FIELD OF THE INVENTION

The invention provides chemical probes, kits containing the chemical probes, and methods of their use.

BACKGROUND

The study of reactive cysteines in proteomic research requires a diversity of chemical probes and reporter probes, particularly for the concurrent identification and determination of concentration ratios of different cysteine-containing peptides. Accordingly, it is of interest to create chemical probes a) for isotopic labeling such cysteines and b) having multiple orthogonal chemical reactivity modalities for affinity capture-mediated purification of labeled peptides. The ability to multiplex experiments performed under different conditions is useful for reducing inter-experimental variations and improving operational efficiency.

There is a need for new chemical reporter probes targeting reactive cysteines in proteomic analysis.

SUMMARY OF THE INVENTION

In general, the invention provides chemical reporter probes that may be used in the field of proteomics. In one aspect, the invention provides a compound of formula (I):

or a salt thereof,

where

R ¹ is an affinity capture agent;

R ² is H, an isobaric label, or a fluorophore; and

each of Y ¹ and Y ² is independently an optionally substituted C ₂ -6 alkylene.

In some embodiments, the affinity capture agent is biotin, desthiobiotin, maltose, glutathione, or an IgG Fc fusion protein. In certain embodiments, the affinity capture agent is biotin or desthiobiotin.

In particular embodiments, Y ¹ is an optionally substituted C3 alkylene. In further embodiments, Y ¹ is -CH(CONH2)-CH2-. In yet further embodiments, Y ² is an optionally substituted C3 alkylene. In still further embodiments, Y ² is -CH2-CH(CONH2)-.

In other embodiments, the compound is a compound of formula (IA):

or a salt thereof,

where R ² is H, an isobaric label, or a fluorophore.

In yet other embodiments, R ² is an isobaric label. In still other embodiments, the isobaric label is an iTRAQ label or a TMT label.

In some embodiments, R ² is a fluorophore selected from the group consisting of rhodamine fluorophores and BODIPY fluorophores.

In certain embodiments, R ² is H.

In another aspect, the invention provides a kit including the compound of the invention, or a salt thereof, and directions for using the compound or a salt thereof for labeling a protein having a reactive cysteine.

In yet another aspect, the invention provides a method of labeling a protein having a reactive cysteine by reacting, under Huisgen reaction conditions, the compound of the invention, in which R ² is an isobaric label, or a salt thereof, with a protein having a reactive cysteine labeled with an alkyne-containing probe to form a protein labeled with an isobaric label.

In still another aspect, the invention provides a method of identifying a cysteine protease inhibitor by:

(i) incubating a candidate compound with a cysteine protease having a reactive cysteine to produce a test sample;

(ii) reacting the test sample with an alkyne-containing probe to produce an alkynylated sample comprising an alkynylated cysteine protease, wherein the alkynylated cysteine protease has an alkynylated reactive cysteine;

(iii) reacting, under Huisgen reaction conditions, the alkynylated sample with the compound of the invention, or a salt thereof, to produce a conjugate sample comprising a conjugated cysteine protease;

(iv) contacting the conjugate sample with a solid support coated with an agent having affinity for the affinity capture agent to produce a conjugated cysteine protease immobilized on the solid support;

(v) cleaving disulfide bonds in the conjugated cysteine protease immobilized on the solid support; and

(vi) detecting the isobaric label, thereby determining whether the candidate compound is a cysteine inhibitor.

In some embodiments, the candidate compound is identified as a cysteine inhibitor if the proportion of the isobaric label detected in step (vi) to the total amount of the cysteine protease is same as or exceeds the proportion of the isobaric label to the total amount of the cysteine protease, as detected according to the same method with the exception that a known cysteine protease inhibitor is used instead of the candidate compound. In certain embodiments, the candidate compound is identified as a cysteine inhibitor if the amount of the isobaric label detected in step (vi) is directly correlated to the amount of the candidate compound used in the method. In particular embodiments, the solid support is a bead. In further embodiments, the cysteine protease is in a biological sample. In yet further embodiments, the biological sample is a cellular lysate. In still further embodiments, the cellular lysate is a lysate of primary cells or cultured cells.

In other embodiments, the Huisgen reaction conditions comprise the reacting a Cu(ll) catalyst, the alkynylated sample, and the compound of any one of claims 1 to 8, or a salt thereof. In yet other embodiments, the Cu(ll) catalyst is copper (II) sulfate.

