane such as:

[0050] In the foregoing illustrative disulfanyl-cyclophanes and calixarenes, each R may independently be a covalent bond to X, X', X", L² and/or L³, or a water soluble group (typically, any X, X' X" or L³ group will be attached via a nitrogen to the CO group shown, giving an amide linkage).

[0051] Specific examples of compounds of the invention include, but are not limited to:

and salts thereof.

[0052] Probe compounds of the invention can be made in accordance with known techniques or variations thereof that will be apparent to those skilled in the art. For examples, synthetic receptors can be made as described in M. Waters and L. James, Synthetic Receptors for Identification of Protein Posttranslation Modifications, US Patent Application Publication No. US 2012/0190586 (published July 26, 2012). Such receptors can be coupled to detectable groups and/or cross-linking groups directly or via intervening linking groups by any suitable technique, typically by substitution reactions such as nucleophilic aromatic substitution reactions, amide bond formation, "click" reactions (e.g., [3+2] cycloaddition reactions such as the Huisgen 1 ,3 -dipolar cycloaddition, including the Cu(I) catalyzed embodiment thereof), etc.). See, e.g., Pinkin et al, Org. Biomol. Chem. 2015, DOI:

10.1039/c5ob01649e D. Methods of use.

[0053] As noted above, a method of detecting a modified amino acid in a protein or peptide may be carried out by:

(a) contacting a protein or peptide to a probe compound as described above under conditions in which the cyclic oligomer binds to the modified amino acid, and then

(b) detecting the coupling of the detectable group to the protein or peptide to thereby detect a modified amino acid in the protein or peptide. Detection may be carried out in accordance with known techniques, depending upon the particular detectable group and detection system employed.

[0054] In some embodiments, the contacting step is carried out under conditions in which a nucleophilic amino acid on the protein or peptide adjacent the modified amino acid (e.g., within 2, 5, 10 or 20 amino acid residues thereof) reacts with the crosslinking group; and the detecting step is carried out by detecting the coupling of the detectable group to the protein or peptide through the nucleophilic amino acid, the presence of the detectable group on the protein or peptide indicating the presence of a modified amino acid in the protein or peptide.

[0055] In embodiments where the probe compound is of Formula IV (A-L²-X"), wherein A is the cyclic oligomer synthetic receptor, X" is a catalytic detection-inducing group, and L² is a linking group or a covalent bond; the method further comprising contacting a second and third reagent to the protein or peptide. The second reagent comprises a compound of Formula V: RO₂C-L⁴-Z, where RO₂C- is a reactive ester that covalently binds to all lysines in the protein or peptide, L⁴ is a linking group or covalent bond, and Z is a reactive group. The third reagent comprises a compound of Formula VI: B-L⁵-Nuc, where B is a detectable group, L⁵ is a linking group or covalent bond, and Nuc is a nucleophilic group that cross- reacts with reactive group Z in a reaction catalyzed by catalytic detection-inducing group X".

[0056] The foregoing labeling methods provide a means for selective covalent modification of a protein or peptide containing a modified amino acid residue (i.e., methylated lysine and methylated arginine). These probes can be used in any application for detection of a particular post-translational modification is desired. In some embodiments:

[0057] Probes of this nature can supplement or replace the use of antibodies in various assay formats including Western blots, peptide microarrays, immunoprecipitation, protein and peptide enrichment for proteomics applications, and other assays.

[0058] The synthesis and function of these probes is highly reproducible given that they are discrete chemical compounds, unlike polyclonal antibodies. [0059] These probes have the potential to be used in intracellular labeling and imaging applications in live cells, whereas antibodies are limited to labeling cell surface antigens.

[0060] These probes have the potential to be used in proteomics applications for sequence- independent, global characterization of post-translational modifications.

[0061] Probes of this nature can be used for sample enrichment of PTMs in mass spectrometry proteomics.

[0062] Elements, aspects and illustrative embodiments of the present invention are explained in greater detail in the following non-limiting Examples.

EXAMPLE 1

[0063] A synthetic receptor for the PTM trimethyl lysine (Kme3) was transformed into a chemical probe capable of selective turn-on fluorescence labeling of histone peptides containing Kme3. In the labeling scheme, receptor binding to Kme3 places the electrophile in close proximity to a nucleophilic lysine on the target peptide, facilitating covalent labeling (Fig.2). The utility of this labeling strategy is demonstrated for the development of a turn-on fluorescence assay for screening inhibitors of histone deacetylase activity. This work represents the first example of synthetic-receptor directed affinity labeling and provides impetus for the use of synthetic receptors as agents for site-selective chemical modification of proteins mediated by PTMs.

