NL2012026C2 - Building blocks for introducing michael acceptors in selected peptide sequences. - Google Patents

Building blocks for introducing michael acceptors in selected peptide sequences. Download PDF

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NL2012026C2
NL2012026C2 NL2012026A NL2012026A NL2012026C2 NL 2012026 C2 NL2012026 C2 NL 2012026C2 NL 2012026 A NL2012026 A NL 2012026A NL 2012026 A NL2012026 A NL 2012026A NL 2012026 C2 NL2012026 C2 NL 2012026C2
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Huib Ovaa
Monique Mulder
Oualid Farid El
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Stichting Het Nl Kanker Inst Antoni Van Leeuwenhoek Ziekenhuis
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
    • C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/64—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and sulfur atoms, not being part of thio groups, bound to the same carbon skeleton
    • C07C323/66—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and sulfur atoms, not being part of thio groups, bound to the same carbon skeleton containing sulfur atoms of sulfo, esterified sulfo or halosulfonyl groups, bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C391/00—Compounds containing selenium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
    • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
    • C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
    • C07C2603/18—Fluorenes; Hydrogenated fluorenes

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Description

BUILDING BLOCKS FOR INTRODUCING MICHAEL ACCEPTORS IN SELECTED PEPTIDE SEQUENCES
Field of the Invention
The present invention concerns the field of site- and chemo-selective modification of peptides and proteins. In particular, the invention provides new building blocks that can be used for synthesising modified peptides and proteins. The moiety introduced using the building block of the invention can be converted to a Michael Acceptor. The present invention concerns these building blocks per se as well as the processes for preparing them. The present invention also concerns the processes of introducing Michael Acceptors in selected peptide or protein sequences as well as the intermediate and end products obtained in the respective steps of such processes.
Background of the Invention
Proteases selectively catalyze the hydrolysis of peptide bonds and can be divided in several main classes: threonine proteases, serine proteases, cysteine proteases, aspartic proteases and metalloproteases. Threonine, serine and cysteine proteases have many common active site features, including an active site nucleophile and a general base. This particular feature is an attractive target for covalent modification of the active site in order to bind and/or inhibit the enzyme.
Threnonine proteases harbor a threonine (Thr) residue within the active site. Threonine proteases use the secondary alcohol of their N-terminal threonine as a nucleophile to perform catalysis. The threonine must be N-terminal since the terminal amide of the same residue acts as a general base by polarising an ordered water which deprotonates the alcohol to increase its reactivity as a nucleophile. Catalysis takes place in two steps. Firstly the nucleophile attacks the substrate to form a covalent acyl-enzyme intermediate, releasing the first product. Secondly the immediate is hydrolyzed by water to regenerate the free enzyme and release the second product.
Within the family of threonine proteases, the catalytic subunits of the proteasome constitute the most prominent members. The proteasome (also referred to as multicatalytic protease (MCP), multicatalytic proteinase, multicatalytic proteinase complex, multicatalytic endopeptidase complex, 20S, 26S, or ingensin) is a large, multiprotein complex present in both the cytoplasm and the nucleus of all eukaryotic cells. It is a highly conserved cellular structure that is responsible for the ATP-dependent proteolysis of most cellular proteins. The 26S proteasome is able to degrade proteins that have been marked by the addition of ubiquitin molecules. Typically, ubiquitin is attached to the ε-amino groups of lysines in a multistep process utilizing ATP and El (ubiquitin activating) and E2 (ubiquitin-conjugating) enzymes. Multi-ubiquitinated substrate proteins are recognized by the 26S proteasome and are degraded. Numerous regulatory proteins are substrates for ubiquitin dependent proteolysis. Many of these proteins function as regulators of physiological as well as pathophysiological cellular processes. Alterations in proteasome activity have been implicated in a number of pathologies including neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, as well as occlusion/ischaemia reperfusion injuries, and aging of the central nervous system. The ubiquitin-proteasome pathway also plays a role in neoplastic growth. The regulated degradation of proteins such as cyclins, CDK2 inhibitors, and tumor suppressors is believed to be important in cell cycle progression and mitosis. Tumor suppressor p53 degradation is known to be carried out via the ubiquitin-proteasome pathway, and disrupting p53 degradation by inhibition of the proteasome is a possible mode of inducing apoptosis. The proteasome is also required for activation of the transcription factor NFkB by degradation of its inhibitory protein, IkB. NFkB has a role in maintaining cell viability through the transcription of inhibitors of apoptosis. Blockade of NFkB activity has been demonstrated to make cells more susceptible to apoptosis.