Definitions

"Alkyl" refers to a linear, saturated, acyclic, monovalent hydrocarbon radical or branched, saturated, acyclic, monovalent hydrocarbon radical, having from one to twelve carbon atoms, preferably one to eight carbon atoms or one to six carbon atoms, and which is attached to the rest of the molecule by a single bond. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, 1 -methylethyl

(/so-propyl), n-butyl, n-pentyl, 1 ,1 -dimethylethyl (f-butyl), 3-methylhexyl, 2-methylhexyl, and the like. An optionally substituted alkyl radical is an alkyl radical that is optionally substituted, valence permitting, by one, two, three, four, or five substituents independently selected from the group consisting of halo, cyano, oxo, -OR ¹⁴ , -0C(0)-R ¹⁴ , -N(R ¹⁴ )2, -C(0)0R ¹⁴ , -C(0)N(R ¹⁴ ) ₂ , -N(R ¹⁴ )C(0)0R ¹⁵ , -N(R ¹⁴ )C(0)R ¹⁵ ,

-N(R ¹⁴ )S(0)tR ¹⁵ (where t is 1 or 2), -S(0) _t 0R ¹⁵ (where t is 1 or 2), -S(0) _P R ¹⁵ (where p is 0, 1 , or 2) and -S(0)tN(R ¹⁴ ) ₂ (where t is 1 or 2), where each R ¹⁴ is independently hydrogen or alkyl; and each R ¹⁵ is independently alkyl.

“Alkylene” refers to an alkyl group, in which one hydrogen atom was replaced by a valency. An optionally substituted alkylene is an alkylene that is optionally substituted as described for alkyl.

“Balance group” refers to a fragment of an isobaric label having at least one isotopically enriched atom at a predetermined position within the balance group. Typically, a balance group is excised from the isobaric label upon fragmentation in MS/MS. A non-limiting example of a balance group is carbonyl.

“Carbonyl” refers to the group -CO-.

“Carboxylic acid activation group for reaction with amino groups” refers to a monovalent leaving group useful for substitution reactions at a carbonyl with an amine nucleophile. Non-limiting examples of a carboxylic acid activation group for reaction with amino groups include succinimidyloxy and

sulfosuccinimidyloxy.

“Isobaric label” refers to a compound or a monovalent group of formula A-B-C, where A is a reporter group mass tag, B is a balance group, and C is a carboxylic acid activation group for reaction with amino groups or a valency. When C is a carboxylic acid activation group for reaction with amino groups, the isobaric label is an unconjugated isobaric label. When C is a valency, the isobaric label is a monovalent group. Each of the balance group and the reporter group mass tag includes isotopically enriched atoms such that molecules of the isobaric labels have the same total molecular weight. Nonlimiting examples of isobaric labels include isotopically enriched versions of N-succinimidyl 4- methylpiperazin-1 -ylacetate and N-succinimidyl 3-[2-(2,6-dimethylpiperidin-1 -yl)acetylamino]propionate. “Isotopically enriched” refers to an atom or a group of atoms present at a predetermined position in a molecule in an isotopic abundance exceeding the natural isotopic abundance of a given element. For example, a hydrogen atom may be isotopically enriched in ² H, a nitrogen atom may be isotopically enriched in ¹⁵ N, and an oxygen atom may be isotopically enriched in ¹⁷ 0 or ¹⁸ 0.

“Reporter group mass tag” refers to a fragment of an isobaric label having at least one isotopically enriched atom at a predetermined position within the reporter group mass tag group. Typically, a reporter group mass tag is separated from the isobaric label upon fragmentation in MS/MS.

“Salts” refer to compounds having at least two ions and a net charge of zero. The ions are cations and anions. When the compound of formula (I) is positively charged, its salt includes an anionic counterion. When the compound of formula (I) is negatively charged, its salt includes a cationic counterion. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid.

Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate,

benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme illustrating an exemplary method of the invention for labeling a protein having a reactive cysteine. In step 1 , a protein sample is reacted with an alkyne-containing probe to produce an alkyne-labelled protein. In step 2, the alkyne-labeled protein is then reacted under Huisgen reaction conditions with an exemplary chemical reporter probe of the invention to produce a conjugate of the chemical probe and the protein. In step 3, the resulting conjugate is purified using affinity capture methods such as biotin moieties binding to streptavidin-coated solid-support systems. In step 4, trypsin digest on the solid support, followed by disulfide bond cleavage using dithiothreitol, provides an iTRAQ- labelled peptide including a portion of the protein, the portion containing the reactive cysteine.

FIG. 2 is a scheme illustrating an exemplary method of preparing a chemical probe and reaction with a reporter probe of the invention.

FIG. 3 is a drawing showing the components of an exemplary isobaric label molecule— an iTRAQ molecule— that can be used in the preparation of chemical reporter probes of the invention. This isobaric label molecule includes three components: a carboxylic acid activation group for reaction with amino groups (e.g., an N-hydroxy succinic acid ester), a balance group, and a reporter group mass tag.