[0064] Design and Synthesis of a Probe for Labeling of Kme?: The first generation of the probe was designed to label histone peptides containing both Kme3 and a pendant unmethylated lysine. The probe features a trisulfonato calix [4] arene (CX4) synthetic receptor for Kme3 conjugated via a short flexible linker to a fluorogenic nitrobenzoxadiazole (NBD) group. The CX4-ONBD probe was prepared by assembling three separate

components: propargyl ONBD compound 1, CX4-Ar receptor 2, and amino- azide

linker 3 (Scheme 1). First, the alkyne functionalized NBD compound 1 was prepared by treating NBD chloride with propargyl alcohol in the presence of tertiary amine base. The synthetic receptor 2 and the flexible amino-azide linker 3 were prepared with minor alterations to published procedure. After preparing each of the three components,

trisulfonated CX4-Ar receptor 2 was functionalized with linker 3 using carbodiimide coupling to produce azdde-functionalized 4. Finally, copper catalyzed azide— alkyne cycloaddition (CuAAC) was used to conjugate alkyne 1 to azide 4, generating the CX4- ONBD probe 5 which was purified by reversed-phase HPLC.

[0065] Characterization of Binding by Isothermal Titration Calorimetry: The binding affinity of the CX4-ONBD probe 5 to a histone 3 (H3) peptide containing Kme3 (Table 1) was measured using isothermal titration calorimetry (1TC). Under phosphate buffer conditions at pH 7.4 with no added salt (Table 2, entry 1), the CX4-ONBD probe had a of 4.8 uM for K9me3R14. In 10 mM borate buffer with 100 roM NaCl (pH 8.6),

receptor 4 exhibited about 20-fold selectivity for K9me3K14 over K9K14 with dissociation constants of 18.3 uM and 390 μΜ, respectively (Table 2, entries 2 and 3). This suggests that the affinity labeling is not achieving the maximum selectivity possible. Several factors likely contribute to this, including the flexibility of the linker that reduces the degree of

preorganization as well as the fact that the unmethylated peptide has twice the number of reactive lysines, which reduces the selectivity.

Table 1. Peptides Used for ITC, Fluorescence Labeling, and Deacetylase Activity Screening

Table 2. Binding and Thermodynamic Data from ITC Experiments

[0066] Optimization of Receptor-Mediated Labeling: To evaluate the degree of receptor-mediated labeling, probe 5 was incubated with either K9me3K14 or K9K14.

Initially, concentrations well above the K_d were chosen for both probe and peptide to ensure complete host-guest complexation. Each peptide was incubated with probe 5 in 10 mM potassium phosphate buffer (pH 7.4) at room temperature for 24 h (Table 3), and the labeling reactions were analyzed by HPLC. Reaction yields after 24 h revealed slow reactivity of the CX4-ONBD probe toward the representative hi stone peptides with modest 2: 1 selective labeling for K9me3K14 over K9K14 (29% and 12% labeling yield, respectively). Increasing the pH of the buffer used in the labeling reaction to 8.6 resulted in a marked rate acceleration. Unfortunately, this rate acceleration was accompanied by a complete loss in labeling selectivity for K9me3K14 over the K9K14 peptide (75% and 70% labeling yield,

respectively).

Table 3. Optimization of Labeling Reaction Conditions of CX₄-ONBD Probe 5 with H3 Peptides

[0067] To circumvent unselective labeling, conditions with different concentrations of NaCl were screened. Varying NaCl concentration from SO mM to 150 mM revealed two general trends: the overall labeling yield decreases slightly at higher concentrations of salt, while selectivity for K9me3K14 over K9K14 improves with increasing salt Under conditions containing 40 uM probe and 20 uM peptide, the labeling selectivity improved to nearly 6-fold for K9me3K14 over K9K14.

[0068] Distance-Dependence of Labeling Rate: To probe the distance-dependence of labeling, we also investigated the reaction of probe 5 with biologically relevant histone peptides with varying numbers of amino acids separating the Kme3 binding site and the pendant reactive amine (Table 4). Reactions were performed using 40 μΜ 5and 20 uM peptide in 25 mM bicine buffer (137 mM NaCl, 2.7 mM KC1, 1 mM MgCl₂, pH 8.02), and fluorescence measurements were taken after 24 h. Probe 5 reacted with K4me3K9 to the greatest extent, suggesting a preference for a spacing of four amino acids between Kme3 and the reactive amine (Fig. 3). In contrast, K36me3K37 and K27K36me3, the two peptides with the shortest and longest spacing, respectively, showed considerably slower labeling.

