WO2010014236A2 - Acylhydrazone-based cleavable linkers - Google Patents

Abstract

The present invention provides cleavable linker compounds of Formula (I): wherein: X is a cleavable linker comprising an acylhydrazone; and Y and R are each independently selected from the group consisting of covalent coupling groups and members of a specific binding pair. Solid supports having such cleavable linkers coupled thereto, and methods of using the same, are also described.

Description

ACYLHYDRAZONE-BASED CLEAVABLE LINKERS

Harold L. Kohn and Ki Duk Park

This invention was made with US government support under grant no. 5R01NS054112 from the National Institute of Neurological Disorders and Stroke. The US Government has certain rights to this invention.

Background of the Invention

In recent years significant progress has been made in the identification of protein- ligand, protein-drug, and protein-protein interactions (Berggard et al., 2007; Walsh et al., 2006). Nonetheless, the complexity of the proteome, the low abundance of many proteins, and the temporal and spatial distribution of proteins make protein target identification difficult. However, new methods have been advanced to identify proteins involved in biochemical and regulatory pathways. Common to many of these is the selective capture of the tagged protein of interest from the biological milieu using affinity-based chromatography (Scriba et al., 2004). Since the biotin-avidin association is among the tightest non-covalent interactions known (K₃ = 10¹⁴-10¹⁵ M^"1) (Green et al., 1990), biotinylated tags and (strept)avidin supports have been extensively used in affinity-based chromatographic strategies (Wilchek et al., 1990). This method's utility has been diminished by the very inefficient release of the intact biotinylated protein from the streptavidin support under mild conditions (Holmberg et al., 2005; Marie et al., 1990), a situation that makes it difficult to identify low abundance proteins.

Several methods have been advanced to address the difficulty of the biotinylated product release step. They include using either biotin analogs (Hirsch et al., 2002; Zeheb et al., 1986) or protein-engineered streptavidin mutants (Howarth et al., 2006; Malmstadt et al., 2003; Morag et al., 1996; Wu et al., 2005), structural modifications that weaken the biotin- (strept)avidin interaction. While these methods improve the release of biotinylated molecules, the reduced association constant for the complex does not allow the application of stringent wash conditions to remove nonspecific protein interactions from the resin and adversely affects the capture efficiency of the biotin-tagged adducts. Another approach has been to use conditions that disrupt the biotin-(strept)avidin complex (Holmberg et al., 2005; Jenne et al., 1999; Tong et al., 1992). Unfortunately, several of these conditions (e.g., 1% aqueous SDS/heat) are harsh and generally lead to the release of abundant proteins that nonspecifically bind to the matrix or streptavidin. Furthermore, the recovery yields of the biotinylated materials are still low and the captured molecules are often either destructed or denatured. A third strategy has been to use biotinylated agents that incorporate a cleavable site between the biotin and the bound (bio)molecule. Examples include linkers that contain a proteolytic (Dieterich et al., 2007; Speers et al., 2005) or a chemically labile (e.g., disulfide (Finn et al., 1985; Marie et al., 1990; Shimkus et al., 1985), acid-sensitive (van der Veken et al., 2005), base-sensitive (Ball et al., 1997; Kazmierski et al., 1995), nucleophile-sensitive (Lin et al., 1991), photolytic (Bai et al., 2004; Thiele et al., 1994), reductive (Verhelst et al., 2007)) site.

Summary of the Invention

A first aspect of the invention is a compound of Formula I:

Y-X-R (I) wherein:

X is a cleavable linker comprising an acylhydrazone; and

Y and R are each independently selected from the group consisting of covalent coupling groups and members of a specific binding pair.

A further aspect of the invention is a support (e.g., a solid support) useful for binding a compound of interest from a mixture, wherein the compound of interest is a first member of a binding pair and the support comprises a second member of the binding pair coupled to the support by a cleavable linker, the improvement comprising: employing a compound as herein above and below as the cleavable linker (e.g. where R is the first member of the binding pair). The cleavable linker can be covalently coupled or specifically bound (e.g., by biotin-avidin binding) to the support.

Further aspects of the invention include methods of using the cleavable linkers and supports described herein.

The present invention is explained in greater detail in the drawings herein and the specificaiton set forth below. The disclosures of all United States patent references cited herein are to be incorporated herein by reference in their entirety. Brief Description of the Drawings

Figure 1. General Strategy of Cleavable Linker for Isolation, Identification and Detection of Target Proteins.

Figure 2. Synthesis of Acylhydrazone Linker 1 and Exchange of Hydrazone Linkage in 1 with Acethydrazide (7).

Figure 3. Comparison of the Efficiency of Protein Release from BSA Adducts (9 and 11) Modified with either Cleavable Linker 1 or Non-cleavable Linker 10. A: Chemical structure of non-cleavable linker 10 and two different BSA samples (9 and 11) modified with either cleavable linker 1 or non-cleavable linker 10. B: All reactions were run in aqueous 50 mM HEPES (pH 5.8) using optimized condition (7 (10 mM) and SDS (20 mM) at 50 ⁰C (1 h)) (data not shown). Lanes marked "s" correspond to the supernatant removed from the bead mixture after the initial treatment, while the lanes marked "b" refer to the sample obtained after the beads were washed and then treated with loading buffer and heated at 95 ⁰C for either 5 min (Ab) or 15 min (lane Bb). For reaction B, 5- to 10-fold sample volumes from the recovered Bb were loaded to facilitate comparison. The proteins were visualized by silver staining.

Figure 4. Use of 1 for Adduct Capture and Validation of the Release and Reduction Steps.

Figure 5. Use of 1 for Enolase Capture, Release, and Detection. A: Scheme of enolase capture, release, and detection. The single cysteine unit in enolase (19) was treated with 13 to give approximately 30% of alkyne functionalized enolase 20 based on MS (data not shown) and then converted to enolase 21 under Cu(I) -mediated cycloaddtion condition. B: Use of fluorescein hydrazide 22 for detection and isolation of streptavidin-bound modified enolase 21. Lane 1, 23 (the enolase recovered after incubation of enolase 21 with streptavidin beads in aqueous 50 mM HEPES (pH 5.8), followed by washing of the beads, and then treatment of the beads with 22 (1.5 mM) using an optimized condition (p-anisidine [10 mM], 37 °C, 4 h); lane 2, 24 (reduction of 23 with NaCNBH₃ at pH 3.8); lane 3, control (all reactions were the same as 23 except the cleavable linker was excluded in the cycloaddition step). The proteins were visualized by fluorescent detection by excitation at 488 nm and detection at 520 nm.

Figure 6. Proteomic Target Search in Mouse Liver Proteome with the Cleavable Linker 1 Using 25 and Comparison with Non-cleavable Linker 10. A: Chemical structures of 25 and 26. B: After labeling with by 25 (5 μM or 25 μM) followed by Cu(I)-mediated cycloaddition with 26, the signal was detected by in-gel fluorescence scanning. Using 5 μM 25, ALDH-I (arrow) was selectively labeled. At 25 μM 25, ALDH-I (arrow) and an abundant protein(s) (asterisk) in liver lysate were labeled. C: After labeling with 25 (25 μM), probe-labeled proteins were captured by streptavidin beads after Cu(I)-mediated cycloaddition to either cleavable linker 1 or non-cleavable linker 10. The beads were washed and treated using a mild cleavage condition (7 (100 mM), /7-anisidine (10 mM) in aqueous 50 mM HEPES (pH 5.8) (37 °C, 4 h)). Lanes marked "s" correspond to the supernatant removed from the bead mixture after the initial treatment, while the lanes marked "b" refer to the sample obtained after treatment of the remaining beads with loading buffer (95 ⁰C, 5 min). The proteins were visualized by silver staining. D: Comparison of the release efficiency of 25 labeled target protein (ALDH-I) after Cu(I)-mediated cycloadditon to either cleavable linker 1 and non- cleavable linker 10 by western blot using anti-ALDH-1 antibody.

Detailed Description of the Preferred Embodiments 1. Definitions.

"Alkyl" as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. "Loweralkyl" as used herein, is a subset of alkyl, in some embodiments preferred, and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like. Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups

"Cycloalkyl" as used herein alone or as part of another group, refers to a saturated or partially unsaturated cyclic hydrocarbon group containing from 3, 4 or 5 to 6, 7 or 8 carbons (which carbons may be replaced in a heterocyclic group as discussed below). Representative examples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. These rings may be optionally substituted with additional substituents as described herein such as halo or loweralkyl. The term "cycloalkyl" is generic and intended to include heterocyclic groups as discussed below unless specified otherwise. Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups.

"Alkenyl" as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms (or in loweralkenyl 1 to 4 carbon atoms) which include 1 to 4 double bonds in the normal chain. Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4- pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like. Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups.

"Alkynyl" as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms (or in loweralkynyl 1 to 4 carbon atoms) which include 1 triple bond in the normal chain. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2- butynyl, 4-pentynyl, 3- pentynyl, and the like. The term "alkynyl" or "loweralkynyl" is intended to include both substituted and unsubstituted alkynyl or loweralkynyl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above. Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups.

"Heterocyclo" as used herein alone or as part of another group, refers to an aliphatic (e.g., fully or partially saturated heterocyclo) or aromatic (e.g., heteroaryl) monocyclic- or a bicyclic-ring system. Monocyclic ring systems are exemplified by any 3 to 8 membered ring containing 1 , 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur. The 5 membered ring has from 0-2 double bonds and the 6 membered ring has from 0-3 double bonds. Representative examples of monocyclic ring systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic and heterocyclic ring systems fused to an aryl group as defined herein, a cycloalkyl group as defined herein, or another monocyclic or heterocyclic ring system as defined herein. Representative examples of bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like. Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups.

"Aryl" as used herein alone or as part of another group, refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings. Representative examples of aryl include, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. The term "aryl" is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above. Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups.

"Heteroaryl" as used herein is as described in connection with heterocyclo above. Such groups can be unsubstituted or substituted with one or more (e.g., one, two, three four, etc.) independently selected electron-donating or electron-withdrawing groups.

"Arylalkyl" as used herein alone or as part of another group, refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2- phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.

"Heteroarylalkyl" as used herein alone or as part of another group, refers to a heteroaryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

"Heterocycloalkyl" as used herein alone or as part of another group, refers to a heterocyclo group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. "Electron-withdrawing" and "electron donating" refer to the ability of a substituent to withdraw or donate electrons relative to that of hydrogen if the hydrogen atom occupied the same position in the molecule. These terms are well understood by one skilled in the art and are discussed in Advanced Organic Chemistry, by J. March, John Wiley and Sons, New York, N. Y., pp. 16-18 (1985), incorporated herein by reference. Examples of such electron withdrawing and electron donating groups or substituents include, but are not limited to halo, nitro, cyano, carboxy, loweralkenyl, loweralkynyl, loweralkanoyl (e.g., formyl), carboxyamido, aryl, quaternary ammonium, aryl (loweralkanoyl), carbalkoxy and the like; acyl, carboxy, alkanoyloxy, aryloxy, alkoxysulfonyl, aryloxysulfonyl, and the like; hydroxy, alkoxy or loweralkoxy (including methoxy, ethoxy and the like); loweralkyl; amino, lower alkylamino, di(loweralkyl) amino, aryloxy (such as phenoxy), mercapto, loweralkylthio, lower alkylmercapto, disulfide (loweralkyldithio) and the like; 1-piperidino, 1-piperazino, 1- pyrrolidino, acylamino, hydroxyl, thiolo, alkylthio, arylthio, aryloxy, alkyl, ester groups (e.g., alkylcarboxy, arylcarboxy, heterocyclocarboxy), azido, isothiocyanato, isocyanato, thiocyanato, cyanato, and the like. One skilled in the art will appreciate that the aforesaid substituents may have electron donating or electron withdrawing properties under different chemical conditions. Moreover, the present invention contemplates any combination of substituents selected from the above-identified groups. See US Patent No. 6,133,261 and 5,654,301 ; see also US Patent No. 4,711 ,532.

"Acyl" as used herein alone or as part of another group refers to a -C(O)R radical, where R is any suitable substituent such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl or other suitable substituent as described herein.

"Alkanoyl" refers to the group -C(O)R', wherein R' is lower alkyl. Hence "alkanoyl" groups are particular examples of "acyl" groups, as described above.

"Halo" or "halogen," as used herein refers to -Cl, -Br, -I or -F.

"Oxy" as used herein refers to an -O- group.

"Sulfonyl," as used herein, refers to an -SO₂ - group.

"Thio" as used herein refers to an -S- group.

"Support" as used herein may be any suitable support, including but not limited to controlled pore glass, oxalyl-controlled pore glass, silica-containing particles, polymers of polystyrene, copolymers of polystyrene, copolymers of styrene and divinylbenzene, copolymers of dimethylacrylamide and N,N'-bisacryloylethylenediamine, soluble support medium such as dendrimers, PEPS, lipid particles such as liposomes, etc. See, e.g., US Patent No. 6,653,468; see also US Patent Nos. 7,202,264 and 6,664,372.

"Solid support" as used herein is meant to comprise any solid (flexible or rigid) substrate (e.g., a particulate or non-particulate substrate) onto which it is desired to apply an array of one or more binding agents. The substrate may be biological, non-biological, organic, inorganic or a combination thereof, and may be in the form of particles, strands, precipitates, gels, sheets, tubings, spheres, containers, capillaries, pads, slices, films, plates, slides, etc, having any convenient shape, including disc, sphere, circle, etc. The substrate surface supporting the array may have any two-dimensional configuration and may include, for example steps, ridges, kinks, terraces and the like and may be the surface of a layer of material different from that of the rest of the substrate. See, e.g., US Patent No. 7,563,587.