Isotopes of nitrogen and carbon are distributed in the isobaric tag such that the molecular weights of each isobaric tag in a plurality of isobaric tags are same, provided that the molecular weights of the reporter tags are different. Upon ionization in a mass spectrometer, individual molecules of a peptide labeled with such tags have the same molecular weight. Upon extraction and further fragmentation by the mass spectrometer, the labeled peptide releases reporter tags, and the relative signal intensity ratios of the reporter tags, sorted by molecular weight, allow for relative quantification of the labeled peptide and, in particular, the amino acid residues to which they were attached.

FIG. 4 is a drawing showing the components of an exemplary isobaric label molecule— a TMT molecule— that can be used in the preparation of chemical reporter probes of the invention. This isobaric label molecule includes three components: a carboxylic acid activation group for reaction with amino groups (e.g., an N-hydroxy succinic acid ester), a balance group, and a reporter group mass tag.

Isotopes of nitrogen and carbon are distributed in the isobaric tag such that the molecular weights of each isobaric tag in a plurality of isobaric tags are same, provided that the molecular weights of the reporter tags are different. Upon ionization in a mass spectrometer, individual molecules of a peptide labeled with such tags have the same molecular weight. Upon extraction and further fragmentation by the mass spectrometer, the labeled peptide releases reporter tags, and the relative signal intensity ratios of the reporter tags, sorted by molecular weight, allow for relative quantification of the labeled peptide and, in particular, the amino acid residues to which they were attached.

DETAILED DESCRIPTION

In general, the invention provides chemical reporter probes for proteomic analysis and methods of their use. A chemical reporter probe may be a compound of formula (I):

or a salt thereof,

where

R ¹ is an affinity capture agent;

R ² is H, an isobaric label, or a fluorophore; and

each of Y ¹ and Y ² is independently an optionally substituted C2-6 alkylene.

An isobaric label may be, e.g., an iTRAQ label or a TMT label.

A non-limiting example of the compound of formula (I) is a compound of formula (IA):

or a salt thereof, where R ² is as described for the compound of formula (I).

The labeling of reactive cysteines in proteomics requires a reporter probe that contains several features for measuring the reactivity of covalent fragments through reactive competition with such a probe: (a) an affinity capture agent (e.g., biotin), (b) a cleavage site for separating the probe from the affinity capture agent after purification, and (c) a group (e.g., -N3) for isotopically labeling a peptide. In a preferred embodiment of this invention, the chemical reporter probe is a compound of formula (IA).

Advantageously, the disulfide link in the chemical reporter probes of the invention permits a convenient and chemically orthogonal cleavage of the affinity capture agent upon treatment under reducing conditions, e.g., upon treatment with dithiothreotol (DTT).

Advantageously, the chemical probes of the invention are small and utilize a chemically orthogonal disulfide cleavage process, thus, providing a simple proteomic analysis approach.

Affinity Capture Agent

Chemical reporter probes described herein include an affinity capture agent. Affinity capture agent/agent having affinity for the affinity capture agent pairs that may be useful in the present invention include, e.g., biotin/streptavidin, desthiobiotin/streptavidin, glutathione/glutathione-S-transferase (GST) domain, IgG Fc fusion protein/Protein A/G, and maltose/maltose-binding protein fusion. Accordingly, an affinity capture agent may be, e.g., biotin, desthiobiotin, glutathione, IgG Fc fusion protein, or maltose. An agent having affinity for the affinity capture agent may be, e.g., streptavidin, glutathione-S-transferase (GST) domain, Protein A/G, or maltose-binding protein fusion. Typically, biotin and desthiobiotin bind to streptavidin, glutathione binds to glutathione-S-transferase (GST) domain, IgG Fc fusion protein binds to Protein A/G, and maltose binds to maltose-binding protein fusion. An affinity capture agent can be used to sequester proteins labeled using chemical probes of the invention. For example, a particle (e.g., a bead) coated with an agent having affinity for the affinity capture agent may be contacted with the compound of formula (I), or its conjugate to a protein, to immobilize the compound of formula (I) , or its conjugate to a protein. The immobilized compound of formula (I), or its conjugate to a protein, is thus separated into a different phase from the reaction mixture. The particle may be a magnetic particle (e.g., a magnetic bead). Magnetic particles may facilitate separation of the conjugates containing a compound of formula (I) and a protein from the reaction mixture. Isobaric Labels

Chemical reporter probes described herein may include an isobaric label, e.g., an iTRAQ label or a TMT label. The iTRAQ or TMT labels may be attached to the -Nhh group in a chemical probe of the invention (e.g., R ³ is an iTRAQ label or a TMT label). Chemical reporter probes including isobaric labels described herein may be useful, e.g., in assays described herein. Advantageously, isobaric labels allow the chemical reporter probes of the invention to be used in a multiplexed format. In multiplexed assays, iTRAQ labels are used in the chemical reporter probes to label different protein samples by attaching the chemical reporter probes to individual samples of the target proteins conjugated to an alkyne containing probe.