Interestingly, the peptide containing K4me3 and a free N-terminal amine (with a spacing of 2 amino acids) was the second most reactive in the series of peptides that were screened. This may be explained in part by the increased acidity of the alpha amino group relative to the ε amine of the lysine side chain, making it a better nucleophile at pH 8. Unexpectedly, probe 5 reacted with K23K27me3 (three amino acid spacing) the slowest, indicating that there may be subtleties in sequence and binding conformation that affect labeling rate in addition to amino acid spacing. While these results show some dependence of the rate of reaction on distance, all sequences tested gave a turn-on fluorescence response which demonstrates the generality of 5 for labeling relevant histone H3 peptides.

Table 4. Peptides Used for Distance Dependence Labeling Experiments

[0069] Receptor-Mediated Turn-On Fluorescence Labeling of Kme3 Peptides: The turn-on fluorescence labeling of histone peptides by 5 was explored by monitoring the fluorescence emission at 520 nm (excitation at 485 nm). Using optimized conditions of 40 μΜ probe 5 and 20 μΜ peptide in 10 mM phosphate buffer (100 mM NaCl, pH 8.6), measurements were taken every hour, and reactions were allowed to proceed under ambient temperature for 24 h (Fig. 4). A pronounced turn-on fluorescence response was observed for labeling of K9me3K14 with 5 (diamonds). On the other hand, the turn-on response for labeling with the K9K14 peptide (triangles) was significantly lower, even though the peptide contains two reactive lysine residues. Trials with probe 5 alone appear to show only a slight increase in fluorescence signal over time, presumably due to slow hydrolysis of the O-NBD group. [0070] Receptor-Mediated Turn-On Fluorescence Assay for HDAC Activity:

Reactions between acetylated Kme3 peptides (200 μΜ) and HDAC1

were carried out in 25 mM bicine buffer, 137 mM NaCl, 2.7 mM KC1, 1 mM MgCl₂ atpH 8.02 and 37 °C. After 1 h, reactions were diluted with the bicine reaction buffer to quench the reaction before addition to a 96-well plate containing 5. Fluorescence labeling was performed with a final concentration of 40 μΜ 5 and 20 uM peptide at ambient temperature for 24 h. Samples containing only 5 and HDAC1 were used as negative controls and showed a slow increase in fluorescence over time, likely due to the presence of the primary amine Tris in the stock buffer of the commercial enzyme. H3 peptides K4me3K9 and K27K36me3 were used as positive controls for detennining the maximum extent of fluorescence labeling possible for peptides deacetylated by HDAC 1.

[0071] Enzymatic assays with both the K4me3K9ac peptide and the K27acK36me3 peptide provide measurable turn-on fluorescence above background, and comparable fluorescence to the positive control (Fig. 5). Indeed, the fact that the K27acK36me3 peptide achieves the full fluorescence intensity of the positive control, whereas the K4me3K9ac peptide does not regain full fluorescence suggests mat K27acK36me3 is a more active substrate for HDAC1. Thus, even though different spacing results in different maximum fluorescence at a given time point, the assay can still be used to measure substrate reactivity relative to positive controls. Moreover, analysis of the fluorescence output after 6 h exhibits identical results as the data at 24 h, despite low conversion, indicating that shorter assay times are feasible (Fig. 7).

[0072] Additionally, the CX4-ONBD showed no reactivity toward the hydroxamate group of the SAHA inhibitor, which further displays the probe's selectivity toward amine nucleophiles (Fig. 6).