Cysteine proteases are a class of proteases having as a common feature a cysteine (Cys) residue within the active site. Cysteine proteases use the thiol group as a nucleophile to perform catalysis. The first step in the reaction mechanism by which cysteine proteases catalyze the hydrolysis of peptide bonds is deprotonation of the thiol in the enzyme's active site by an adjacent amino acid with a basic side chain, usually a histidine residue. The next step is nucleophilic attack by the deprotonated cysteine's anionic sulfur on the substrate carbonyl carbon. In this step, a fragment of the substrate is released with an amine terminus, the histidine residue in the protease is restored to its deprotonated form, and a thioester intermediate linking the new carboxy-terminus of the substrate to the cysteine thiol is formed. Therefore they are also sometimes referred to as thiol proteases. The thioester bond is subsequently hydrolyzed to generate a carboxylic acid moiety on the remaining substrate fragment, while regenerating the free enzyme by an adjacent amino acid with a basic side chain, usually a histidine residue.
Among the family of cysteine proteases are deubiquitinating proteases, cathepsins, SUMO proteases, calpains and caspases. Cysteine proteases play multifaceted roles, virtually in every aspect of physiology and development. In humans they are responsible for apoptosis, MHC class II immune responses, pro-hormone processing, and extracellular matrix remodeling important to bone development. The ability of macrophages and other cells to mobilize elastolytic cysteine proteases to their surfaces under specialized conditions may also lead to accelerated collagen and elastin degradation at sites of inflammation in diseases such as atherosclerosis and emphysema.
Not surprisingly, given the importance of the various proteases in many biological processes, considerable effort has been put into the development of agents capable of covalently modifying the enzyme active sites. Such agents will have utility in biological research (so-called activity-based probe or ABP), as diagnostic agents, and, potentially, as therapeutic agents (protease inhibitors).
Michael Acceptor groups, such as α-β unsaturated carbonyl moieties, have been known for some time as ‘electrophilic trap’, capable of interacting with protease catalytic site nucleophiles to result in (irreversible) covalent bond formation and, hence, enzyme inhibition.
In order to provide useful ABP’s and/or (therapeutic) protease inhibitors, agents need to be designed that bind their target protease with high affinity and selectivity. Specificity is not defined by the nature of the catalytic centre alone; on either side of the catalytic centre there is a series of binding sites that favour particular amino acids in their substrates. These amino acid sequences are referred to as the recognition sequence. Prior art approaches to develop protease ABP’s or inhibitors include the use of peptide sequences corresponding to a (non-prime side or prime side) cleavage fragment, so that a Michael Acceptor group can be introduced at the terminus of the peptide. Bogyo et al. (Chemistry & Biology, June 1998, 5:307-320), for example, describe various ‘non-prime side’ proteasome probes containing a ‘terminal’ vinyl sulfone moiety. Pfizer et al. (International Journal of Peptide Research and Therapeutics, Vol. 13, Nos. 1-2, June 2007, pp. 93-104) describe various non-prime side papain-like cysteine proteases.
Conceivably, approaches based on the complete recognition sequence, i.e. ABP’s or inhibitors containing a Michael Acceptor scaffold flanked by both prime side and non-prime side recognition sequences (or fragments), result in enhanced affinity and/or selectivity. Such approaches however are seriously hampered by synthetic difficulties. These difficulties, for one thing, are a consequence of the fact that Michael Acceptor groups are (by their nature) highly reactive.
Hence, while techniques for introducing a Michael Acceptor in specific proteinase substrates are known in the art, there remains room for significant improvement in this area. In particular, it would be highly desirable to have ‘building blocks’ available with general utility for introducing Michael Acceptors in ‘any’ given peptide sequence. Building blocks are needed, in other words, that can be employed in conventional peptide synthesis without interfering in the reactions typically involved therein, so as to facilitate the synthesis of a wide range of ABP’s and/or protease inhibitors, including primed side probes/inhibitors, with minimal differences compared to the natural protease substrate.
It is the objective of the present invention, to accomplish this.
Summary of the Invention
The present invention provides a new and generally applicable approach for introducing (scaffolds containing) Michael Acceptors in peptides and proteins, which is particularly easy and versatile and allows for the synthesis of primed-side protease ABP’s and/or inhibitors.
In accordance with the invention, certain novel aminocarboxylic acid and aminosulfonic acid compounds incorporating a β-thiocarbonyl or β-selenocarbonyl moiety are provided that can be employed as building blocks in protein or peptide synthesis. The β-thiocarbonyl or β-selenocarbonyl group can selectively be transformed (/// situ) into an α-β unsaturated carbonyl moiety that functions as a Michael Acceptor. The building blocks of the present invention are easily synthesized in high yield from commercially available compounds involving a relatively low number of synthetic steps, as will be illustrated herein.