Typically, unconjugated isobaric labels (e.g., iTRAQ labels and TMT labels) include three components: a carboxylic acid activation group for reaction with amino groups (e.g., an N-hydroxy succinic acid ester), a balance group, and a reporter group mass tag. Non-limiting examples of unconjugated iTRAQ labels include isotopically enriched versions of N-succinimidyl 4-methylpiperazin-1- ylacetate (see FIG. 3). Non-limiting examples of unconjugated TMT labels include isotopically enriched versions of N-succinimidyl 3-[2-(2,6-dimethylpiperidin-1-yl)acetylamino]propionate (see FIG. 4). Atomic isotopes (e.g., nitrogen and carbon isotopes) are distributed between the reporter group mass tags and the balance groups such that the molecular weights of a plurality of isobaric labels are same, the molecular weights of the reporter group mass tags in the plurality of isobaric labels are different, and the molecular weights of the balance groups are different. Upon ionization in a mass spectrometer, a plurality of the same protein species labeled with different isobaric labels is ionized, resulting in a parent molecular ion having the same molecular weight. Upon fragmentation, the reporter group mass tags are liberated from the labeled proteins, and the ratios of reporter group mass tags of different molecular weights provide the relative abundance of the same protein in different samples. FIG. 3 illustrates an exemplary, unconjugated isobaric label that is an unconjugated iTRAQ label. FIG. 4 illustrates an exemplary, unconjugated isobaric label that is an unconjugated TMT label.

Fluorophores

Chemical reporter probes described herein may include a fluorophore. Non-limiting examples of fluorophores include rhodamine fluorophores and BODIPY fluorophores. Non-limiting examples of unconjugated rhodamine fluorophores include rhodamine B, rhodamine 6G, rhodamine 123,

carboxymethylrhodamine (TAMRA), tetramethylrhodamine (TMR), TRITC, sulforhodamine 101 , Texas Red, rhodamine red, Alexa Fluor, DyLight, and HiLyte fluor. A further non-limiting example of a rhodamine dye is 9-[2-[[(3-carboxypropyl)methylamino]carbonyl]phenyl]-3,6-bis (dimethylamino)- xanthylium. Non-limiting examples of BODIPY fluorophores include BODIPY FL, BODIPY FL-X, BODIPY R6G, BODIPY 493/503, BODIPY 530/550, BODIPY TMR-X, BODIPY 558/568, BODIPY 564/570,

BODIPY 576/589, BODIPY 581/591 , BODIPY TR-X, BODIPY 630/650-X, AND BODIPY 650/665-X. Further non-limiting examples of unconjugated BODIPY fluorophores include are [1-[3-[5-[(3,5-dimethyl- 2H-pyrrol-2-ylidene)methyl]-1 H-pyrrol-2-yl]-1-oxopropoxy]-2,5-pyrrolidinedionato]difluoro -boron, (T-4)-(T- 4)-[1-[3-[5-[(3,5-Dimethyl-2H-pyrrol-2-ylidene-KN)methyl]-1 H-pyrrol-2-yl-KN]-1-oxopropoxy]-2,5- pyrrolidinedionatojdifluoroboron, and BODIPY FL NHS Ester. Fluorophores may be useful for the visualization of captured and/or purified peptide fragments, for example, in gels (e.g., acrylamide gels). Methods for the separation and visualization of peptide fragments using acrylamide gels are known in the art.

Methods of Use

Chemical reporter probes described herein may be used to label a protein having a reactive cysteine. The method typically includes reacting, under copper (ll)-mediated Huisgen reaction conditions, a chemical probe of the invention with a protein having a reactive cysteine labeled with an alkyne- containing probe (e.g., N-5-hexyn-1-yl-2-iodo-acetamide or 1-(2-propyn-1-yl)-1 H-pyrrole-2,5-dione) to produce a protein covalently linked to the chemical probe of the invention. When the chemical reporter probe of the invention includes an isobaric label (e.g., an iTRAQ label or a TMT label), the protein covalently linked to a chemical reporter probe of the invention is a protein labeled with an isobaric label (e.g., an iTRAQ label or a TMT label).

Protein labeling with an isobaric label (e.g., an iTRAQ label or a TMT label) described herein may be used to identify a candidate compound as a cysteine protease inhibitor. Typically, a method of identifying a cysteine protease inhibitor using chemical probes of the invention includes the following steps:

(i) incubating a candidate compound with a cysteine protease (e.g., cellular lysate proteins derived from a tissue sample or a cellular sample) to produce a test sample;

(ii) reacting the test sample with an alkyne-containing probe to produce an alkynylated sample (e.g., as shown in Figure 1 , Step 1);

(iii) reacting, under Huisgen reaction conditions, the alkynylated sample with the chemical probe of the invention to produce a conjugate sample;

(iv) contacting the conjugate sample with a solid support coated with an agent having affinity for the affinity capture agent to produce proteins immobilized on the solid support;

(v) cleaving disulfide bonds in the proteins immobilized on the solid support (e.g., by reacting the proteins immobilized on the solid support with a disulfide reducing agent (e.g., dithiothreitol or dithioerythrito I)); and

(vi) detecting the isobaric label to determine whether the candidate compound is a cysteine protease inhibitor.