[0073) Inhibition Assays and IC50 Determination with Receptor-Mediated Turn-On Fluorescence Assay: After establishing selective turn-on fluorescence labeling of a Kme3 histone peptide, the labeling methodology was applied toward the development of a chemical assay for histone deacetylase (HDAC) activity. The ability to measure enzyme inhibition with probe 5 was deterrnined using two different HDACs, HDAC1 and HDAC3/NCOR1 (Fig. 8). Enzymatic reactions with K4me3K9ac were run in the absence or presence of SAHA. As can be seen in Fig. 4, in both cases, a significant difference in turn-on

fluorescence was observed, indicating that this probe can be used to qualitatively observe enzyme inhibition, and that the assay can be used for multiple HDACs. [0074] To further validate the utility of our turn-on fluorescence assay for HDAC inhibitor screening, a dose-response experiment for the inhibition of HDAC3/NCOR1 by SAHA (Fig. 9) was carried out. For these experiments, inhibitor was serially diluted into 50 mM bicine buffer (137 mM NaCl, 2.7 mM KC1, 1 mM MgCl₂, 0.5 mg/mL BSA, pH 8.0), and 11.2 nM HDAC3/NCOR1 was allowed to react with 100 μΜ K4me3K9ac for 4 h at 37 °C. After quenching each reaction by the addition of 10 uM SAHA, samples were added to a 96 well plate containing probe 5, giving a final concentration of 40 uM 5, 75 uM peptide, and 8.4 nM HDAC3/NCOR1. Fluorescence measurements were made after 24 h and were used to generate a dose-response curve. An IC50 value of 1.3 ± 0.3 uM was determined for the inhibition of HDAC3/NCOR1 by SAHA.

EXAMPLE 2

[0075] This example describes a probe containing a synthetic receptor linked to a UV- excitable benzophenone group (Fig. 10).

[0076] Benzophenone can crosslink any C-H bond that is in close enough proximity. The structure and synthesis of Probe 2 are shown in Scheme 2.

Scheme 2: Synthesis of Probe 2

[0077] The probe uses a disulfide-linked macrocycle as a synthetic receptor for trimethyl lysine and benzophenone as a photo-affinity group. After incubating Probe 2 in the presence of a histone H3 peptide containing trimethyl lysine and irradiating with UVA light for 1 hour, there was a decrease in the concentration of un-modified probe (Fig. 11). This indicates successful crosslinking.

EXAMPLE 3

[0078] This example shows a probe compound in which the detectable group coupled to the synthetic receptor is a catalytic group (Case 4 in Figure IE above).

EXPERIMENTAL

[0079] Synthesis of 1: 4-chloro-nitrobenzo[c][l,2,5]oxadiazole (0.100 g, 0.50 mmol) was dissolved in 5 mL CH₂CI₂ under a nitrogen atmosphere. To this solution was added a mixture of propargyl alcohol and DIPEA in 5 mL CH₂CI₂, and stirring was continued for 72 h. The solvent was removed under reduced pressure, and the crude product was purified by column chromatography (EtOAc/Hexanes 1:1) to give the product as a yellow-brown solid (0.053 g, 48%). 1HNMR (CDCI₃, 400 MHz): 8.558 (d, 1H, C-H), 6.880 (d, 1H, C-H), 5.129 (d, 2H, C-H), 2.720 (t, 1H, C-H).

[0080] Synthesis of 2: Synthesized according to published procedure (Daze et al. Org. Lett. 74:1512 (2012)) but with a modified purification procedure. After completion of the reaction, solvent was removed under reduced pressure. The residue was taken up in

MeOH/H₂O (1:1), and palladium was removed by QuadraPure® TU resin. The resin (0.5 g) was added to the solution and heated to reflux under nitrogen for 24 h. The mixture was filtered, and the solvent was removed under reduced pressure. The crude product was used without further purification.

[0081] Synthesis of 3: Prepared according to published procedure (Bonger et al, Bioorg. Med Chem. 75:4841 (2007)).

[0082] Synthesis of 4: EDC-HC1 (0.087 g, 0.45 mmol) and N-hydroxysuccinimide (0.050 g, 0.43 mmol) were added to a solution of 2 (0.176 g, 0.22 mmol) in 5 mL DMF/H₂O (1:1) under a nitrogen atmosphere, and the mixture was stirred for 1 h. 3 (0.100 g, 0.46 mmol) was added, and the solution was allowed to stir at room temperature overnight The solvent was removed under reduced pressure, and the resulting residue was used in the next step without further purification. MS (calculated): 983.18 [M-H]. HUMS (observed, ESI-) 983.17311 [M-H].