"Covalent coupling group" as used herein includes, but is not limited to, electrophilic affinity bait groups, photochemical affinity bait groups, and reactive groups.

"Electrophilic affinity bait group" as used herein includes, but is not limited to, NCS, C(O)H, and acyl halide, haloketones, alkyl halide, and alkyl sulphonate bait groups.

"Photochemical affinity bait group" as used herein includes, but is not limited to, aryl azide, diazirine, and benzophenone bait groups.

"Reactive groups" as used herein includes, but is not limited to, halo, sulfonate, aldehyde, ketone, ester, and activated ester reactive groups.

"Specific binding pair" (abbreviated "sbp") as used herein describes a pair of molecules (each being a member of a specific binding pair) which are naturally derived or synthetically produced. One of the pair of molecules has a structure (such as an area or cavity) on its surface that specifically binds to (and is therefore defined as complementary with) a particular structure (such as a spatial and polar organization) of the other molecule, so that the molecules of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs (without any limitation thereto) are antigen- antibody, antibody-hapten, biotin-avidin, ligand-receptor (e.g., hormone receptor, peptide- receptor, enzyme-receptor), carbohydrate-protein, carbohydrate-lipid, lectin-carbohydrate, nucleic acid-nucleic acid (such as oligonucleotide-oligonucleotide). See, e.g., US Patent No. 7,563,587; 7,506,556.

"Nucleic acid" refers to a deoxyribonucleotide polymer (DNA) or ribonucleotide polymer (RNA) in either single- or double-stranded form, and also encompasses synthetically produced analogs that can function in a similar manner as naturally occurring nucleic acids. While natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, nucleotides or bases. These include, for instance, peptide nucleic acids (PNAs) as described in, e.g., U.S. Pat. No. 5,948,902 and the references cited therein; pyranosyl nucleic acids (p-NAs) as described in, e.g., WO 99/15540 (p-RNAs), WO 99/15539 (p-RNAs), and WO 00/1 101 1 (p-DNAs); locked nucleic acids (LNAs), as described in, e.g., U.S. Pat. No. 6,316,198; and phosphothionates and other variants of the phosphate backbone of native nucleic acids. See, e.g., US Patent No. 7,563,587.

"Oligonucleotide" refers to single stranded nucleotide multimers of from about 5 to about 100 nucleotides. See, e.g., US Patent No. 7,563,587.

"Antibody" as used herein means an immunoglobulin which may be naturally, or partly or wholly synthetically, produced, and also includes active fragments thereof, including Fab antigen-binding fragments, univalent fragments and bivalent fragments. The term also covers any protein having a binding domain which is homologous to an immunoglobulin binding domain. Such proteins can be derived from natural sources, or partly or wholly synthetically produced. Exemplary antibodies are the immunoglobulin isotypes and the Fab, Fab', F(ab')₂, scFv, Fv, dAb, and Fd fragments. See, e.g., US Patent No. 7,563,587.

2. Linker compounds and supports.

As noted above, the present invention provides compounds of Formula I:

Y-X-R (I) wherein:

X is a cleavable linker comprising an acylhydrazone; and

Y and R are each independently selected from the group consisting of covalent coupling groups and members of a specific binding pair.

In some embodiments, Y is a covalent coupling group {e.g., N₃) and R is a member of a specific binding pair {e.g., avidin).

In some embodiments, the cleavable linker further comprising a polyalkylene oxide group {e.g., poly(ethylene glycol)).

In some embodiments, the compound has the structure of Formula II:

wherein:

Y and R are as given above;

A, B, C, and X are each independently selected from the group consisting of O, S, N(H), N(R), N(R')(R")⁺, CH₂, C(R')(R"), single covalent bond, and double covalent bond;

F' is selected from the group consisting of hydrogen, lower alkyl, and aryl (which can be unsubstituted or substituted with one or more electron-donating or electron-withdrawing groups);

W is selected from the group consisting of a single covalent bond, O, S, N(H), and N(R');

R and R" are each independently selected loweralkyl; n is 0 or 1-4; and m is 0 or 1-4.

In some embodiments of the foregoing, R is selected from the group consisting of ssDNA, RNA, antigenic peptides, N-terminal polyhistidines, covalent coupling groups, and a group of the Formula:

In some embodiments of the foregoing, Y is selected from the group consisting of N₃, CCH, electrophilic affinity bait groups, photochemical affinity bait groups, reactive groups, peptides, proteins, nucleic acid, carbohydrates, lipids, modified drugs, inhibitors, enzyme substrates or mimics, and organic groups.

A particular example of the foregoing is a compound having the formula:

The linker compounds of the present invention can be coupled to supports in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art. Thus, a further aspect of the invention is a support (e.g., a solid support) useful for binding a compound of interest from a mixture, wherein the compound of interest is a first member of a binding pair and the support comprises a second member of the binding pair coupled to the support by a cleavable linker, the improvement comprising employing a compound as described herein as the cleavable linker (e.g. where R is the first member of the binding pair). The cleavable linker can be covalently coupled or specifically bound (e.g., by biotin-avidin binding) to the support.

3. Methods of use.

In use, the present invention provides a method of releasing a compound of interest bound to a support from that support, wherein the compound of interest is bound to the support through a cleavable linker. The method employs a cleavable linker comprising an acylhydrazone group therein (e.g., a cleavable linker as described herein and above), and is carried out by cleaving the acylhydrazone by contacting an acylhydrazide and/or an anionic detergent (e.g., sodium dodecyl sulfate; SDS) thereto.

In some embodiments of the foregoing, the contacting step is carried out under near neutral pH conditions (e.g., pH 4, 5 or 6 up to pH 8 or 9).

In some embodiments of the foregoing, the acylhydrazide is contacted to the acylhydrazone linker in solution in an amount of at least 1, 10 or 50 mM.

In some embodiments of the foregoing, the acylhydrazide is contacted to the acylhydrazone in solution in an amount up to 500 or 1000 mM.

In some embodiments of the foregoing, the anionic detergent is contacted to the acylhydrazone in solution in an amount of at least 0.5, 1, 5 or 10 mM. In some embodiments of the foregoing, the anionic detergent is contacted to the acylhydrazone in solution in an amount up to 50 or 100 mM.

In some embodiments of the foregoing, the contacting step is carried out at a temperature of at least 4, 10 or 20 ⁰C.

In some embodiments of the foregoing, the contacting step is carried out at a temperature of up to 40 or 50 °C.

In some embodiments of the foregoing, the contacting step is carried out for a time of at least 10, 20 or 30 minutes.

In some embodiments of the foregoing, the contacting step is carried out for a time of up to 2 or 4 hours.

In some embodiments of the foregoing,at least 50, 60, 70 or 80 percent by weight of the compound of interest is released following the contacting step.

In some embodiments of the foregoing, the acylhydrazide comprises a detectable group that reacts with the cleavable linker in the cleaving step, whereby the compound of interest can be detected after the cleaving step by detecting the detectable group coupled thereto.

Cleavable linkers of the present invention can be implemented in a variety of additional ways, such as in the methods, compounds and products described in US Patent Application Publication No. 20090181860 (Applied Biosystems) in place of the disulfide bond cleavable linkers described therein. Hence the present invention provides, among other things, a collection of at least two distinguishably labeled oligonucleotide probe families (optionally wherein probes in each probe family comprise a constrained portion and an unconstrained portion, optionally wherein each position in the constrained portion is at least 2-fold degenerate, and and optionally wherein probes in each family comprise a scissile internucleoside linkage). In some embodiments, each probe comprises a terminus that is not extendable by ligase. In some embodiments, each probe comprises a detectable moiety at a position between the scissile linkage and the terminus that is not extendable by ligase. In some embodiments, the scissile linkage is a phosphorothiolate linkage. In some embodiments, the collection comprises 2, 3, or 4 or more probe families. The detectable moiety is preferably attached by a cleavable linker, and optionally is photobleachable. The cleavable linker preferably comprises an acylhydrazone as described herein, and in use is cleaved by an acylhydrazide as described herein. The present invention is explained in greater detail in the following non-limiting Examples.

Experimental

We looked for an easy-to-prepare, cleavable linker that would, under mild conditions, efficiently release the (bio)molecules tethered to a support and for which destruction and/or denaturation of the (bio)molecules would be minimal. Ideally, the cleavage reaction would allow the incorporation of a traceable tag (i.e., isotopic, radioactive, fluorescent) that permits the detection and identification of the captured (bio)molecule by chromatographic and spectroscopic methods when it is released.

We here describe our test system, the biotin/streptavidin complex in which the protein of interest is attached to the biotin by an acylhydrazone cleavable linker (Figure 1). Acylhydrazones have been used extensively in drug delivery systems (Sawant et al., 2006), in monitoring conjugation reactions (Flinn et al., 2004), and as structural units in the construction of dynamic combinatorial libraries (Goral et al., 2001). Studies show that in most instances, acylhydrazones are stable under near neutral-to-basic pH conditions (pH ~8- 10), but they undergo both hydrolysis (Flinn et al., 2004; Smith et al., 2007) and hydrazone exchange with hydrazides (King et al., 1986) in moderately acidic solutions (pH -4-5). Herein, we show that biotinylated probes with an imbedded acylhydrazone unit are readily cleaved in high yields at near neutral pH values with acylhydrazides that contain a traceable tag providing (bio)molecules that can be identified in proteomic searches by conventional analytical techniques. We expect that incorporation of an acylhydrazone unit within a linker will be generalized, allowing for the selective release of tagged molecules from non- covalently or covalently bound supports.

A. RESULTS

Design and Chemical properties of an Illustrative Cleavable Linker. Compound 1 was selected and synthesized as our test linker (Figure 2). It contained an acylhydrazone cleavage site, a biotin unit, a terminal azide on 1 to permit capture with an alkyne-modified protein using a copper(I)-mediated cycloaddition reaction ("click chemistry") (Agard et al., 2006; Dieterich et al., 2007; MacKinnon et al., 2007; Rostovtsev et al., 2002; Speers et al., 2003; Tornoe et al., 2002), and a polyethylene glycol linker to increase its water solubility and to minimize adverse steric interactions with the immobilized streptavidin during protein capture (Marie et al., 1990). The success of 1 to capture biomolecules hinged upon its ability to undergo Cu(I)-mediated cycloaddition without disrupting the acylhydrazone linkage, its stability in aqueous solutions, and its ability to undergo cleavage with acylhydrazides. We first showed that the acylhydrazone linkage in 1 was stable to Cu(I)-mediated cycloaddition conditions (Scheme 1, data not shown), and remained intact at pH 7.4 and above but at pH 5.8 underwent consumption (38%) at 22 °C (2 h) (data not shown). Significantly, 1 efficiently reacted with acethydrazide (7) (20-100 equiv) at 22 °C to give 8 (Figure 2). For example, at pH 5.2 and 6.2 the reaction was essentially complete within 1 h at 22 °C, using as little as 20 equiv of 7 (HPLC analysis) (data not shown). The ease of this imine exchange was markedly faster than that reported by King and coworkers for a similar transformation (King et al., 1986). Next, we examined the stability of the acylhydrazone linker in the presence of detergents (SDS, NP-40, Triton X-100) since many proteomic protocols employ these agents to facilitate protein solubilization. HPLC analysis showed that when SDS (0.5 and 1%) was included in the buffered solutions (pH 7.4) there was a rapid loss of 1, while similar losses were not observed with the detergents NP-40 (1%) and Triton X-100 (1%) (data not shown).

Use of 1 for Protein Capture, Release, and Detection. To evaluate the utility of 1 for protein capture and release, we prepared BSA sample 9 (Scheme 2, proposed general structure of the photoadduct; the structure of the azide photoadduct has not been determined) (Figure 3A) after photocross-linking with photoactivable lacosamide probe followed by click chemistry with 1 (data not shown). The BSA sample 9 was captured by streptavidin-agarose beads and divided into equal aliquots. Each portion was either treated with or without 7 (50, 200 mM) at different pH values (5.8, 7.4) to release the captured proteins. We also tested whether either p-anisidine or aniline would facilitate acylhydrazone cleavage since these aryl amines have been reported to catalyze oxime ligation, hydrazone formation and transimination (acylhydrazone exchange) at moderate pH values (Dirksen et al., 2006a; Dirksen et al., 2006b). Consistent with our HPLC findings (data not shown), we observed increased linker cleavage at pH 5.8 compared with at pH 7.4, upon the addition of 7, and upon the inclusion of either />-anisidine or aniline with 7 (data not shown). We then examined the effect of 20 mM SDS (0.5%) on the cleavage reaction of 9 in aqueous pH 5.8 solutions at 22 ⁰C. In agreement with our HPLC studies (data not shown), we found that 20 mM SDS facilitated linker cleavage, and that inclusion of both SDS and 7 provided slightly enhanced levels of cleavage than with either one alone (data not shown). To examine the recovery efficiency of protein capture and release of our method, we quantified the percentage yield of the immobilization, flowthrough/wash, and elution steps using model protein 9. The release efficiency could be readily determined from the ratio of the amount eluted under mild cleavage conditions to that captured by streptavidin prior to cleavage. Significantly, more than 85% of the protein captured by streptavidin-agarose beads was released with 7 using mild conditions (7 (100 mM), SDS (20 mM) in aqueous 50 mM HEPES (pH 5.8) (50 °C, 1 h)). Under harsh cleavage conditions (SDS loading buffer (2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bromophenol blue) (95 ⁰C, 5 min)), protein recovery was near-quantitative (see experimental details below). These harsh conditions are expected to completely cleave the acyl hydrazone bond.