Cellular lysate proteins typically contain reactive cysteine residues. Reactive cysteines represent so-called“hot spots” that are often related to the function of the biological function and reactivity of the protein or peptide of interest. Identification of such“hot spots” may be useful for identification of drug targets, for diagnostic characterization of active proteins, and for identification of small molecules that can inhibit the action of such proteins and peptides by binding at reactive cysteine site(s). The degree of reactivity of different cysteines within a protein is a function of the microenvironment of the cysteine.

The presence of an inhibitor small molecule may hinder the reaction of an active cysteine with a cysteine reaction probe, e.g., the reaction shown in FIG. 1 , Step 1. Should the small molecule hinder this reaction, then the reaction of the chemical reporter probe will either not occur or occur to a lower extent. The step of determining whether the candidate compound is a cysteine protease inhibitor may include measuring the dependence of the change in the ratio of proteins (e.g., cysteine proteases) labeled with an isobaric label (e.g, an iTRAQ label or a TMT label) to the total proteins in the tissue sample or cellular sample on the concentration of the candidate compound in step (i). Reduction in the ratio with concomitant increase in the concentration of the candidate compound in step (i) indicates that the candidate compound is a cysteine protease inhibitor. Preferably, the proteins immobilized on the solid support in step (iv) are treated under the tryptic digest conditions according to methods known in the art.

Advantageously, isobaric labels can allow multiplexed assays. For example, steps (i), (ii), and (iii) can be performed in a plurality of reaction vessels with different, individual candidate compounds matched to each reaction vessel and/or different tissue or cellular samples matched to each reaction vessel based on the identity of the isobaric labels. After step (iii), the samples from the plurality of the reaction vessels can be pooled and analyzed together starting with step (iv). At step (vi), the candidate compound may be identified as a cysteine protease inhibitor by detecting the isobaric label. For example, the candidate compound may be identified as a cysteine protease inhibitor if the amount of the isobaric labels detected in step (vi) (e.g., normalized to the total amount of the cysteine protease) is the same as or is lower than the amount of the isobaric labels (e.g., normalized to the total amount of the cysteine protease), as detected according to the same method with the exception that a known cysteine protease inhibitor is used in place of the candidate compound. Alternatively, the candidate compound may be identified as a cysteine inhibitor if the amount of the isobaric label detected in step (iv) is directly correlated to the amount of the candidate compound used in the method. At step (iv), the isobaric labels permit correlation of the obtained data or reporter group mass of the isobaric tags as obtained by fragmentation of extracted molecular ions (e.g., MS/MS data) with individual reaction vessels and, therefore, with individual candidate compounds and/or individually treated samples of protein and peptides derived from tissues or cellular samples. The methods of isobaric label detection and analysis are known in the art.

Cellular lysate samples (e.g., from cellular or tissue samples) may be used as sourced of cysteine proteases of interest. The cellular lysate samples may be produced from cells of animal origin, including but not limited to cells obtained from mammals. The cells may be primary cells, e.g., primary cells isolated from an animal tissue (e.g., mammalian tissue (e.g., human tissue)). The cells may be cultured cells (e.g., cultured animal tissue cells (e.g., cultured mammalian tissue cells (e.g., cultured human tissue cells))). The cells may be normal cells or cells from a subject having a disease or disorder (e.g., a cancer, autoimmune disorder, neurodegenerative disorder, or infection (e.g., viral infection, bacterial infection, fungal infection, or parasitic infection)).

Methods of Preparation

A non-limiting example of the preparation of a chemical probe of the invention is illustrated in FIG.

2.

The following examples are meant to illustrate the invention. They are not meant to limit the invention in any way. EXAMPLES

Example 1. Preparation of Compounds

Reagents were purchased from Aldrich, Sigma, TCI (Shanghai) Development, Chembon Pharmaceutical Co., Ltd, zhangjiagang aimate huaxue youxiangongsi, Changzhou Qinuo BioTech Co.

Ltd, and Shanghai Weiyuan Fine Fluorine Technology Development Co., Ltd or other suppliers as indicated below and used without further purification. Reactions using microwave irradiation for heating were conducted using a Biotage Initiator†. The purification of multi-milligram to multi-gram scale was conducted by methods known to those skilled in the art such as elution of silica gel flash column;

preparative flash column purifications were also affected in some cases by use of disposal pre-packed silica gel columns (Welch/Agela) eluted with a Biotage CombiFlash system.