[0083] Synthesis of 5: The crude residue containing 4 (0.037 g, 0.038 mmol) and 1 (0.049 g, 0.22 mmol) were dissolved in 5 mL DMF/100 mM Sodium acetate, pH 5.5 (4:1). Copper iodide (0.001 g, 0.005 mmol) was added, and the solution was allowed to stir at room temperature for 48 h. The solvent was removed under reduced pressure, and the resulting residue was purified by semi-preparative reversed-phase HPLC (solvent A: 10 mM NH4OAC in 9:1 H₂O:CH₃CN; solvent B: 10 mM NH₄OAC in 9:1 CH₃CN:H₂O) using the gradient: 0- 100% B from 0-60 min with a flow rate of 4.0 mL/min. MS (calculated) 1202.21 [M-H]. HRMS (observed, ESI-) 1202.20580 [M-H].

[0084] Synthesis of 6: Prepared according to published procedure (Corbett a/.,

Chemistry 14:2153 (2008)).

[0085] Synthesis of 7: Triphenylmethanol (2.91 g, 11.18 mmol) and 6 (1.00 g, 5.37 mmol) were dissolved in 25 mL diethyl ether/THF (3:2) under an atmosphere of nitrogen. Once dissolved, boron trifluoride diethyl etherate (3 mL, 24.31 mmol) was added dropwise, and the solution was stirred for 10 min. The solvent was then removed under reduced pressure, and the residue was taken up in 100 mL diethyl ether. The organic layer was washed with 1 N NaOH (1 x 200 mL), H₂O (1 x 200 mL), and brine (1 x 200 mL). The organic fraction was dried over MgSO₄, and the solvent was removed under reduced pressure to give the product as a white solid (3.3 g, 92%).

[0086] Synthesis of 8: Dicyclohexylcarbodiimide (0.48 g, 2.32 mmol), N- hydroxysuccinimide (0.27 g, 2.35 mmol), and 7 (1.5 g, 2.24 mmol) were dissolved in 15 mL , CH₂CI2 under an atmosphere of nitrogen, and the solution was allowed to stir overnight. The reaction mixture was filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (100% CH₂CI2) to afford the product as a white solid (1.1 g, 65%).

[0087] Synthesis of 9: 4-Benzoyl L-phenylalanine (0.25 g, 0.93 mmol) and 8 (0.35 g, 0.46 mmol) were dissolved in 10 mL DMF under a nitrogen atmosphere. To this solution was added DIPEA (0.48 mL, 2.76 mmol), and the solution was allowed to stir overnight at room temperature. The reaction mixture was diluted with 100 mL H₂O, and the crude product was extracted into CH₂Cl₂ (3 x 50 mL). The organic layer was washed with H₂O (4 x 100 mL) and brine (1 x 100 mL). The organic fraction was dried over MgSO₄, and the solvent was removed under reduced pressure to give a white solid (0.4 g, 95%) which was used without further purification.

[0088] Synthesis of 10: 9 was added to a flask under a nitrogen atmosphere, and a solution of trifluoroacetic acid (5.25 mL), triisopropylsilane (0.30 mL, 1.46 mmol), H₂O (0.30 mL), and CH₂CI2 (0.30 mL) was added. The solution was stirred for 3 h, and the solvent was removed by bubbling through nitrogen. The resulting residue was triturated in hexanes (10 x 10 mL) and dried under nitrogen to give the product as a light yellow solid (0.19 g, quantitative).

[0089] Monomer A: Prepared according to published procedure (Corbett et td., Chemistry 14:2153 (2008)).

[0090] Synthesis of A₂B-BPA: A preparative scale dynamic combinatorial library was set up using butyl trimethylammonium iodide as a guest to template A₂B-BPA formation.

Monomer A (8.9 mg, 0.025 mmol), monomer 10 (10.9 mg, 0.025 mmol), and butyl trimethylammonium iodide (24.0 mg, 0.099 mmol) were dissolved in 10 mL 50 mM sodium borate buffer (pH 8.5). The library was allowed to reach equilibration after 3 days, and A₂B- BPA was purified by semi-preparative reversed phase HPLC (solvent A: 10 mM NH₄OAc in H₂O; solvent B: 10 mM NH₄OAc in 9:1 CH₃CN:H₂O) using the gradient: 0-100% B from 0- 60 min with a flow rate of 4.0 mL/min.

[0091] Peptide Synthesis. Peptides were synthesized on a 0.6 mmol scale by hand using Fmoc protected amino acids (amino acids with reactive functional groups contained an appropriate protecting group). Coupling reagents were HOBt/HBTU in DMF. All peptides were acylated at the N-terminus with a solution of 5% acetic anhydride and 6% 2,6-lutidine in DMF. Cleavage was performed using a cocktail of 95% TF A/2.5%

triisopropylsilane/2.5% H₂O for 3 hours. Peptides were purified by semipreparative reverse- phase HPLC on a C18 column at a flow rate of 4 mL/min. Peptides were purified with a linear gradient of A and B (A: 95% H₂O/5% C¾CN with 0.1% TFA, B: 95% CH₃CN/5% H₂O with 0.1 % TFA).