Since SDS can affect protein structure, function, and mass spectrometric identification, we identified conditions for the protein recovery step that excluded SDS as well as those that contained 20 mM SDS (0.5%) (data not shown). We found that 7-mediated cleavage of streptavidin bound 9 led to high protein recovery rates after 4 h in pH 5.8 and pH 7.4 (37 °C) solutions without 20 mM SDS. Specifically, we observed that at pH 5.8, inclusion of /7-anisidine in the reaction solution led to efficient cleavage (-97%) at 37 °C after 4 h. Correspondingly, high protein recovery rates (-93—98%) were also obtained at 50 °C after 1 h in acidic solutions (pH 4.2 and 5.8) upon the inclusion of 20 mM SDS with 7. Reducing the temperature from 50 ⁰C (-93%) to 37 °C (-72%) to 22 ⁰C (-48%) for the pH 5.8 reactions led to progressively lower cleavage yields.

To investigate the efficiency of cleavable linker 1 for protein capture and release compared with a non-cleavable linker, we synthesized linker 10 (Scheme 3, Sun et al., 2006). We compared streptavidin-bound BSA 9 containing 1 with streptavi din-bound BSA 11 containing 10 (Figure 3A). Non-cleavable linker 10 cannot undergo acylhydrazone cleavage, thus protein release can only occur by disruption of the biotin-(strept)avidin complex (Holmberg et al., 2005; Jenne et al., 1999; Tong et al., 1992). Both streptavidin-bound BSA 9 and 11 were treated for 1 h at pH 5.8 using 7 (100 mM) and SDS (20 mM) at 50 ⁰C (Figure 3B). The extent of modified BSA recovery was determined by analyzing the supernatant and bead samples. Inspection of Figure 3B showed that very low levels of protein recovery were obtained from the initial supernatant from the BSA sample 11 modified with the non- cleavable linker 10 (Figure 3B). Correspondingly, we observed high protein recovery from the supernatant from BSA 9 containing the cleavable linker 1 (Figure 3B, lane As). Consistent with reports in the literature (Holmberg et al., 2005; Tong et al., 1992), we observed low levels of protein recovery from the streptavidin-bound BSA 11 containing the non-cleavable linker 10 after the bead was treated with SDS-loading buffer at 95 ⁰C for 15 min (Figure 3B, lane Bb). We estimated that the relative efficiencies of protein recovery from the cleavable linker BSA adduct 9 under moderate conditions (pH 5.8, 7 (100 mM), SDS (20 mM), 50 °C, 1 h) compared with the non-cleavable linker BSA adduct 11 after heating (95 ⁰C) with SDS-loading buffer for 15 min to be ~20:l, based on comparison of the silver-stained protein lanes in Figure 3B for As and Bb.

Structural Validation of the Capture, Release and Reduction Steps and Detection of Protein Using Traceable Tag. An important structural feature in the acylhydrazide- mediated cleavage of linker 1 is the formation of a new acylhydrazone (e.g., 8). We anticipated that incorporating a traceable acylhydrazone tag (i.e., isotopic, radioactive, fluorescent) in the captured (bio)molecule would facilitate its detection and identification. The key chemical steps of the acylhydrazone-based cleavable linker methodology were validated by HPLC and mass spectrometry beginning with jV-acetyl-L-cysteine methyl ester (12) (Figure 4). Reacting 12 with N-(propyl-2-ynyl)maleimide (13) gave 14 as a mixture of diastereomers. Compound 14 was then treated with 1 using CuBr-mediated cycloaddition conditions to provide 15. The ¹H NMR, ¹³C NMR, and mass spectral data were in agreement with the proposed adduct (data not shown). Treatment of 15 with an ~1 :1 mixture of 7 and 7- di- (p-anisidine (10 mM), pH 5.7, 37 ⁰C, 2 h) gave the corresponding exchanged acetylhydrazone 16 and 16-J₃ as the major product (HPLC /_R 12.8 min, data not shown). Mass spectral analysis of the HPLC purified 12.8 min product showed a diagnostic "doublet" at m/z 608.2 and 61 1.2 for 16 and 16-dj [M + Na]⁺, respectively (data not shown). Correspondingly, sequential conversion of 15 to imine 16 and 16-di (pH 5.8) followed by NaCNBH₃ reduction (pH 3.8) led to the appearance of a new peak in the HPLC (data not shown). Isolation of the new product by HPLC and mass spectral analysis gave a "doublet" pattern at m/z 610.2 and 613.2 consistent with the reduced acethydrazides 17 and 17-rf₃ [M+Na]⁺, respectively (Figure 4). The facility of this reduction is important since it provides a chemically stable product not readily susceptible to acid hydrolysis and which is suitable for mass spectral analysis. When the NaCNBH₃ reduction was conducted at higher pH values (pH -7.4) 17 and 17-d^ were not observed but rather we isolated the anisidine derivative 18 (m/z 637.3 [M+H]⁺) (Figure 4). This finding suggested that at near neutral pH values 16 and 16-d₃ underwent transimination with anisidine and then reduction to give 18 as the major product. These results demonstrated that the acylhydrazide-mediated cleavage of the linker provided a traceable tag in the cleaved product that can be monitored by mass spectrometry.

Similarly, we showed that fluorescein hydrazide treatment of enolase sample 21 modified with cleavable linker 1 (Figure 5A) led to the appearance of a strong fluorescent band corresponding to the expected enolase adducts both before and after NaCNBH₃ (pH 3.8) treatment (Figure 5B). Since only fluorescently labeled proteins are released this methodology can significantly increase both the detection sensitivity and purification efficiency of the proteins of interest in proteomic analyses.

Use of Cleavable Linker 1 in Proteomic Target Searches: Capture and Identification. Next, we tested whether cleavable linker 1 can be applied to capture proteins of interest from a natural proteome. The Cravatt laboratory showed that alkyne phenyl sulfonate ester 25 (Speers et al., 2004; Weerapana et al., 2008) (Figure 6A) selectively reacted with aldehyde dehydrogenase- 1 (ALDH-I) in the mouse soluble liver lysate. We asked whether the use of 25 and cleavable linker 1 would permit efficient capture and removal of ALDH-I from the mouse soluble liver proteome using streptavidin beads, and whether the release of the captured ALDH-I from the streptavidin support with 7 would proceed with minimal release of non-specific "background" proteins that bind to the streptavidin resin (Fonovic et al., 2007).

Accordingly, mouse soluble liver lysate (pH 8.0) was incubated with 25 (5, 25 μM) at room temperature (1 h) and then treated with rhodamine azide (RhN₃, 26), TCEP, TBTA and CuSO₄. The lysate was separated by SDS-PAGE and the labeled proteins visualized by in-gel fluorescence (Figure 6B). In agreement with earlier reports (Speers et al., 2004), only a single protein band was observed at ~50 kDa when 5 μM of 25 was used (Figure 3A, lane 1). Increasing the concentration of 25 to 25 μM led to increased intensity of this band and the appearance of lower molecular weight bands (Figure 6B, lane 3). The in-gel detection of the -50 kDa band was consistent with the selective tagging of ALDH-I by activity probe 25 as detailed in the literature (Speers et al., 2004).

The experiment was repeated with 25 (25 μM) substituting either cleavable linker 1 or non-cleavable linker 10 for 26. The labeled complexes were captured by adding streptavidin- agarose beads. After extensive wash with buffer, the recovered beads from both the biotinylated cleavable 1 and the non-cleavable 10 probes were treated with 7 (100 mM) and p-anisidine (10 mM), pH 5.8 at 37 °C (4 h), conditions designed to release proteins from cleavable linker 1. The supernatants from these reactions were recovered. The remaining beads were treated with SDS-loading buffer, heated at 95 °C (5 min) and the supernatants recovered. The recovered protein samples were loaded on SDS-PAGE and the resolved bands visualized by silver staining (Figure 6C) and analyzed by western blot (Figure 6D). The silver stained gel lane for the 7-treated supernatant from cleavable linker 1 was remarkably clean displaying only two prominent bands at -50 and -44 kDa (Figure 6C, lane As). This result is consistent with that shown in Figure 6B. Based on the result from Coomassie blue staining of the untreated whole lysate, we suspect that the 44 kDa band corresponds to abundant protein(s) (data not shown). Significantly, the lane from the supernatant recovered from the remaining beads treated with loading buffer (95 ⁰C, 5 min) showed these two bands along with significant amounts of other bands (Figure 6C, lane Ab), indicating that many background proteins are indeed non-specifically captured by the streptavidin-agarose beads. Correspondingly, the 7-treated supernatant from non-cleavable linker 10 displayed only a weak band at -44 kDa and no band at -50 kDa (Figure 6C, lane Bs) while the remaining beads lane showed significant amounts of the -44 kDa and -50 kDa bands corresponding to an abundant protein(s) and ALDH-I, respectively, and many other bands attributed to background proteins (Figure 6C, lane Bb). To confirm that the selectively captured 50 kDa protein by the cleavable linker approach is indeed ALDH-I, we performed western blot analysis using an ALDH-I antibody (Figure 6D). We observed a prominent band consistent for ALDH-I in the supernatant recovered from cleavable linker 1 upon treatment with 7 but not in the corresponding supernatant from non-cleavable linker 10 (Figure 6D, lanes As versus Bs). Analysis of the SDS loading buffer-treated beads from both reactions showed the presence of ALDH-I (Figure 6D, lanes Ab and Bb). The silver stained and western blot gels documented that the supernatant recovered from the cleavable linker 1 after 7-mediated cleavage was highly enriched with target protein ALDH-I and was devoid of most background proteins present in the lysate. Correspondingly, ALDH-I was not detected in the supernatant from the non-cleavable linker 10 (Figures 6C and 6D, lane Bs), but it was observed after harshly heating (95 °C, 5 min) the recovered streptavidin resin with SDS- loading buffer (Figures 6C and 6D, lane Bb), a condition that results in the release of many background proteins.

B. DISCUSSION

We selected 1 as our prototype acylhydrazone linker because of its structural properties and its expected solubility in water. The acylhydrazone moiety in 1 is the cleavage site, and it provided the necessary balance between stability and reactivity. We found that under mildly acidic conditions (pH 5.8) the acylhydrazone unit underwent cleavage while at near neutral (pH 7.4) and basic (pH 10) conditions it remained intact (data not shown). This finding is in general agreement with earlier reports (Goral et al., 2001 ; King et al., 1986). The observed pH-dependency for 1 hydrolysis led us to use moderately basic (pH 7.4-8.5) conditions for storage, handling, and chemical transformations where cleavage of the linker was not desired. We identified additives that promoted cleavage of the acylhydrazone group. Using the findings of Dirksen et al. (2006a & 2006b), we confirmed that both aniline andp- anisidine facilitated acylhydrazide exchange (pH 5.8, 7.4) (data not shown). A surprising result was that the anionic detergent SDS fostered acylhydrazone cleavage (data not shown). Correspondingly, use of non-anionic detergents, NP-40 and Triton X-100, in place of SDS did not promote cleavage. The chemical factors responsible for the SDS-mediated process have not been determined. Specifically, we do not know if SDS serves as either a specific or general base in the hydrolysis step, or SDS-mediated acylhydrazone loss is due to direct attack of the hydrazone linkage by SDS, or if SDS-mediated loss is due, in part, to the formation of micelles that facilitates hydrazone modification (Camilo et al., 2004; Gogoi et al., 2005; Wang et al., 2005). Most important for our study was our demonstrating that 7 induced hydrazone exchange in 1. Acethydrazide exchange of acylhydrazones has been described by King et al. (1986). The reaction was reported to proceed to near completion within 2 days at pH 4.7 and 5.2 (25 ⁰C), but at pH 7.0 substantial amounts of unreacted starting material (-40%) remained after 8 d. We found that with 1 the reaction proceeded to near completion at pH 5.2 and 6.2 within 1 h at 22 ⁰C and that high yields of exchange were observed at pH 7.4 after 7 h at 22 °C (data not shown). Similarly efficient cleavage for the acylhydrazone group was observed in the BSA modified product 9 bound to the streptavidin support, and the reaction was catalyzed by the additive p-anisidine, which permitted efficient cleavage at pH 7.4 within 2 h (data not shown). The facility with which 7 cleaves 1 at near neutral pH values compared with the earlier findings (King et al., 1986) suggests that structural changes surrounding the acylhyrazone linkage may affect cleavage rates.

To test the utility of 7-mediated cleavage of acylhydrazone linkers for protein capture and isolation, we used two protein-based model systems. Since linker 1 contained a terminal azide unit we installed an alkyne moiety (moieties) within the test proteins to permit Cu(I)- mediated cycloaddition reaction. Accordingly, BSA was randomly modified with the photoaffinity lacosamide probe to give an alkyne functionalized BSA (data not shown). Correspondingly, the single cysteine unit in S. cerevisicte enolase (19) (Chin et al., 1981 ; Holland et al., 1981) permitted the Michael addition to N-(prop-2-ynyl)maleimide (13) to give alkyne functionalized enolase 20 (Figure 5). Together these modified proteins permitted us to test the utility of acylhydrazone linker 1. The biotinylated BSA products 9 and 11 obtained after Cu(I)-mediated cycloaddtion with 1 and 10, respectively, were captured with streptavidin beads. Utilizing the information learned in our model studies with 1, we identified a series of conditions that led to efficient cleavage of the acyhydrazone linker in BSA 9, allowing protein release from the streptavidin support. Using 100 mM 7 and 20 mM SDS, we observed efficient cleavage and modified BSA release within 1 h at 50 ⁰C in pH 5.8 solutions (data not shown). High yields of modified BSA release were also observed within 4 h in both pH 5.8 (22 °C) and pH 7.4 (37 °C) solutions containing 7 (100 mM) and /7-anisidine (10 mM). Excluding SDS in the cleavage protocol may facilitate subsequent mass spectrometric detection of the recovered protein and permit us to identify multi-protein complexes that maintain their bioactive conformations. Finally, we expect that the acylhydrazone group generated after linker cleavage can serve as a convenient aldehyde- protecting group that can be removed with mild acid thereby permitting further protein tagging and modification. Correspondingly, 7-mediated cleavage of 11 led to the release of very low levels of BSA (Figure 3B). When we compared the relative amounts of BSA recovered from streptavidin-bound BSA 9 (containing cleavable linker 1) with streptavidin- bound BSA 11 (containing non-cleavable linker 10) we observed a ~20-fold increases in BSA recovery with 9. This increased amount is significant since BSA release from 9 proceeded under mild conditions while harsh conditions were required for 11. Using the modified BSA protein 9 we showed that 7-mediated acyl hydrazone cleavage under mild conditions led to the release of more than 85% of the protein captured by the streptavidin-agarose beads (experimental details below).