For the purpose of judging compound identity and purity, typically the analytical LC-MS (liquid chromatography/mass spectroscopy) system was used consisted of a Waters ZQ™ platform with electrospray ionization in positive ion detection mode with an Agilent 1100 series HPLC with autosampler. The column was usually a Water Xterra MS C18, 3.0 ^c 50 mm, 5 pm. The flow rate was 1 mL/min, and the injection volume was 10 pL. UV detection was in the range 210-400 nm. The mobile phase consisted of solvent A (water plus 0.06% TFA) and solvent B (acetonitrile plus 0.05% TFA) with a gradient of 100% solvent A for 0.7 min changing to 100% solvent B over 3.75 min, maintained for 1.1 min, then reverting to 100% solvent A over 0.2 min.

For some separations, the use of super critical fluid chromatography may also be useful. Super critical fluid chromatography separations were performed using a Mettler-Toledo Minigram system with the following typical conditions: 100 bar, 30 °C, 2.0 mL/min eluting a 12 mm AD column with 40% MeOH in super critical fluid CO2. In the case of analytes with basic amino groups, 0.2% isopropyl amine was added to the methanol modifier.

Alternatively, compound 14 has also been purified by reverse phase HPLC using methods known in the art. In some cases, preparative HPLC purification was conducted using PE Sciex 150 EX Mass Spec controlling a Gilson 215 collector attached to a Shimadzu preparative HPLC system and a Leap auto-injector. Compounds were collected from the elution stream using LC/MS detection in the positive ion detection: The elution of compounds from C-18 columns (2.0 X 10 cm eluting at 20 mL/min) was effected using appropriate linear gradation mode over 10 minutes of Solvent (A) 0.05% TFA/H20 and Solvent (B) 0.035% TFA/acetyl nitrile. For injection on to HPLC systems, the crude samples were dissolved in mixtures of methanol, acetyl nitrile and DMSO.

Compounds were characterized either by ¹ H-NMR using a Bruker ADVANCE III HD 400MHz Spectrometer or Bruker AVANCE 300MHz Spectrometer.

LIST OF ABBREVIATIONS DCM dichloromethane

DIEA diisopropylethylamine

DMF A/,A/-dimethylformamide

DMSO dimethylsulfoxide

EDCI 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide

EtOAc ethyl acetate HATU 1 -[bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, also known as N-[(Dimethylamino)-1 H-1 ,2,3-triazolo-[4,5-b]pyridin-1 -ylmethylene]-

N-methylmethanaminium hexafluorophosphate N-oxide

HOAc Acetic acid

HPLC high pressure chromatography

MeOH methyl alcohol

MW microwave

N2 nitrogen

NH4OH ammonium hydroxide

rt room temperature

TFA trifluoroacetic acid

Synthesis of Compound 11

10 11

To a stirred solution of (2S)-2-amino-3-[[(2S)-2-amino-2-carboxyethyl]disulfanyl]prop anoic acid (3.00 g, 12.485 mmol, 1 .00 equiv..) in MeOH (50.00 ml_) was added SOCI2 (5.94 g, 49.940 mmol, 4.00 equiv.) dropwise at 0 °C under N2 atmosphere. The resulting mixture was stirred for 5 h at 76 °C under N2 atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under reduced pressure. The resulting solid was diluted with 30 ml_ of DCM. The precipitated solids were collected by filtration and washed with DCM (3x10 ml_). The resulting solid was dried with infrared lamp. This resulted in methyl (2S)-2-amino-3-[[(2S)-2-amino-3-methoxy-3- oxopropyl]disulfanyl]propanoate, dihydrochloride (3 g, 70.41 %) as a white solid. Compound 1 1 (ES, m/z)\ [M+1 -2HCI] ⁺ = 269.2

Synthesis of Compound 12

Me0 ₂ C S ^— S C0 ₂ Me 1 ) NH4OH, rt, 2 h H NOC S-S CONH ₂

11 12

To a stirred solution of ammonia water (30.00 ml_) was added methyl (2S)-2-amino-3-[[(2S)-2- amino-3-methoxy-3-oxopropyl]disulfanyl]propanoate dihydrochloride (3.00 g, 8.791 mmol, 1.00 equiv.) at room temperature. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was dried by lyophilization. The residue was added to the mixture of EA (10 ml_) and HCI in 1 ,4-dioxane (4 mol/L, 10 ml_). The solution was stirred for 1 h at room temperature. The precipitated solids were collected by filtration and washed with EA (2x10 ml_). The resulting solid was dried with infrared light. This resulted in (2S)-2-amino-3-[[(2S)-2-amino-2-carbamoylethyl]disulfanyl]pr opanamide dihydrochloride (2 g, 73.10%) as a yellow solid. Compound 12 (ES, m/z)\ [M+1 -2HCI] ⁺ = 239.1 Synthesis of Compound 13