(0092] Methylated peptides were synthesized with either 2 equivalents of Fmoc- Lys(Boc)(Me)-OH purchased from BaChem or Fmoc-Lys(Me)2-OH HC1 purchased from Anaspec and coupled for 5 hours. The trimethyllysine containing peptides were synthesized by reacting corresponding dimethylated peptides (0.06 mmol scale) prior to cleavage from the resin with MTBD (10 equiv.) and methyl iodide (10 equiv.) in DMF (5 mL) for 5 hours with bubbling N₂ in a peptide flask. After washing the resin with DMF and CH₂C1₂ and drying, the peptide was cleaved as normal.

[0093] Fluorescence Labeling. Fluorescence labeling experiments were performed in black 96 well optical bottom plates (non-treated, non-sterile, polystyrene) using a POLARstar Omega (BMG Lantech, Inc.) plate reader. A fluorescence optic with a 485 nm excitation filter and a 520 nm emission filter was used. Labeling reactions were carried out at ambient temperature in 10 mM sodium phosphate buffer (pH 7.40), and Titer Tops® sealing film was used to prevent evaporation.

[0094] Photo-crosslinking Experiments. Photo-crosslinking experiments were performed in quartz cuvettes using a light box fitted with 8 UVA lamps (320-400 nm). Experiments were carried out in 10 mM sodium phosphate buffer (pH 7.4), and samples were irradiated for 1 hour. Samples were analyzed by analytical reversed phase HPLC after irradiation (solvent A: 0.1% TFA in 95:5 H₂O:CH₃CN; solvent B: 0.1% TFA in 95:5 CH₃CN:H₂O) using the gradient: 0-100% B from 0-60 min with a flow rate of 1.0 mL/min.

[0095] Isothermal titration calorimetry. ITC binding experiments were conducted using a Microcal AutoITC200. Titrations were performed at 25 °C in 10 mM potassium phosphate, pH 7.4 or 10 mM sodium borate, 100 mM NaCl, pH 8.6. The concentration of 5 was determined by measuring the UV-vis absorbance at 373 nm, using a NanoDrop2000 with a xenon flash lamp, 2048 element linear silicon CCD array detector, and 1 mm path length. The concentration of 4 was determined from UV-vis absorbance at 315 nm. Solutions of 0.9-

I .2 mM of peptide were titrated into a 81-85 μΜ solution of 4 or 5, using 2.0 μL increments every 3 min. Heats of dilution were subtracted prior to fitting. Binding curves were produced using the supplied Origin software and fit using one-site binding models.

[0096] In vitro HDAC assay. Enzymatic reactions with HDAC1 were performed at 37 °C in 25 mM bicine buffer, 137 mM NaCl, 2.7 mM KC1, 1 mM MgCl₂, pH 8.02, with 0.1 μg/μL. HDAC1 and 200 μΜ acetylated peptide. After 1 h, the reactions were diluted with buffer before adding to a 96- well plate containing probe 5 to give final concentrations of 20 μΜ peptide, 40 μΜ probe 5, and 10 ng/μL, HDAC1 (reactions containing SAHA were diluted to contain 5 μΜ SAHA). Fluorescence labeling was monitored every four for 24 h.

[0097] For in vitro inhibition experiments with HDAC3/NCOR1 , a 5.5 dilution series (starting with 92 uM SAHA) was prepared in 50 mM bicine buffer, 137 mM NaCl, 2.7 mM KC1, 1 mM MgC-2, 0.5 mg/mL BSA (pH 8.02). Enzymatic reactions were performed using

I I.2 nM HDAC3/NCOR1 (purchased from Enzo Life Scienes) and 100 μΜ acetylated peptide at 37 °C. After 4 h, the reactions were quenched with 10 μΜ SAHA before adding to a 96-well plate containing probe 5 to give final concentrations of 77 μΜ peptide, 40 μΜ probe 5, and 8.6 nM HDAC3/NCOR1. Fluorescence labeling was monitored after 24 h.