The cleavage step was validated by treatment of the modified iV-acetyl-L-cysteine methyl ester adduct 15 with 7 and 1-dτ, (Figure 4). Mass spectrometric analysis of both the cleaved acetylhydrazone adducts 16 and 16-J₃, and the corresponding NaCNBH₃-reduced adducts 17 and 17-c?₃ showed a characteristic mass spectrometric signature pattern ([M] + [M+3]) for the desired products (data not shown). Our finding that NaCNBH₃-reduction of the reaction mixture containing 16, and 7 and 1-dτ, at pH -7.4 provided />-anisidine adduct 18 indicated that employment of a mixture of non-deuterated and deuterated aniline derivatives in the cleavage step can similarly provide a diagnostic mass spectral pattern useful for protein identification.

An important component in the design of the acyhydrazone linker was incorporating a traceable tag upon protein release from the support. We further documented this design feature using fluorescent hydrazide 22 for acylhydrazone linker cleavage in streptavidin bound enolase 21 (Figure 5). With 22 we showed that acylhydrazone exchange of streptavidin-bound enolase 21 led to a protein band in the gel that was readily visualized on fluorescent imaging. Since only the tagged proteins were fluorescently labeled and released, this approach could significantly increase both the detection sensitivity and purification efficiency of the proteins of interest in proteomic analyses. Finally, we recognized that the acylhydrazone group in the protein-released product can undergo reduction with NaCNBH₃ to the corresponding acylhydrazide, a chemically stable product not readily susceptible to acid hydrolysis. Thus, we reduced the hydrazone group in enolase 23 with NaCNBH₃ in acid to give 24. This reduction step provides a convenient opportunity to incorporate a radioactive tag in the protein through the use of [³H]-NaCNBH₃, which can aid protein detection and identification studies.

The utility of cleavable linker 1 was tested in a proteomic search. Cravatt and coworkers have elegantly demonstrated that ALDH-I was selectively labeled in the mouse soluble liver lysate by phenyl sulfonate ester 25 at pH 8.0. The specificity of this activity probe for ALDH-I provided a stringent test for our linker. We asked if linker 1 selectively reacted with 25-modified ALDH-I by Cu(I)-mediated cycloaddition and whether, upon streptavidin bead capture and treatment of the resin with 7 under mild conditions, the supernatant would be enriched in ALDH-I and be devoid of background proteins. Using cleavable linker 1, we demonstrated that the supernatant recovered after treating the streptavidin beads with 7 under mild conditions was enriched in ALDH-I and was remarkably clean of background proteins (Figure 6C). The silver stained gel for this sample showed only two prominent protein bands, the first at -50 kDa corresponding to ALDH-I and the second at ~44 kDa, which we suspect is an abundant protein(s) in the lysate that was modified by the 25 μM 25. The absence of background proteins in Figure 6C, lane As, is important since proteomic target identification studies have been hampered by the presence of such proteins in samples containing the target(s) (Fonovic et al., 2007). As anticipated, ALDH-I was not detected in the supernatant after treatment of the beads with 7 when the non-cleavable linker 10 was employed in the Cu(I)-mediated cycloaddition reaction, but it was observed after harshly heating (95 ⁰C, 5 min) the recovered streptavidin resin with loading buffer containing 2% SDS and 1% mercaptoethanol, a condition that results in the release of many background proteins that nonspecifically bound to streptavidin resin. The recovered yield for ALDH-I in the supernatant was somewhat lower than that observed for cleaved BSA 9 under similar conditions (Figure 6), and may reflect the different nonspecific matrix binding properties of these two proteins.

Our studies demonstrated that incorporating an acylhydrazone group within the biotin-containing linker permits efficient linker cleavage from the streptavidin support. Some advantages associated with this method include: (1) the ease of linker synthesis; (2) stability of the linker to bioorthogonal coupling transformations (e.g., Cu(I) -mediated cycloaddition reactions); (3) linker stability at near neutral and moderately basic pH values; (4) chemoselective cleavage of the linker with acylhydrazides with minimal release of background proteins non-specifically bound to (streptavidin) supports; (5) high recovery yields of the cleaved proteins; and (6) ability to incorporate a traceable tag (e.g., isotopic, fluorescent) in the captured protein to facilitate detection and identification. Finally, the mild conditions needed for acylhydrazone cleavage offers the possibility that non-denatured proteins or protein complexes can be captured and then released. Countering these advantages is the need to avoid moderately acidic pH values during protein capture to prevent premature linker cleavage and the recognition that linker cleavage leads to a permanent structural modification in the protein.

The acylhydrazone linker compares favorably with previously introduced cleavable linkers. Like 1, each linker has advantages and disadvantages to consider prior to selection and use. For instance, photolytic-based cleavable linkers typically undergo efficient release, but upon cleavage, they release reactive carbonyl compounds (aldehydes, ketones) that can react with the protein (Bai et al., 2004; Thiele et al., 1994). Acid-sensitive linkers that require trifluoroacetic acid (TFA) can lead to non-specific cleavage and TFA use requires its removal prior to MS analysis (van der Veken et al., 2005). Linkers that rely on proteolytic enzymes (e.g., trypsin) provide a selective cleavage method but require that a designed peptide sequence be installed within the linker that is efficiently cleaved (Dieterich et al., 2007; Fonovic et al., 2007; Speers et al., 2005). The use of proteolytic enzymes for cleavage may prevent the isolation of the intact protein. Proteomic experiments utilizing disulfide linkers require the use of buffers that are devoid of reducing reagents (Finn et al., 1985; Fonovic et al., 2007; Marie et al., 1990; Shimkus et al., 1985). Moreover, these disulfide linkers can be cleaved under cellular conditions. This concern for premature cleavage of the linker is not an issue for the diazobenzene linker advanced by Bogyo and coworkers and where Na₂S₂O₄ serves as the chemoselective reductant (Fonovic et al., 2007; Verhelst et al., 2007).

Our procedure compliments the widely used method that couples on-bead digestion and mass spectrometry (Chrestensen et al., 2004). This on-bead method benefits from its simplicity and ease of use. Unlike the on-bead method, our procedure allows the chemoselective release of the protein of interest, the incorporation of an isotopic tag that facilitates mass spectrometric identification, the isolation of the intact protein, and the opportunity to identify the site of protein adduction.

C. EXPERIMENTAL PROCEDURES I

Synthesis of Compounds 3, 4, 7-</₃, 8, 10, 11, 13-15, 16, 16-rf₃, 17 and 17-rf₃, 27, 30, and 32-34. See Experimental Procedures II below.

Preparation of iV-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy) acetaldehyde Biotinyl- hydrazone (1). To an anhydrous CH₂Cl₂ solution (2 mL) of oxalyl chloride (0.10 mL, 1.10 mmol) maintained at -78 °C under Ar was slowly added freshly distilled DMSO (0.19 mL, 3.29 mmol). The mixture was stirred (30 min), and then dry 4 (0.20 g, 0.91 mmol) was added via syringe. The mixture was stirred for an additional 30 min at -78 °C, and then Et₃N (0.78 mL, 6.57 mmol) was added (10 min) and the mixture was stirred (15 min) at this temperature and warmed to ambient temperature. The reaction solution was diluted with CH₂Cl₂ (10 mL) and washed with H₂O (2 x 10 mL). The organic layer was dried (Na₂SO₄), concentrated, and the crude aldehyde 5 (0.19 g, 0.88 mmol) was dissolved in CH₂Cl₂ (1 mL) and added to a solution of 6 (0.15 g, 0.58 mmol) in THFVH₂O (1 : 1, 5 mL). The reaction mixture was allowed to stir at room temperature (16 h), the solvent was evaporated in vacuo, and then the crude product purified by column chromatography (SiO₂; 1/9 MeOH/CHCl₃) to yield 0.20 g (74%) of 1 as a white solid: mp 134-135 ⁰C; R₁= 0.35 (1/9 MeOH/CHCl₃); IR (nujol) 2924, 2101, 1667, 1557, 1459 cm^"1; ¹H NMR (CD₃OD) (minor conformer in parenthesis (a mixture of conformers similar to other hydrazones (Syakaev et al. 2006))) δ 1.45-1.52 (m, C(6)H2), 1.58-1.79 (m, C(7)H₂, C(8)H₂), 2.27 (t, J = 7.4 Hz, C(9)H₂ (2.65 (t, J = 7.5 Hz, C(9)H₂))), 2.71 (d, J- 12.6 Hz, C(5)HH'), 2.93 (dd, J= 4.7, 12.6 Hz, C(5)HH'), 3.18-3.25 (m, C(2)H), 3.37 (t, J = 4.8 Hz, N₃CH₂), 3.65-3.69 (m, 2 OCH₂CH₂O, N₃CH₂CH₂), 4.18 (d, J = 5.3 Hz, OCH₂CHN (4.15 (d, J= 5.3 Hz, OCH₂CHN))), 4.31 (dd, J- 4.2, 7.6 Hz, C(3)H), 4.49 (dd, J = 4.7, 7.6 Hz, C(4)H), 7.49 (t, J= 5.3 Hz, OCH₂CHN (7.33 (t, J= 5.3 Hz, OCH₂CHN))); ¹³C NMR (CD₃OD) (minor conformer in parenthesis) δ 26.0 (26.6) (C(6)), 29.6 (C(7)), 29.9 (30.0) (C(8)), 33.2 (35.2) (C(9)), 41.2 (C(5)), 51.9 (N₃CH₂), 57.1 (57.2) (C(2)), 61.8 (C(3)), 63.5 (C(4)), 71.2, 71.3, 71.4, 71.5, 71.7, 71.8 (3 CH₂OCH₂), 145.6 (149.3) (OCH₂CHN), 166.3 (C(2')), 172.7 (177.9) (C(IO)); HRMS (ESI) 458.2182 [M + H⁺] (calcd. for Ci₈H₃₂N₇O₅S 458.2186); Anal. (C₁₈H₃iN₇O₅S, 0.25 H₂O) Calcd.: C, 46.79%; H, 6.87%; N, 21.22%; S, 6.94%. Found: C, 46.67%; H, 6.82%; N, 20.88%; S, 6.88%.

Treatment of Enolase (19) with Maleimide 13 to Afford Enolase 20. To a 200 μM solution (1 mL) of S. cerevisiae enolase (19) (Sigma, E6126) in aqueous 50 mM HEPES (pH 7.4) was added a 200 mM solution (0.1 mL) of 13 (2.7 mg, 20.0 //mol) in 5% CH₃CN/aqueous 50 mM HEPES (pH 7.4). The solution was incubated at room temperature (2 h) and then the reaction solution was diluted with aqueous 50 mM HEPES buffer (pH 7.4) to 5 mL and passed through NAP-5 columns pre-equilibrated with 50 mM HEPES buffer (pH 7.4). The eluents (~10 mL) were combined and stored at 4 °C.

Cycloaddition Reaction Between Enolase 20 and Cleavable Linker 1 to Afford Enolase 21 (Method A). To an aqueous 50 mM HEPES solution containing 20 that was obtained after passage through the NAP-5 column (1 mL), was sequentially added a 20 mM solution (50 //L) of 1 (0.44 mg, 1.0 /rniol) in 5% CH₃CN/aqueous 50 mM HEPES (pH 7.4), a 20 mM solution (50 μL) of biotin hydrazide (6) (0.26 mg, 1.0 //mol) in 5% CH₃CN/aqueous 50 mM HEPES (pH 7.4), and CuBr (0.2 mg, 1.4 //mol). The reaction mixture was rotated using Roto-shake (8 rpm, Scientific Industries Inc., Model No. SI-1100, Bohemia, NY) at room temperature (1 h), then divided in two equal portions and passed through separate NAP- 5 columns pre-equilibrated with HEPES buffer (pH 7.4) to give an aqueous solution of enolase 21. The eluents were combined (~2 mL) and stored at 4 °C. Use of Fluorescent Hydrazide 22 for Detection and Isolation of Streptavidin- bound Modified Enolase 21 and Reduction of Imine 23 to 24 (Method B). An aliquot of enolase 21 (200 μL) in aqueous 50 mM HEPES buffer (pH 7.4) was added to an immobilized streptavidin slurry (1 mL) (High Capacity Streptavidin Agarose Resin, Pierce, Rockford, IL) and rotated using a shaker (15 rpm) at 4 °C (90 min). The streptavidin beads were washed with aqueous 15 mM HEPES buffer (pH 7.4) (10 x 0.8 mL). The beads were centrifuged (1000 rpm, 1 min), and the supernatant removed. An aqueous 50 mM HEPES solution (100 μL, pH 5.8) was added and the beads treated with fluorescent hydrazide 22 (Alexa fluor hydrazide, Al 0436, Molecular Probes, 85.5 μg, 0.15 μmol; final concentration 1.5 mM) and /7-anisidine (123.0 μg, 1 μmol; final concentration 10 mM). The reaction mixture was gently shaken at 37 °C (4 h) and then the supernatant (100 //L) containing 23 was collected.