To a stirred solution of 5-[(3aS,4S,6aR)-2-oxo-hexahydro-1 H-thieno[3,4-d]imidazol-4-yl]pentanoic acid (5.00 g, 20.466 mmol, 1.00 equiv.) and 1-hydroxypyrrolidine-2,5-dione (2.36 g, 20.466 mmol, 1.00 equiv.) in DMF (50.00 ml_) was added EDCI (4.71 g, 24.559 mmol, 1.20 equiv.) at room temperature under N2 atmosphere. The resulting mixture was stirred for 16 h at room temperature. The solution was poured into 200 ml_ of ice water and the mixture kept still for 3 h at 0 °C. The precipitated solids were collected by filtration and washed with water (2x20 ml_). The solids were dried with infrared lamp. This resulted in 2, 5-dioxopyrrolidin-1-yl 5-[(3aS, 4S, 6aR)-2-oxo-hexahydro-1 H-thieno [3, 4-d] imidazol-4- yljpentanoate (3.5 g, 50.10%) as a white solid. Compound 13 (ES, m/z)\ [M+1] ⁺ = 342.2

Synthesis of Compound 14

To a stirred solution of (2S)-2-amino-3-[[(2S)-2-amino-2-carbamoylethyl]disulfanyl]pr opanamide dihydrochloride (2.00 g, 6.426 mmol, 1.00 equiv.) and DIEA (3.32 g, 25.704 mmol, 4.00 equiv.) in DMSO (20.00 ml_) was added 2,5-dioxopyrrolidin-1-yl 5-[(3aS,4S,6aR)-2-oxo-hexahydro-1 H-thieno[3,4- d]imidazol-4-yl]pentanoate (2.19 g, 6.426 mmol, 1.00 equiv.) at room temperature. The resulting mixture was stirred for 16 h at room temperature. The resulting mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel 120 g; mobile phase, CH3CN in water (0.1 % TFA), 5% to 30% gradient in 10 min; 60 mL/min; detector, UV 220 nm. This resulted in 5- [(3aS,4S,6aR)-2-oxo-hexahydro-1 H-thieno[3,4-d]imidazol-4-yl]-N-[(1 S)-2-[[(2S)-2-amino-2- carbamoylethyl]disulfanyl]-1-carbamoylethyl]pentanamide; trifluoroacetic acid (1.1 g, 11.83%) as a white solid. Compound 14 (ES, m/z)\ [M+1 -TFA] ⁺ = 465.2 Synthesis of Compound 16

To a stirred solution of (2S)-6-azido-2-[[(tert-butoxy)carbonyl]amino]hexanoic acid (0.20 g, 0.732 mmol, 1 .10 equiv.) and DIEA (0.26 g, 1 .996 mmol, 3.00 equiv.) in DMSO (10.00 mL) was added HATU (0.33 g, 0.865 mmol, 1 .30 equiv.) at room temperature under N2 atmosphere. The resulting mixture was stirred for 10 min. To the above mixture was added 5-[(3aS,4S,6aR)-2-oxo-hexahydro-1 H-thieno[3,4- d]imidazol-4-yl]-N-[(1 S)-2-[[(2S)-2-amino-2-carbamoylethyl]disulfanyl]-1 -carbamoylethyl]pentanamide; trifluoroacetic acid (1 .10 g, 0.665 mmol, 1 .00 equiv., 40%) at room temperature. The resulting mixture was stirred for additional 3 h at room temperature. The resulting mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel, 120 g; mobile phase, CH3CN in water (0.05% FA), 20% to 70% gradient in 8 min; 70 mL/min; detector, UV 220 nm. This resulted in tert- butyl N-[(1 S)-1 -[[(1 S)-2-[[(2S)-2-[5-[(3aS,4S,6aR)-2-oxo-hexahydro-1 H-thieno[3,4-d]imidazol-4- yl]pentanamido]-2-carbamoylethyl]disulfanyl]-1 -carbamoylethyl]carbamoyl]-5-azidopentyl]carbamate (250 mg, 44.39%) as a white solid. Compound 16 (ES, m/z): [M+1 ] ⁺ = 719.3. ¹ H-NMR of Compound 16 (300 MHz, DMSO-de, ppm): d 8.30 (d, J = 8.6 Hz, 1 H), 8.01 (d, J = 8.3 Hz, 1 H), 7.36 (d, J = 15.9 Hz, 3H), 7.16 (s, 1 H), 7.01 (d, J = 7.0 Hz, 1 H), 6.38 (d, J = 18.3 Hz, 2H), 4.52-4.48 (m, 2H), 4.33-4.29 (m, 1 H), 4.16- 4.12 (m, 1 H), 3.93-3.86 (m, 1 H), 3.29-3.27 (m, 2H), 3.21 -1 .07 (m, 3H), 2.90- 2.80 (m, 3H), 2.58 (d, J = 12.4 Hz, 1 H), 2.15 (t, J = 7.1 Hz, 2H), 1 .62-1 .48 (m, 8H), 1 .36 (s, 13H).