[0098] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A compound useful for detecting a modified amino acid in a protein or peptide, said compound comprising:

(a) a cyclic oligomer synthetic receptor that binds to said modified amino acid,

(b) optionally, an electrophilic cross-linking group covalently coupled to said cyclic oligomer,

(c) optionally a detectable group covalently coupled to either said cyclic oligomer or said cross-linking group;

subject to the proviso that either (i) said electrophilic cross-linking group covalently coupled to said cyclic oligomer, or (ii) said detectable group covalently coupled to either said cyclic oligomer or said cross-linking group, is present.

2. The compound of claim 1 having the Formula:

wherein:

A is a cyclic oligomer synthetic receptor;

X is an electrophilic cross-linking group;

X' is a photoactivatable cross-linking group;

X" is a fluorescent electrophilic cross-linking group or a catalytic detection-inducing group;

B is a detectable group; and

L² and L³ are each an independently selected linking group or a covalent bond.

3. The compound of claim 2 having the structure:

wherein:

each R¹ is an independently selected aryl, alkylaryl, alkylarylalkyl, heteroaryl, alkylheteroaryl, or al kylheteroary lalkyl ;

each L¹ is an independently an alkyl, alkenyl, disulfide, thioether, ether, ester, amide, dithioacetal, or combination thereof; and

n is from 2 or 3 to 5, 10 or 20.

4. The compound of any one of claims 1 to 3, wherein said detectable group is a dye, fluorophore, enzyme label, luminescent group, radiodetectable group, or specific binding ligand.

5. The compound of claim 1 to 4, wherein said electrophilic cross-linking group is an aryl ether, ester, alkyl sulfonate, aldehyde, ketone, alpha,bcta unsaturated carbonyl, disulfide, alkyl halide, ketone, isocyanate, isothiocyanate, acyl imidazole, alkyne, alkene, or azide.

6. The compound of claim 2 to 5, wherein L² and L³ are independently selected alkyl, peptide, or polyalkylene oxide.

7. The compound of claim 3 to 6, wherein:

each R¹ is independently selected from phenyl, naphthyl, and 9,10-dihydro-9,10- ethenoanthracyl,

each L¹ is independently selected from -S-S-, -CHR-, -OCHR-, or -SCHR-, where R is H, alkyl, or aryl.

8. The compound of claim 2 to 7, wherein said cyclic oligomer is selected from the group consisting of:

and salts thereof;

wherein each R is independently a covalent bond to X, X', X", L² and/or L³, or is a water soluble group.

and salts thereof.

10. The compound of claim 1 , wherein said compound is:

or a salt thereof.

11. The compound of claim 1, wherein said compound is:

or a salt thereof.

12. A method of detecting a modified amino acid in a protein or peptide, comprising:

(a) contacting a protein or peptide to a compound of any preceding claim under conditions in which said cyclic oligomer binds to said modified nucleic acid, and then

(b) detecting the coupling of said detectable group to said protein or peptide to thereby detect a modified amino acid in said protein or peptide.

13. The method of claim 12, wherein:

said contacting step is carried out under conditions in which a nucleophilic amino acid on said protein or peptide adjacent said modified amino acid reacts with said crosslinking group; and

said detecting step is carried out by detecting the coupling of said detectable group to said protein or peptide through said nucleophilic amino acid, the presence of said detectable group on said protein or peptide indicating the presence of a modified amino acid in said protein or peptide.

14. The method of claim 12 or 13, wherein said modified amino acid is a methylated lysine or methylated arginine.

15. The method of any one of claims 12 to 14, wherein said nucleophilic amino acid is a lysine, methyllysine, glutamic acid, aspartic acid, or cysteine.

16. The method of claim 12 or 13, wherein said compound is of Formula IV (A-L²-X"), wherein A is said cyclic oligomer synthetic receptor, X" is a catalytic detection-inducing group, and L² is a linking group or a covalent bond; said method further comprising contacting a second and third reagent to said protein or peptide;

said second reagent comprising a compound of Formula V: RO₂C-L⁴-Z, where RO₂C- is a reactive ester that covalently binds to all lysines in said protein or peptide, L⁴ is a linking group or covalent bond, and Z is a reactive group; and

said third reagent comprises a compound of Formula VI: B-L⁵-Nuc, where B is a detectable group, L⁵ is a linking group or covalent bond, and Nuc is a nucleophilic group that cross-reacts with reactive group Z in a reaction catalyzed by catalytic detection-inducing group X''.