The supernatant (50 μL) was treated with a 10 mM solution Of NaCNBH₃ (50 μL) in 50 mM NaOAc buffer (pH 3.8) and rotated using Roto-shake (8 rpm) at room temperature (2 h) to give a mixture containing 24. Both the supernatant containing 23 and the reaction mixture containing 24 were loaded on a 10% SDS-PAGE gel. Labeled proteins were visualized using a typhoon 9400 scanner (Amersham Bioscience) with excitation at 488 nm and detection at 520 nm.

Proteome Sample Preparation, Probe Labeling, Cycloaddition Reaction, and In- GeI Fluorescence Scanning. Compound 25 and azide-rhodamine reporter tag (RhN₃, 26) were prepared according to previous reports (Speers et al., 2004). Mouse liver lysates (43 μL of 2.0 mg/mL protein in PB (pH 8.0)), prepared as described in Experimental Procedures II below, were treated with 5 μM or 25 μM 25 at room temperature (1 h). To incubated lysates with 25 were sequentially added 26 (100 μM), TCEP (1 mM), TBTA (100 μM) and CuSO₄ (1 mM). Samples were vortexed and allowed to react at room temperature (1 h). Proteins were separated by SDS-PAGE after addition of 4χ SDS-PAGE loading buffer and visualized in-gel using a typhoon 9400 scanner (Amersham Bioscience) with excitation at 555 nm and detection at 580 nm.

Capture and Release of Probe-labeled Proteins from Mouse Liver Lysate. Mouse soluble liver lysate (400 μL) was treated with 25 (3.08 μg, 10 nmol, 25μM) at room temperature (1 h) and passed through a NAP-5 column to exchange buffer to an aqueous 50 mM HEPES buffer (pH 7.4) and then divided in two 250 μL aliquots. Utilizing Method A, to each aliquot was added a 5 mM solution (10 μL) of either 1 or 10 (22 μg, 50 nmol) in 5% CH₃CN/aqueous 50 mM HEPES (pH 7.4), a 5 mM solution (10 μL) of biotin hydrazide (6) (13 μg, 50 nmol) in CH₃CN/aqueous 50 mM HEPES (pH 7.4), and CuBr (0.2 mg, 1.4 μmol). Using Method B, immobilized streptavidin slurry (300 μL) was added and the beads were treated with 7 (0.78 mg, 10 μmol) and/»-anisidine [final concentration 10 mM] at 37 ⁰C (4 h). The supernatants were collected and then the remaining streptavidin beads were boiled (5 min) with SDS-PAGE loading buffer (2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bromophenol blue). The samples were loaded on two SDS-PAGE (10%) gels and the proteins visualized by silver staining and detected for ALDH-I by western blot using anti- ALDH-I antibody (ab23375, Abeam) as detailed in the Experimental Procedures II below.

D. EXPERIMENTAL PROCEDURES II

General Methods. Melting points were determined with a Thomas-Hoover melting point apparatus. Infrared spectra (IR) were run on a ATI Mattson Genesis Series FTIR™ spectrometer. Absorption values are expressed in wave-numbers (cm^"1). Proton (¹H NMR, 300 MHz) and carbon (¹³C NMR, 75 MHz) nuclear magnetic resonance spectra were taken on a Varian Gemini 2000 spectrometer. The high-resolution mass spectrum was performed on a Bruker Apex-Q 12 Tesla FTICR by Dr. M. Crowe at the University of North Carolina- Chapel Hill, and the low-resolution mass spectra were done on a BioToF-II-Bruker Daltonics spectrometer by Dr. S. Habibi. Microanalysis was provided by Atlantic Microlab, Inc. (Norcross, GA). TLC was performed on silica gel 60F₂₅₄ glass plates. Analytical HPLC was performed using Waters 2690 and Waters 600 instrument at 254 nm (Waters 996 and Waters 2487 instrument), and a Waters μBondapak C-18 column (3.9 x 300 mm, Waters Corp. Cat. No. WAT027324) was utilized and a gradient mobile phase (0/100 CH₃CN/H₂O - 50/50 CH₃CN/H₂O) was employed for 30 min.

2-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)ethyl p-Tosylate (3). An aqueous NaOH (0.69 g, 17.13 mmol) solution (4 mL) was added to a THF solution (4 mL) of tetraethylene glycol (2) (21.95 g, 113 mmol) at 0 °C and then a THF solution (13 mL) of/?-toluenesulfonyl chloride (2.08 g, 10.93 mmol) was added while stirring (1 h). After stirring at 0 °C (2 h), the reaction mixture was poured into ice H₂O (65 mL). The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ (3 x 50 mL). The combined organic layers were washed twice with H₂O (50 mL). The organic layer was dried (Na₂SO₄) and evaporated in vacuo to yield 3.80 g (99%) of crude 3 as a yellow oil (Kishimoto et al., 2005): R_f = 0.55 (EtOAc); ¹H NMR (CDCl₃) δ 2.59 (s, CH₃), 3.73-3.87 (m, 3 CH₂OCH₂, CH₂OH), 4.30-4.32 (m, CH₂OTs), 7.48 (d, J= 8.4 Hz, 2 ArH), 7.94 (d, J= 8.4 Hz, 2 ArH); ¹³C NMR (CDCl₃) δ 21.8 (CH₃), 61.9 (CH₂OH), 68.9, 69.4, 70.5, 70.6, 70.8, 70.9, 72.6 (3 CH₂OCH₂, CH₂OTs ),128.2, 130.0, 133.2, 145.0 (C₆H₄).

2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethanol (4). NaN₃ (1.40 g, 21.55 mmol) was added to an EtOH solution (60 mL) of 3 (3.00 g, 8.62 mmol). After stirring at 70 °C (18 h), H₂O (50 mL) was added and the solvent was concentrated to ~l/3 volume in vacuo. The remaining solution was extracted with EtOAc (3 x 50 mL). The combined organic phases were dried (Na₂SO₄) and evaporated in vacuo, and the crude product purified by column chromatography (SiO₂; 1/9 MeOH/CHCl₃) to yield 1.63 g (86%) of 4 as a yellow oil (Goncalves et al., 2005): R_f= 0.50 (EtOAc); ¹H NMR (CDCl₃) δ 2.60 (s, OH), 3.40 (t, J= 5.1 Hz, N₃CH₂), 3.60-3.63 (m, CH₂OH), 3.67-3.70 (m, 2 OCH₂CH₂O, N₃CH₂CH₂), 3.72-3.75 (m, CH₂CH₂OH); ¹³C NMR (CDCl₃) δ 50.8 (N₃CH₂), 61.9 (CH₂OH), 70.2, 70.5, 70.7, 70.8, 70.9, 72.6 (3 CH₂OCH₂).

Synthesis of Acethydrazide-rf₃ (7-</₃). Hydrazine (6.0 mL, 0.19 M) was cooled to 0 °C and acetyl chloride-d₃ (4.2 mL, 0.06 M) was added over 2 min. After stirring at 0 ⁰C (1 h), the reaction mixture was dissolved in EtOH (10 mL) and filtered. The organic layer was concentrated in vacuo and the crude product purified by column chromatography (SiO₂; 1/4 MeOH/CHCl₃) to yield 3.62 g (80%) of 7-^₃ as a white solid: mp 61-64 °C (mp for acethydrazide: 53-58 ⁰C); R_f = 0.50 (1/4 MeOH/CHCl₃); ¹H NMR (DMSO-c/₆) δ 4.14 (s, NHNH₂), 8.95 (s, NHNH₂): ¹³C NMR (DMSO-^₆) δ 19.3-20.3 (CD₃C(O)), 168.8 (C(O)).

Exchange of Hydrazone Linkage in 1 with Acethydrazide (7) and Acethydrazide- di (7-rf₃) Compound 1 (0.06 g, 0.14 mmol) was dissolved in a 5% CH₃CN/ aqueous 50 mM HEPES solution (5 mL, pH 5.6) and a 1:1 mixture of 7 and l-d₃ (0.54 g, 6.77 mmol) was added. The reaction solution was allowed to stir at room temperature (2 h), the solvent was evaporated in vacuo, and then the crude product purified by column chromatography (SiO₂; 1/9 MeOH/CHCl₃) to yield 0.30 g (81%) of a mixture of 8 and 8-d₃ as a yellow oil; R_f= 0.50 (1/9 MeOH/CHCl₃); ¹H NMR (CDCl₃) (minor conformers (for similar production of conformers, see Syakaev et al., 2006) in parenthesis) δ 2.25 (s, 1/2 C(O)CH₃ (2.07 (s, 1/2 C(O)CH₃))), 3.40 (t, J= 5.0 Hz, N₃CH₂), 3.66-3.70 (m, 2 OCH₂CH₂O, N₃CH₂CH₂), 4.16 (d, J = 5.4 Hz, OCH₂CHN (4.28 (d, J = 3.3 Hz, OCH₂CHN), 4.23 (d, J = 4.8 Hz, OCH₂CHN), (4.34 (d, J = 2.7 Hz, OCH₂CHN))), 7.25 (t, J = 5.0 Hz, OCH₂CHN (6.57 (t, J = 3.5 Hz, OCH₂CHN), 7.49 (t, J = 5.0 Hz, OCH₂CHN))), 9.62 (s, NH (9.28 (s, NH), 10.05 (s, NH))); ¹³C NMR (CDCl₃) (minor conformer in parenthesis) δ 20.4 (1/2 CH₃C(O)), 50.9 (N₃CH₂), 70.2, 70.3, 70.4, 70.7, 70.8, 70.9 (3 CH₂OCH₂), 143.4 (147.3) (OCH₂CHN), 173.8 (C(O)); HRMS (ESI) 296.1337 [M + Na⁺] (calcd. for C₁₀H₁₉N₅O₄Na 296.1335), 299.1525 [M + Na⁺] (calcd. for Ci₀Hi₆D₃N₅O₄Na 299.1520).

Hydrazone Exchange of 1 Using Acethydrazide (7) to Give 8. Compound 1 (0.06 g, 0.14 mmol) was dissolved in a 5% CH₃CN/aqueous 50 mM HEPES solution (5 mL, pH 5.6) and 7 (0.53 g, 6.77 mmol) was added. The reaction solution was allowed to stir at room temperature (2 h), the solvent was evaporated in vacuo, and then the crude product purified by column chromatography (SiO₂; 1/9 MeOH/CHCl₃) to yield 0.03 g (84%) of 8 as a yellow oil; Rf = 0.50 (1/9 MeOH/CHCl₃); ¹H NMR (CDCl₃) (minor conformers in parenthesis) δ 2.25 (s, C(O)CH₃ (2.06 (s, C(O)CH₃))), 3.40 (t, J = 5.0 Hz, N₃CH₂), 3.66-3.70 (m, 2 OCH₂CH₂O, N₃CH₂CH₂), 4.16 (d, J= 5.1 Hz, OCH₂CHN (4.28 (d, J= 3.0 Hz, OCH₂CHN), 4.23 (d, J = 5.1 Hz, OCH₂CHN), 4.34 (d, J = 2.7 Hz, OCH₂CHN))), 7.27 (t, J = 5.1 Hz, OCH₂CHN (7.48 (t, J = 5.1 Hz, OCH₂CHN))), 10.14 (s, CHNNH (9.60 (s, CHNNH); ¹³C NMR (CDCl₃) (minor conformer in parenthesis) δ 20.4 (21.8) (CH₃C(O)), 50.9 (N₃CH₂), 70.2, 70.3, 70.4, 70.7, 70.8, 70.9 (3 CH₂OCH₂), 143.5 (147.1) (OCH₂CHN), 174.0 (166.9) (C(O)); HRMS (ESI) 274.1516 [M + H⁺] (calcd. for Ci₀H₂₀N₅O₄ 274.1515); Anal. (C₁₀H₁₉N₅O₄) Calcd.: C, 43.95%; H, 7.01%; N, 25.63%. Found: C, 43.68%; H, 7.09%; N, 24.96%.

iV-(Prop-2-ynyl)maleimide (13). A solution Of Ph₃P (2.67 g, 10.3 mmol) in THF (40 mL) was cooled to -78 °C under Ar, and diethyl diazodicarboxylate (1.88 mL, 10.3 mmol) was added over 2 min. After the reaction mixture was stirred (5 min), propargyl alcohol (0.61 mL, 10.3 mmol) was added over 5 min and the reaction mixture was allowed to stir (5 min). Maleimide (1.00 g, 10.3 mmol) was added to the reaction mixture as a solid and the resulting mixture was allowed to remain at -78 °C (5 min), and then stirred at ambient temperature (48 h). The solvent was evaporated in vacuo, and then the crude product purified by column chromatography (SiO₂; 1/3 EtOAc/hexanes) to give 0.40 g (30%) of 13 (Karlen et al., 1970) as a yellow oil: R_f = 0.70 (1/3 EtOAc/hexanes); ¹H NMR (CDCl₃) δ 2.16 (t, J = 2.6 Hz, CCH), 4.22 (d, J = 2.6 Hz, CH₂C), 6.71 (s, 2 CHC(O)); ¹³C NMR (CDCl₃) δ 26.9 (CH₂C), 71.7 (CCH), 77.1 (CCH ), 134.6 (2 CHC(O)), 169.4 (2 CHC(O)).