Synthesis of Compound 1

To a stirred solution of tert-butyl N-[(1 S)-1 -[[(1 S)-2-[[(2S)-2-[5-[(3aS,4S,6aR)-2-oxo-hexahydro- 1 H-thieno[3,4-d]imidazol-4-yl]pentanamido]-2-carbamoylethyl]d isulfanyl]-1 -carbamoylethyl]carbamoyl]-5- azidopentyl]carbamate (14.00 mg, 0.019 mmol, 1 .00 equiv) in DCM (0.50 mL) was added TFA (0.25 mL) at room temperature. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was concentrated under vacuum. The residue was dried by freezing. This resulted in (2S)-N- [(1 S)-2-[[(2S)-2-[5-[(3aS,4S,6aR)-2-oxo-hexahydro-1 H-thieno[3,4-d]imidazol-4-yl]pentanamido]-2- carbamoylethyl]disulfanyl]-1 -carbamoylethyl]-2-amino-6-azidohexanamide (22.5 mg) as a gray solid. LC- MS-PH-AIS-PPA-087-0 (ES, m/z)\ [M+1 ] ⁺ = 619.1

Example 2. Labeling of Proteins Using Compounds of Formula (I)

A compound of Formula (I) may be used for labeling proteins containing a reactive cysteine. For example, the labeling protocol may be as follows: a compound of Formula (I) is first treated with 2 to 3 reactive equivalents of an isobaric labeling reagent, e.g., a commercially available iTRAQ or TMT labeling reagent. Such reactions may be performed in, e.g., individual Eppendorf tubes at room temperature for, e.g., 1 hour, followed by quenching with excess piperidine. The volatiles may be removed from the samples. If desired, the resulting residue may be re-dissolved in phosphate-buffered saline (PBS).

Example 3. Assays Utilizing a Compound of Formula (I)

A compound of Formula (I) may be used in the proteomic study of proteins containing a reactive cysteine and identification of therapeutically active agents. In one aspect of this invention, the following general protocol may be used.

1) Proteome samples are labelled with alkyne containing probes in DMSO solutions in, e.g.,

separate Eppendorf tubes, under the predetermined conditions (e.g., varied probe

concentrations, varied reaction times, varied reactivity of a competing ligand, and/or experiment replicate).

2) To each, separate tube containing the proteome analysis sample then is added the PBS sample from Example 2.

3) To the mixture produced in Step 2 is added a copper (II) salt (e.g., copper (II) sulfate) to mediate the reaction of the azide group of the reporter probe of Formula (I) with the alkyne group of the cysteine-reactive probe.

4) At this point, it is possible to combine the samples for subsequent, common treatment and

manipulations.

5) The sample is then purified by loading to streptavidin beads, followed by washing with PBS.

6) The adhered samples are then treated on-bead under the tryptic digest conditions according to methods known to those skilled in the art.

7) Treatment of the bead-adhered peptides under the disulfide reducing conditions (e.g,,

dithiothreitol) and subsequent washing elutes a mixture of eustatically labelled peptides.

8) This mixture of eustatically labelled peptides is then analyzed by chromatographic and/or MS/MS methods for the identification and quantification of isobarically labelled peptides using methods known to those skilled in the art.

The procedure described above may be used, for example, to identify a cysteine protease inhibitor. In these methods, a cellular lysate containing a cysteine protease, e.g., from a cellular or tissue sample, is first incubated with known or putative cysteine protease inhibitors at predetermined concentrations. For each concentration, a compound of Formula (I) is derivatized with a different isobaric labelling reagent (e.g., iTRAQ or TMT). The protocol is then followed as above. Varying levels of the reporter ion for each identified peptide by MS/MS methods informs if the known or putative cysteine protease inhibitor has effectively competed with a compound of Formula (I) and if so, at what concentration and at which residue. Compounds which bind to reactive cysteine residues or in a hindering manner at the site of such reactive cysteines will hinder the reaction of the cysteine reactivity- based probe and thereby provide less opportunity for the reporter probe of this reaction to couple to the acetylene moiety of the reactivity probe. As a result, a smaller mass reporter signal will be observed in this sample. In this manner, cysteine protease inhibitors may be ranked according to their affinity for specific cysteine residues within proteins of therapeutic interest.

OTHER EMBODIMENTS

Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

Other embodiments are in the claims.