Addition of iV-Acetyl-L-cysteine Methyl Ester (12) to jV-(Prop-2-ynyl)maleimide (13) to Give 14. N-(Prop-2-ynyl)maleimide (13) (0.35 g, 2.59 mmol) and iV-acetyl-L-cysteine methyl ester (12) (0.69 g, 3.89 mmol) were dissolved in a 5% CH₃CN/aqueous 50 mM HEPES (5 mL, pH 7.2) and allowed to stir at room temperature (1 h). The reaction solution was concentrated in vacuo and purified by column chromatography (SiO₂; 3/1 EtOAc/hexanes) to yield 0.70 g (87%) of 14 as a white solid as an ~ 1 : 1 diastereomeric mixture: mp 93-95 °C; R₁ = 0.35 (3/1 EtOAc/hexanes); ¹H NMR (CDCl₃) (diastereomer in parenthesis) δ 2.07 (s, CH₃C(O) (2.08 (s, CH₃C(O))), 2.23-2.26 (m, CCH), 2.46-2.59 (m, CHCHH'S), 3.02 (dd, J = 7.4, 13.7 Hz, CHCHH'S (3.15-3.28 (m, CHCHH'S))), 3.15-3.28 (m, maleimide-CHH'C(O)), 3.44-3.57 (m, maleimide-CHH'C(O)), 3.79 (s, OCH₃ (3.79 (s, OCH₃))), 3.85 (dd, J = 4.1, 9.5 Hz, maleimide-CHC(O) (4.00 (dd, J = 4.1, 9.2 Hz, maleimide-CHC(O))), 4.28 (t, J = 2.1 Hz, CH₂CCH), 4.89-4.96 (m, CHCH₂S), 6.55 (d, J = 7.8 Hz, NH (6.95 (d, J = 7.8 Hz, NH))); ¹³C NMR (CDCl₃) (diastereomer in parenthesis) δ 23.1 (23.2) (CH₃C(O)), 28.3 (28.4) (CH₂CCH), 34.3 (34.9) (CH₂S), 35.8 (36.5) (maleimide- CH₂C(O)), 38.9 (40.3) (maleimide-CHC(O)), 51.3 (53.0) (OCH₃), 52.9 (53.0) (CHCH₂S), 72.0 (72.1) (CCH), 76.2 (76.3) (CCH), 170.2 (170.4), 171.1 (171.3), 172.9 (173.1), 175.6 (175.7) (4 C(O)); HRMS (ESI) 313.0861 [M + H⁺] (calcd. for C₁₃Hi₇N₂O₅S 313.0858).

Cycloaddition Reaction Between 14 and Cleavable Linker 1 Affording 15.

Compound 14 (77.1 mg, 0.25 mmol) and 1 (100.0 mg, 0.22 mmol) were dissolved in 5% CH₃CN/aqueous 50 mM HEPES (pH 7.4) (2 mL), and then fresh CuBr (4.3 mg, 0.03 mmol) was added. The reaction mixture was vigorously stirred at room temperature (30 min), evaporated in vacuo, and then purified by column chromatography (SiO₂; 1/6 MeOH/CHCl₃) to yield 60.3 mg (35%) of 15 as a white sticky foam: R_f= 0.25 (1/9 MeOH/CHCl₃); ¹H NMR (DMSO-(^₆) (diastereomer or minor conformer in parenthesis) δ 1.27-1.38 (m, C(O)H₂), 1.41-1.63 (m, C(7)H₂, C(8)H₂), 1.85 (s, CH₃C(O) (1.86 (s, CH₃C(O))), 2.12 (t, J = 7.2 Hz, C(9)H₂ (2.47 (m, C(9)H₂))), 2.48-2.57 (m, CHCHH'S), 2.57 (d, J = 12.6 Hz, C(5)HH'), 2.79-2.85 (m, C(5)HH'), 2.84-3.28 (m, CHCHH'S, maleimide-CH₂C(O), C(2)H), 3.45-3.55 (m, 2 OCH₂CH₂O), 3.64 (s, OCH₃ (3.65 (s, OCH₃))), 3.79 (t, J = 5.2 Hz, OCH₂CH₂-N-triazole), 3.99-4.15 (m, maleimide-CHC(O), C(3)H, OCH₂CHNNH), 4.28-4.32 (m, C(4)H), 4.47-4.54 (m, OCH₂CH₂-N-triazole, CHCH₂S), 4.62 (s, CH₂-N- maleimide), 6.37, 6.44 (s, N(1')H, N(3')H), 7.30 (t, J = 5.1 Hz, OCH₂CHNNH (7.46 (m, OCH₂CHNNH))), 7.96 (s, triazole-CH), 8.45 (d, J = 2.1 Hz, CH₃C(O)NH (8.47 (d, J = 2.1 Hz, CH₃C(O)NH))), 10.99 (s, NNHC(O) (11.15 (s, NNHC(O)))); ¹³C NMR (CD₃OD) (diastereomer or conformer in parenthesis) δ 22.5, 24.4, 26.5, 29.7 (29.9), 34.1 (34.6), 35.0, 36.9 (37.3), 40.4 (41.7), 41.2, 51.4, 53.2, 54.0, 57.1 (58.5), 61.8, 63.6 (64.4), 70.5, 70.8, 71.0, 71.2, 71.4, 71.6, 71.7, 126.1 (triazole-CH), 144.4, 148.4 (triazole-C, OCH₂CHNNH), 166.2, 172.5, 173.0, 173.4 (173.5), 176.1, 178.1 (178.3) (6 C(O)); MS (ESI) 792.2 [M + Na⁺] (calcd. for C₃₁H₄₇N₉O₁₀S₂Na 792.2).

Exchange of Hydrazone Linkage in 15 with Acethydrazide (7) and Acethydrazide-</₃ (7-rf₃) to Give Imine 16/16-rf₃ and Imine Reduction of 16/16-</₃ with NaCNBH₃ to Give 17 M-d₃. To an aqueous 50 mM HEPES (pH 5.8) solution (1 mL, 5.0 mM) of 15 (3.85 mg, 5.0 μmol) was added 7 and 7-^₃ (1 : 1 mixture, 7.55 mg, 0.1 mM) andp- anisidine (1.23 mg, 10.0 //mol; final concentration 10 mM). The reaction mixture was rotated at room temperature (2 h) and then an aliquot (50 //L) containing 16/16-d₃ was analyzed by HPLC using a //Bondapak C-18 column (3.9 x 300 mm, Waters Corp. Cat. No. WAT027324) and a photodiode array detector (210-340 nm). A gradient mobile phase (0/100 CH₃CN/H₂O-50/50 CH₃CN/H₂O) was employed for 30 min using a flow rate of 1 mL/min. The major peaks were collected and analyzed by ESI-MS.

The reaction solution (0.95 mL) containing 16/16-^₃ was treated with a 200 mM solution of NaCNBH₃ (0.95 mL) in 50 mM NaOAc buffer (pH 3.8) and shaken at room temperature (1 h). The reaction solution containing 17/17-α?₃ was separated by a HPLC and the major peaks collected. The collected solutions were analyzed by ESI-MS. Preparative Reaction: iV-(Prop-2-ynyl)maleimide (13) and iV-(2-(2-(2-(2- Azidoethoxy)ethoxy)ethoxy)acetaldehyde Biotinylhydrazone (1) to Give 27. Compound 13 (13.5 mg, 0.10 mmol) and 1 (22.0 mg, 0.05 mmol) were dissolved in THF/H₂O (1 :5, 1 mL), and then fresh CuBr (1.4 mg, 0.01 mmol) was added. The reaction mixture was vigorously stirred at room temperature (30 min), evaporated in vacuo, and then purified by column chromatography (SiO₂; 1/6 MeOH/CHCl₃) to yield 19.7 mg (67%) of 27 as a yellow sticky foam: R_f = 0.45 (1/6 MeOH/CHCl₃); ¹H NMR (CDCl₃) (minor conformer³ in parenthesis) δ 1.42-1.50 (m, C(6)H₂), 1.60-1.79 (m, C(7)H₂, C(8)H₂), 2.31 (t, J = 7.5 Hz, C(9)H₂ (2.64-2.71 (m, C(9)H₂))), 2.72 (d, J= 12.8 Hz, C(5)HH'), 2.92 (dd, J= 4.8, 12.8 Hz, C(5)HH'), 3.13-3.17 (m, C(2)H), 3.60-3.66 (m, 2 OCH₂CH₂O), 3.86 (t, J = 5.0 Hz, OCH₂CH₂-7V-triazole), 4.14 (d, J = 5.1 Hz, OCH₂CHNNH (4.20 (d, J = 4.8 Hz, OCH₂CHNNH))), 4.28-4.36 (m, C(3)H), 4.51-4.54 (m, C(4)H, OCH₂CH₂-N-triazole), 4.82 (s, CH₂-N-maleimide), 5.37, 6.69 (s, N(I ')H, N(3')H), 6.75 (s, 2 maleimide-CHC(O)N (6.76 (s, 2 maleimide-CHC(O)N))), 7.30 (t, J = 5.1 Hz, OCH₂CHNNH (7.56 (m, OCH₂CHN))), 7.74 (s, triazole-CH (7.76 (s, triazole-CH))), 10.63 (s, NNHC(O) (10.71 (s, NNHC(O)))); ¹³C NMR (CDCl₃) (minor conformer in parenthesis) δ 24.8 (25.6) (C(6)), 28.5 (29.9) (C(7)), 32.1 (31.7) (C(8)), 33.0 (34.3) (C(9)), 40.8 (C(5)), 50.5 (OCH₂CH₂-N-triazole), 55.7 (C(2)), 60.3 (C(3)), 62.1 (C(4)), 69.6 (CH₂-N-maleimide), 70.3, 70.4, 70.5, 70.7, 70.8, 70.9 (3 CH₂OCH₂), 124.1 (triazole-CH), 134.5 (134.6) (maleimide-2 CH), 142.6 (triazole-C), 143.8 (147.3) (OCH₂CHN), 164.1 (C(2')), 170.3 (170.9) (maleimide-2 C(O)), 176.4 (C(IO)); HRMS (ESI) 593.2511 [M + H⁺] (calcd. for C₂₅H₃₇N₈O₇S 593.2506).

Modification of Bovine Serum Albumin (BSA, 28) with (R)-JV-(4-Azidobenzyl)-2- acetamido-3-methoxypropanamide (29) to Give 30 (Method C). To an aqueous 50 raM HEPES (pH 7.4) solution (1 mL, 150 //M) of 28 (10.0 mg, 0.15 //mol) was added a 20 mM solution (0.1 mL) of (i?)-29 (Kohn et al., unpublished work) (0.63 mg, 2.0 μmol) in 5% CH₃CN/aqueous 50 mM HEPES (pH 7.4). The solution was irradiated with 312 nm light (8 W, Spectroline EB-280C, Spectronics Corp., New York, USA) at room temperature (15 min) and then the reaction solution was diluted with 50 mM HEPES buffer (pH 7.4) to 5 mL and passed through NAP-5 columns (GE Healthcare, Buckinghamshire, UK) using 50 mM HEPES buffer (pH 7.4). The NAP-5 columns were pre-equilibrated with HEPES buffer (pH 7.4). The eluent (-10 mL) was stored at 4 ⁰C. Cycloaddition Reaction Between 30 and Cleavable Linker 1 Affording 9 (Method

D). To an aqueous 50 mM HEPES solution containing 30 that was obtained after passage through the NAP-5 column (1 mL), was sequentially added a 20 mM solution (50 //L) of 1 (0.44 mg, 1.0 μmoϊ) in 5% CH₃CN/aqueous 50 mM HEPES (pH 7.4), a 20 mM solution (50 //L) of biotin hydrazide (6) (0.26 mg, 1.0 //mol) in 5% CH₃CN/aqueous 50 mM HEPES (pH 7.4), and CuBr (0.2 mg, 1.4 //mol). The reaction mixture was rotated using Roto-shake (8 rpm, Scientific Industries Inc., Model No. SI-1100, Bohemia, NY) at room temperature (1 h), then divided in two equal portions and passed through separate NAP-5 columns pre- equilibrated with HEPES buffer (pH 7.4). The eluents were combined (~2 mL) and stored at 4 °C.

General Method for Capture of Biotinylated BSA 9 and Release of Modified BSA by Cleavage of the Linker (Method E). An aqueous 50 mM HEPES solution of 9 (2 mL) was added to an immobilized streptavidin slurry (1 mL) (High Capacity Streptavidin Agarose Resin, Pierce, Rockford, IL) and rotated using a shaker (15 rpm) at 4 °C (90 min). The streptavidin beads were washed with aqueous 15 mM HEPES buffer (pH 7.4) (10 x 0.8 mL) and divided into 10 equal portions. The aliquots were centrifuged (1000 rpm, 1 min), the supernatant removed, and 50 mM HEPES solution (100 //L, pH 5.8-7.4) added and the beads treated with 7 (0.39-1.56 mg, 5-20 //mol) in the presence or absence of a catalyst (aniline, p- anisidine [final concentration 10 mM]; SDS [final concentration 20 mM]). The reaction mixture was gently shaken at a specified temperature (22-50 °C) (1-4 h) and then the supernatant collected. The remaining streptavidin beads were washed with 10 mM HEPES buffer (pH 7.4) (3 x 0.15 mL), and then boiled (5 min) with SDS-loading buffer (2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bromophenol blue). The samples were loaded on a 10% SDS-PAGE gel and the proteins visualized by silver staining. The relative intensities of the bands were determined by densitometry.

Quantification of Recovery Efficiency of Streptavidin Bound BSA 9 Using Either Mild or Harsh Conditions (Method F). Utilizing the Methods C and D, and using 28 (10.0 mg, 0.15 //mol), 29 (0.63 mg, 2.0 //mol), and 1 (0.44 mg, 1.0 //mol), an aqueous 50 mM HEPES solution of 9 (2 mL) was obtained and quantified by UV-VIS spectroscopy (Chameleon, Hidex Oy, Turku, Finland) at 595 nm upon addition of Coomassie G-250 Dye (23236, Thermo Scientific). An aliquot (200 μL) of 9 was added to an immobilized streptavidin slurry (0.3 mL) and rotated using a shaker (15 rpm) at 4 ⁰C (90 min). The streptavidin beads were washed with aqueous 15 mM HEPES buffer (pH 7.4) (8 x 0.3 mL). The supernatants were collected (2.4 mL) and the amounts of unbound 9 were quantified by UV-VIS spectroscopy. The resulting streptavidin beads with immobilized 9 were divided into 2 aliquots. Each portion was suspended in 50 mM HEPES solution (50 μL, pH 5.8), followed by treating with either 7 (0.39 mg, 5 μmol) and SDS (0.5%, 20 mM) at 50 °C (1 h) (mild conditions) or SDS-loading buffer (2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bromophenol blue) at 95 °C (5 min) (harsh conditions). The eluents collected using either the mild or the harsh cleavage conditions were quantified by using the Bradford assay after 50- fold dilution to minimize the effect of SDS on protein concentrations. We found that the amount of protein in the reaction solution (BSA 9) after click chemistry with biotinylated cleavable linker 1 was -660 μg. After incubation with streptavidin beads, the amount of unbound protein in the collected flowthrough and wash was -468 μg. Therefore, the total amount of 9 captured on streptavidin beads was -192 μg. The result of dot blot assay (data not shown) showed that the biotinylated BSA 9 was efficiently captured by streptavidin beads. The protein captured by the streptavidin beads were then released upon treating the beads with either mild or harsh conditions. Quantification of the elution fractions indicated that the protein release from the captured 9 on the streptavidin beads was almost quantitative under the harsh cleavage conditions, whereas the recovery was >85% under the mild conditions.

Dot Blot Assay for Quantification of Capture Yield of Biotinylated BSA 9.

Utilizing the Method F, an aqueous 50 mM HEPES solution of 9 (1 μL) and the flowthrough solution (1 μL) obtained after incubation with the streptavidin beads were used to determine the amount of biotinylated BSA 9 captured by the streptavidin beads using a dot blot assay (data not shown). Solutions (serially diluted by 2-, 4-, 8-, and 16-fold, respectively) (1 μL) were loaded to a nitrocellulose membrane (RPN203D, Amersham) and the membrane was washed (10 min) with TBST (25 mM Tris buffer, 150 mM NaCl, 0.1% Tween-20 (pH 7.6)). The membrane was incubated in 5% BSA/TBST solution (50 mL) at room temperature (1 h) and then incubated with a HRP-conjugated streptavidin specific for biotin (N-100, Pierce) in 5% BSA/TBST solution (1.5 mL) at room temperature (1 h). After washing (x 4, 5 min each) with TBST, chemiluminescent reagent (RPN2132, GE Healthcare) was added to the blot and the signal developed in the darkroom.

0-Ditosylate Tetraethylne Glycol (32). Compound 2 (5.00 mL, 28.95 mmol) was added dropwise to a stirred solution of /Moluenesulfonyl chloride (12.15 g, 63.73 mmol), imidazole (0.04 g, 0.65 mmol) and Et₃N (16.15 mL, 108.30 mmol) in CH₂Cl₂ (30 mL) at 0 ⁰C. The reaction mixture was allowed to stir at room temperature (12 h), and then Et₂O (80 mL) was added and the reaction washed with H₂O (5 x 60 mL). The organic layer was dried (Na₂SO₄) and evaporated in vacuo, and then the crude product was purified by column chromatography (SiO₂; 3/1 EtOAc/hexanes) to yield 13.81 g (95%) of 32 as a clear oil (Busch et al., 2002): R_f = 0.65 (3/1 EtOAc/hexanes); ¹H NMR (CDCl₃) δ 2.44 (s, 2 CH₃), 3.53-3.59 (m, 2 OCH₂CH₂O), 3.68 (t, J = 4.8 Hz, OCH₂CH₂OTs), 4.15 (t, J = 4.8 Hz, OCH₂CH₂OTs), 7.34 (d, J= 8.4 Hz, 4 ArH), 7.79 (d, J= 8.4 Hz, 4 ArH); ¹³C NMR (CDCl₃) δ 21.7 (CH₃), 68.8 , 69.4, 70.6, 70.8 (OCH₂CH₂O, OCH₂CH₂OTs), 128.1, 130.1, 133.1, 145.0 (C₆H₄).

l-Azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane (33). NaN₃ (3.88 g, 59.76 mmol) was added to an EtOH solution (200 mL) of 32 (12 g, 23.90 mmol). After stirring at 70 ⁰C (18 h), H₂O (150 mL) was added and the solvent was concentrated in vacuo. The remaining solution (153 mL) was extracted with EtOAc (3 x 150 mL). The combined organic phases were dried (Na₂SO₄) and evaporated in vacuo, and the crude product purified by column chromatography (SiO₂; 1/2 EtOAc/hexanes) to yield 4.1O g (70%) of 33 as a yellow oil (Sun et al., 2007): R_f= 0.50 (1/2 EtOAc/hexanes); ¹H NMR (CDCl₃) δ 3.40 (t, J = 5.1 Hz, 2CH₂N₃), 3.67-3.70 (m, 3CH₂OCH₂); ¹³C NMR (CDCl₃) δ 50.9 (2 OCH₂CH₂N₃), 70.2 (2 OCH₂CH₂N₃), 70.9 (2 OCH₂CH₂O), the remaining peaks were not detected and are believed to overlap with the other resonances.

2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethanamine (34). Diazide 33 (0.51 g, 2.10 mmol) was stirred with 0.65 M aqueous H₃PO₄ (5.00 mL) while Ph₃P (0.48 g, 1.83 mmol) in Et₂O (4.00 mL) was added dropwise over 45 min. After stirring at room temperature under Ar (24 h), the separated aqueous layer was washed with Et₂O (3 x 5.00 mL), and then KOH (0.40 g) was added. After Et₂O was evaporated from the aqueous layer, the solution was cooled to 4 °C (16 h), and Ph₃PO was removed by filtration. KOH (4 M aqueous solution, 10 rtiL) was added and the aqueous solution was extracted with CH₂Cl₂ (8 x 20 mL). The combined organic phases were dried (Na₂SO₄) and evaporated in vacuo, and then the crude product was purified by column chromatography (SiO₂; 10% NH₄OH in EtOH) to yield 0.21 g (45%) of 34 as a yellow oil (Schwabacher et al., 1998): R_f = 0.40 (10% NH₄OH in EtOH); ¹H NMR (CDCl₃) δ 1.63 (br s, NH₂), 2.88 (br m, CH₂NH₂), 3.40 (t, J= 5.1 Hz, N₃CH₂CH₂), 3.52 (t, J = 5.0 Hz, CH₂CH₂NH₂) 3.63-3.70 (m, 2 OCH₂CH₂O, N₃CH₂CH₂); ¹³C NMR (CDCl₃) δ 41.8 (CH₂NH₂), 50.8 (N₃CH₂CH₂), 70.2 (N₃CH₂CH₂), 70.5, 70.7, 70.8, 70.9 (2 OCH₂CH₂O), 73.8 (CH₂CH₂NH₂).

2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethylamino Biotin (10). Et₃N (0.03 mL, 0.20 mmol) was added to a solution of 34 (0.05 g, 0.24 mmol) in DMF (5 mL). After the solution was stirred (30 min), a solution of N-hydroxysuccinimidobiotin (1.00 g, 0.29 mmol) was added. The reaction mixture was allowed to stir at room temperature (12 h), and then evaporated in vacuo and purified by column chromatography (SiO₂; 1/4/16 AcOH/hexanes/acetone) to yield 0.80 g (74%) of 10 as a white solid (Sun et al., 2007): mp 119-120 ⁰C; R₁ = 0.35 (1/4/16 AcOH/hexanes/acetone); ¹H NMR (CD₃OD) δ 1.39-1.49 (m, C(O)H₂), 1.54-1.78 (m, C(7)H₂C(8)H₂), 2.22 (t, J = 7.2 Hz, C(9)H₂), 2.71 (d, J = 12.6 Hz, C(5)HH'), 2.93 (dd, J = 5.0, 12.8 Hz, C(5)HH'), 3.18-3.24 (m, C(2)H), 3.33-3.40 (m, CH₂CH₂NH, N₃CH₂), 3.55 (t, J = 5.6 Hz, OCH₂CH₂NH), 3.60-3.69 (m, 2OCH₂CH₂O, N₃CH₂CH₂O), 4.31 (dd, J- 4.5, 7.8 Hz, C(3)H), 4.49 (ddd, J= 0.9, 5.1, 7.8 Hz, C(4)H); ¹³C NMR (CD₃OD) δ 27.0 (C(6)), 29.6, 29.9 (C(7), C(8)), 36.9 (C(9)), 40.5 (OCH₂CH₂NH), 41.2 (C(5)), 51.9 (N₃CH₂CH₂O), 57.1 (C(2)), 61.8 (C(4)), 63.5 (C(3)), 70.7 (OCH₂CH₂NH), 71.3, 71.4, 71.6, 71.7, 71.8 (2 OCH₂CH₂O, N₃CH₂CH₂O), 166.3 (C(2')), 176.3 (C(O)NHCH₂); HRMS (ESI) 445.2233 [M + H⁺] (calcd. for C₈H₃₃N₆O₅S 445.2226).

Cycloaddition Reaction Between BSA 30 and Non-cleavable Linker 10 Affording BSA 11 and the Release of BSA 11 from Streptavidin Beads. Utilizing Method D, an aqueous 50 mM HEPES solution containing BSA 30 that was obtained after passage through the NAP-5 column (1 mL), 20 mM solution (50 μL) of 10 (0.44 mg, 1.0 μmol) in 5% CH₃CN/aqueous 50 mM HEPES (pH 7.4), and CuBr (0.2 mg, 1.4 //mol) gave the desired solution (~2 mL) containing BSA 11. Using Method E, the beads were treated with 7 (0.78 mg, 10 μmol) and SDS (0.5%, 20 mM) at 50 °C (1 h). The supernatant was collected and then the remaining streptavidin beads were boiled (15 min) with loading buffer (2% SDS, 10% glycerol, 1% mercaptoethanol, 0.01% bromophenol blue [final concentration]). The samples were loaded on a 10% SDS-PAGE gel and the proteins visualized by silver staining.

Preparation of Mouse Soluble Liver Proteosomes. Mouse liver harvested from wild-type C57BL/6 mice and immediately Dounce homogenized in 50 mM sodium/potassium phosphate buffer (pH 8.0) (PB). The lysate was centrifuged at slow speed (1200 x g for 12 min at 4 ⁰C) to remove debris. The supernatant was centrifuged at high speed (100,000 x g for 1 h at 4 ⁰C). The supernatant was collected and stored at -80 °C until use. The total protein concentration was determined by using the Bradford assay.

Scheme 1. Treatment of 1 and 13 under Cu(I)-mediated Cycloaddition Conditions

Acethydrazide (7) 5O mM HEPES, pH 5 8

31

Scheme 2. Use of 1 for BSA Capture, Release, and Detection

Scheme 3. Synthesis of Non-cleavable Linker 10

REFERENCES

Agard, N. J., Baskin, J. M., Prescher, J. A., Lo, A., Bertozzi, C. R. (2005). A comparative study of bioorthogonal reactions with azides. Chem. Biol. 10, 644-648. Bai, X., Kim, S., Li, Z., Turro, N. J., Ju, J. (2004) Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry. Nucleic Acids Res. 32, 535- 541.

Ball, H. L., Mascagni, P. (1997) Application of reversible biotinylated label for directed immobilization of synthetic peptides and proteins: isolation of ligates from crude cell lysates. J. Peptide Sci. 3, 252-260.

Berggard, T., Linse, S., James, P. (2007) Methods for the detection and analysis of protein- protein interactions. Proteomics 7, 2833-2842.

Busch, B. B., Staiger, C. L., Stoddard, J. M., Shea, K. J. (2002) Living polymerization of sulfur ylides. Synthesis of terminally functionalized and telechelic polymethylene. Macromolecules 35, 8330-8337.

Camilo, N. S., Pilli, R. A. (2004) Addition of carbon nucleophiles to cyclic N-acyliminium ions in SDS/water. Tetrahedron Lett. 45, 2821-2823.

Chin, C. C. Q., Brewer, J. M., Wold, F. (1981) The amino acid sequence of yeast enolase. J. Biol. Chem. 256, 1377-1384.

Chrestensen, C. A., Schroeder, M. J., Shabanowitz, J., Hunt, D. F., PeIo, J. W., Worthington, M. T., Sturgill, T. W. (2004) MAPKAP kinase 2 phosphorylates tristetraprolin on in vivo sites including Ser¹⁷⁸, a site required for 14-3-3 binding. J. Biol. Chem. 279, 10176-10184.

Dieterich, D. C, Lee, J. J., Link, A. J., Graumann, J., Tirrell, D. A., Schuman, E. M. (2007) Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nature Protocols 2, 532-540.

Dirksen, A., Dirksen, S., Hackeng, T. M., Dawson, P. E. (2006a) Nucleophilic catalysis of hydrazone formation and transimination: Implications for dynamic covalent chemistry. J. Am. Chem. Soc. 128, 15602-15603.

Dirksen, A., Hackeng, T. M., Dawson, P. E. (2006b) Nucleophilic catalysis of oxime ligation. Angew. Chem. Int. Ed. 45, 7581-7584.

Finn, F. M., Stehle, C. J., Hofmann, K. (1985) Synthetic tools for adrenocorticotropin receptor identification. Biochemistry 24, 1960-1965.

Flinn, N. S., Quibell, M., Turnell, W. G., Monk, T. P., Ramjee, M. K. (2004) Novel chemoselective linkage chemistry toward controlled loading of ligands to proteins through in situ real-time quantification of conjugate formation. Bioconj. Chem. 15, 1010-1020.

Fonovic, M., Verhelst, S. H. L, Sorum, M. T., Bogyo, M. (2007) Proteomics evaluation of chemically cleavable activity-based probes. MoI. Cell. Proteomics 6, 1761-1770.

Gogoi, P., Hazarika, P., Konwar, D. (2005) Surfactant/LVwater: An efficient system for deprotection of oximes and imines to carbonyls under neutral conditions in water. J. Org. Chem. 76», 1934-1936.

Goncalves, M., Estieu-Gionnet, K., Berthelot, T., Lain, G., Bayle, M., Canron, X., Betz, N., Bikfalvi, A., Deleris G. (2005) Design, synthesis, and evaluation of original carriers for targeting vascular endothelial growth factor receptor interactions. Pharm. Res. 22, 141 1- 1422.

Goral, V., Nelen, M. I., Eliseev, A. V., Lehn, J. M. (2001) Double-level "orthogonal" dynamic combinatorial libraries on transition metal template. Proc. Natl. Acad. Sci. USA 98, 1347-1352.

Green, N. M. (1990) Avidin and streptavidin. Methods Enzymol. 184, 51-67.

Hirsch, J. D., Eslamizar, L., Filanoski, B. J., Malekzadeh, N., Haugland, R. P., Beechem, J. M., Haugland, R. P. (2002) Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. 308, 343-357.

Holland, M. J., Holland, J. P., Thill, G. P., Jackson, K. A. (1981) The primary structures of two yeast enolase genes. Homology between the 5' noncoding flanking regions of yeast enolase and glyceraldehyde-3- phosphate dehydrogenase genes. J. Biol. Chem. 256, 1385- 1395.

Holmberg, A., Blomstergren, A., Nord, O., Lukacs, M., Lundeberg, J., Uhlen, M. (2005) The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures. Electrophoresis 26, 501-510.

Howarth, M., Chinnapen, D. J., Gerrow, K., Dorrestein, P. C, Grandy, M. R., Kelleher, N. L., El-Husseini, A., Ting, A. Y. (2006) A monovalent streptavidin with a single femtomolar biotin binding site. Nature Methods 3, 267-273.

Jenne, A., Famulok, M. (1999) Disruption of the streptavidin interaction with biotinylated nucleic acid probes by 2-mercaptoethanol. Biotechniques 26, 249-254.

Kazmierski, W. M., McDermed, J. (1995) Synthesis of the carbonic acid benzotriazol-1-yl- ester-(2-biotinylamino)-9h-fluoren-9-ylmethyl ester: A convenient transient-biotinylation reagent for use in affinity chromatography. Tetrahedron Lett. 36, 9097-9100.

Karlen, B., Lindeke, B., Lindgren, S., Svensson, K. G., Dahlbom, R., Jenden, D. A., Giering, J. E. (1970) Acetylene compounds of potential pharmacological value. XIV. N-{tert- aminoalkynyl)-substituted succinimides and maleimides. Class of central anticholinergic agents. J. Med. Chem. 13, 651-657.

King, T. P., Zhao, S. W., Lam,T. (1986) Preparation of protein conjugates via intermolecular hydrazone linkage. Biochemistry 25, 5114-5719.

Kishimoto, K., Suzawa, T., Yokota, T., Mukai, T., Ohno, H., Kato, T. (2005) Nano- segregated polymeric film exhibiting high ionic conductivities. J. Am. Chem. Soc. 127, 15618-15623.

Lin, W. -C, Morton, T. H. (1991) Two-step affinity chromatography. Model systems and an example using biotin-avidin binding and a fluoridolyzable linker. J. Org. Chem. 56, 6850- 6856. MacKinnon, A. L., Garrison, J. L., Hegde, R. S., Taunton, J. (2007) Photo-leucine incorporation reveals the target of a cyclodepsipeptide inhibitor of cotranslational translocation. J. Am. Chem. Soc. 129, 14560-14561.

Malmstadt, N., Hyre, D. E., Ding, Z., Hoffman, A. S., Stayton, P. S. (2003) Affinity thermoprecipitation and recovery of biotinylated biomolecules via a mutant streptavidin-smart polymer conjugate. Bioconj. Chem. 14, 575-580.

Marie, J., Seyer, R., Lombard, C, Desarnaud, F., Aumelas, A., Jard, S., Bonnafous, J. C. (1991) Affinity chromatography purification of angiotensin II receptor using photoactivable biotinylated probes. Biochemistry 29, 8943-8950.

Morag, E., Bayer, E. A., Wilchek, M. (1996) Reversibility of biotin-binding by selective modification of tyrosine in avidin. Biochem. J. 316, 193-199.

Rostovtsev, V. V., Green, L. G., Fokin, V. V., Sharpless, K. B. (2002) A stepwise huisgen cycloaddition process: Copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41, 2596-2599.

Sawant, R. M., Hurley, J. P., Salmaso, S., Kale, A.; Tolcheva, E., Levchenko, T. S., Torchilin, V. P. (2006) "Smart" drug delivery systems: Double-targeted pH-responsive pharmaceutical nanocarriers. Bioconj. Chem. 17, 943-949.

Schwabacher, A. W., Lane, J. W., Schiesher, M. W., Leigh, K. M., Johnson, C. W. (1998) Desymmetrization reactions: efficient preparation of unsymmetrically substituted linker molecules. J. Org. Chem. 63, 1727-1729.

Scriba, G. K. E. (2004) Molecular biology in medicinal chemistry: Affinity chromatography. Th., Dingermann, ed. (Weinheim, Germany: Wiley- VCH Verlag GmbH), pp. 21 1-241.

Shimkus, M., Levy, J., Herman, T. (1985) A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns. Proc. Natl. Acad. Sci. USA 82, 2593-2597.

Smith, T. R., Clark, A. J., Napier, R., Taylor, P. C, Thompson, A. J., Marsh, A. (2007) FFuunnccttiioonn aanndd ssttaabbiilliittyy ooff aabbsscciissiicc aacciidd ; acyl hydrazone conjugates by LC-MS² of ex vivo samples. Bioconj. Chem. 18, 1355-1359.

Speers, A. E., Adam, G. C, Cravatt, B. F. (2003) Activity-based protein profiling in vivo using a copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125, 4686- 4687.

Speers, A. E., Cravatt, B. F. (2004) Profiling enzyme activities in vivo using click chemistry methods. Chem. Biol. 11, 535-546.

Speers, A. E., Cravatt, B. F. (2005) A tandem orthogonal proteolysis strategy for high-content chemical proteomics. J. Am. Chem. Soc. 127, 10018-10019.

Sun, X-L., Stabler, C. L.₃ Cazalis, C. S., Chaikof, E. L. (2006) Carbohydrate and protein immobilization onto solid surfaces by sequential Diels-Alder and azide-alkyne cycloadditions. Bioconj. Chem. 17, 52-57. Syakaev, V. V.; Podyachev, S. N.; Buzykin, B. L; Latypov, S. K.; Habicher, W. D.; Konovalov, A.I. (2006) NMR study of conformation and isomerization of aryl- and heteroarylaldehyde 4-tert-butylphenoxyacetylhydrazones. J. MoI. Struct. 788, 55-62.

Thiele, C, Fahrenholz, F. (1994) Photocleavable biotinylated ligands for affinity chromatography. Anal. Biochem. 218, 330-337.

Tong, X., Smith, L. M. (1992) Solid-phase method for the purification of DNA sequencing reactions. Anal. Chem. 64, 2672-2677.

Tornoe, C. W., Christensen, C, Meldal, M. (2002) Peptidotriazoles on solid phase: [1,2,3]- triazoles by regiospecific copper(I)-catalyzed 1,3 -dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 61, 3057-3064. van der Veken, P., Dirksen, E. H. C₅ Ruijter, E., Elgersma, R.C., Heck, A. J. R., Rijkers, D. T. S., Slijper, M., Liskamp, R. M. J. (2005) Development of a novel chemical probe for the selective enrichment of phosphorylated serine- and threonine-containing peptides. ChemBioChem 6, 2271-2280.

Verhelst, S. H. L., Fonovic, M., Bogyo, M. (2007) A mild chemically cleavable linker system for functional proteomic applications. Angew. Chem. Int. Ed. Engl. 46, 1284-1286.

Walsh, D. P., Chang, Y. T. (2006) Chemical genetics. Chem. Rev. 106, 2476-2530.

Wang, W., Wang, S. X., Qin, X. Y., Li, J. T. (2005) Reaction of aldehydes and pyrazolones in the presence of sodium dodecyl sulfate in aqueous media. Syn. Commun. 35, 1263-1269.

Weerapana, E., Simon, G. M., Cravatt, B. F. (2008) Disparate proteome reactivity profiles of carbon electrophiles. Nat. Chem. Biol. 7, 405-407.

Wilchek, M., Bayer, E. (1990) Introduction to avidin-biotin technology. Methods Enzymol. 184, 14-45.

Wu, S. C, Wong, S. L. (2005) Engineering soluble monomeric streptavidin with reversible biotin binding capability. J. Biol. Chem. 280, 23225-23231.

Zeheb, R., Orr, G. A. (1986) Use of avidin-iminobiotin complexes for purifying plasma membrane proteins. Methods Enzymol. 122, 87-94.

Claims

THAT WHICH IS CLAIMED IS:

1. A compound of Formula I:

Y-X-R (I) wherein:

X is a cleavable linker comprising an acylhydrazone; and

Y and R are each independently selected from the group consisting of covalent coupling groups and members of a specific binding pair.

2. The compound of claim 1, wherein Y is a covalent coupling group (e.g., N₃) and R is a member of a specific binding pair (e.g., avidin).

3. The compound of claim 1-2, said cleavable linker further comprising a polyalkylene oxide group (e.g., poly(ethylene glycol)).

4. The compound of claim 1-4 having the structure of Formula II:

wherein:

Y and R are as given above;

A, B, C, and X are each independently selected from the group consisting of O, S, N(H), N(R), N(R')(R")⁺, CH₂, C(R')(R"). single covalent bond, and double covalent bond;

F' is selected from the group consisting of hydrogen, lower alkyl, and aryl (which can be unsubstituted or substituted with one or more electron-donating or electron-withdrawing groups);

W is selected from the group consisting of a single covalent bond, O, S, N(H), and N(R');

R' and R" are each independently selected loweralkyl; n is O or 1-4; and m is O or 1-4.

5. The compound of claim 1-4, wherein R is selected from the group consisting of ssDNA, RNA, antigenic peptides, N-terminal polyhistidines, covalent coupling groups, and a group of the Formula:

(biotin).

6. The compound of claim 1-5, wherein Y is selected from the group consisting of N₃, CCH, electrophilic affinity bait groups, photochemical affinity bait groups, reactive groups, peptides, proteins, nucleic acid, carbohydrates, lipids, modified drugs, inhibitors, enzyme substrates or mimics, and organic groups.

7. The compound of claim 1 having the formula:

8. In a support useful for binding a compound of interest from a mixture, wherein said compound of interest is a first member of a binding pair and said support comprises a second member of said binding pair coupled to said support by a cleavable linker, the improvement comprising: employing a compound of claim 1-7 as said cleavable linker (e.g. where R is said first member of said binding pair).

9. The support of claim 8, wherein said cleavable linker is covalently coupled or specifically bound (e.g., by biotin-avidin binding) to said support.

10. The support of claim 8-9, wherein said support is a solid support.

1 1. The support of claim 8-9, wherein said support is a dendrimer or lipid particle.

12. In a method of releasing a compound of interest bound to a support from that support, wherein said compound of interest is bound to said support through a cleavable linker, the improvement comprising: employing a cleavable linker comprising an acylhydrazone group therein, and cleaving said acylhydrazone by contacting an acylhydrazide and/or an anionic detergent (e.g., sodium dodecyl sulfate; SDS) thereto.

13. The method of claim 12, wherein said contacting step is carried out under near neutral pH conditions (e.g., pH 4, 5 or 6 up to pH 8 or 9).

14. The method of claim 12-13 wherein said acylhydrazide is contacted to said acylhydrazone linker in solution in an amount of at least 1, 10 or 50 mM.

15. The method of claim 12-14, wherein said acylhydrazide is contacted to said acylhydrazone in solution in an amount up to 500 or 1000 mM.

16. The method of claim 12-15, wherein said anionic detergent is contacted to said acylhydrazone in solution in an amount of at least 0.5, 1, 5 or 10 mM.

17. The method of claim 12-16, wherein said anionic detergent is contacted to said acylhydrazone in solution in an amount up to 50 or 100 mM.

18. The method of claim 12-17, wherein said contacting step is carried out at a temperature of at least 4, 10 or 20 °C.

19. The method of claim 12-18, wherein said contacting step is carried out at a temperature of up to 40 or 50 °C.

20. The method of claim 12-19, wherein said contacting step is carried out for a time of at least 10, 20 or 30 minutes.

21. The method of claim 12-20, wherein said contacting step is carried out for a time of up to 2 or 4 hours.

22. The method of claim 12-21, wherein at least 50, 60, 70 or 80 percent by weight of said compound of interest is released following said contacting step.

23. The method of claim 12-22, wherein said acylhydrazide comprises a detectable group that reacts with said cleavable linker in said cleaving step, whereby said compound of interest can be detected after said cleaving step by detecting said detectable group coupled thereto.

24. The method of claim 12-22, wherein said cleavable linker is a compound of claim 1-7.

25. The method of claim 12-22, wherein said support including said cleavable linker is a support of claim 8-1 1.