The invention also entails the use of these building blocks for the introduction of Michael Acceptors in a peptide or protein. The building blocks of the present invention can be used to incorporate a Michael Addition scaffold in virtually any peptide sequence in a manner that is relatively facile. An important aspect of the invention resides in the fact that the building blocks do not actually contain an α-β unsaturated carbonyl moiety and, as such, can be exposed to reactants and conditions normally employed in peptide synthesis, without serious difficulties, to introduce a group that, subsequently, can easily be transformed {in situ) into the α-β unsaturated carbonyl moiety.
Novel polypeptides that become available by this approach, as well as the intermediates, are also the subject of this invention.
Using the approach disclosed herein, diUb based DUB ABPs resembling all seven native diUb structures (i.e. K6, Kil, K27, K29, K33, K48 and K63) were successfully synthesized, as will be illustrated in more detail in the examples.
In summary, the present invention concerns certain building blocks, methods for synthesizing them and their use as building block in protein or peptide synthesis. The invention also concerns methods of synthesizing and modifying selected protease substrates using the present building blocks to obtain useful protease ABP’s or protease inhibitors. Furthermore, the invention concerns the intermediate and end-products obtained in such methods. These and other aspects of the invention, as defined in the appending claims, will be described and exemplified in more detail in the following description and examples.
Detailed description of the Invention A first aspect of the invention concerns building blocks represented by formula (la), (lb), (Ha) or (lib) and salts and esters thereof:
Figure NL2012026CD00061
Figure NL2012026CD00071
wherein: X represents a moiety selected from the group consisting of CH2; CH2-CH2; CH2-CH2-CH2; CHRa; CHRa-CH2 ; CH2-CHRa; CHRa-CH2- CH2; CH2-CHRa-CH2; or CH2-CH2- CHRa, wherein Ra represents the side chain of an amino acid; R1 represents (i) hydrogen or (ii) an amine protecting group; R2 represents (i) hydrogen; (ii) a group represented by the formula -S-R4, wherein R4 represents an optionally substituted branched or linear C|-Cr, alkyl or Ci-Cealkenyl; or (iii) a thiol protecting group; and R3represents halogen or hydroxyl;
In an embodiment, the compound 4-amino-3-mercapto-butanoic acid, i.e. the compound according to formula (I), wherein R1 represents hydrogen, R2 represents hydrogen and X represents CH2 is excluded from the scope of the invention.
In an embodiment, compounds according to formula (I), wherein R1 represents hydrogen, R2 represents hydrogen are excluded from the scope of the invention.
In the above formulae R1 may represent hydrogen or an amine protecting group. As will be understood by the skilled person, the present invention provides building blocks in unprotected, partly protected or completely protected form. Since the use of the compounds in peptide synthesis will typically require protection of all functional groups, a particularly preferred embodiment of the invention concerns protected building blocks according to formula (I) or (II), wherein R1 an amine protecting group.
The term ‘amine protecting group’ refers to any organic moiety which is readily attached to an amine nitrogen atom, and which, when bound to the amine nitrogen, renders the resulting protected amine group inert to the reaction conditions to be conducted on other portions of the compound and which, at the appropriate time, can be removed to regenerate the amine group. Examples of such amine protecting groups are known to the person skilled in the art and include, but are not limited to: acyl types such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; aromatic carbamate types such as benzyloxycarbonyl (Cbz) and substituted benzyloxy-carbonyls, l-(p-biphenyl)-l-methylethoxy-carbonyl, and 9- fluorenylmethyloxycarbonyl (Fmoc); aliphatic carbamate types such as tert-butyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl; cyclic alkyl carbamate types such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; alkyl types such as trityl and benzyl; trialkylsilane such as trimethylsilane; and thiol containing types such as phenylthiocarbonyl and dithiasuccinoyl. Preferably the amine protecting group in accordance with the present invention is selected from the group consisting of Cbz; /;-Methoxybenzyl carbonyl; Boc; Fmoc; Benzyl; /;-Methoxybenzyl; 3,4-Dimethoxybenzyl; /;-methoxyphenyl; Tosyl; sulfonamides; allyloxycarbonyl; trityl and methoxytrityl, all of which are, as such, commercially available and well-known in the art. Due to their wide-spread application, the exact chemistry involved in the use of these groups in peptide synthesis has been described extensively and is part of the skilled person’s common general knowledge. Amine protecting groups and protected amine groups are described in, e.g., C. B. Reese and E. Haslam, "Protective Groups in Organic Chemistry," J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapters 3 and 4, respectively, and T. W. Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis," Second Edition, John Wiley and Sons, New York, N.Y., 1991, Chapters 2 and 3.
In the above formulae -R2 may represent an alkylthio or alkenylthio, group represented by the formula -S-R4, -R4 representing substituted or unsubstituted linear or branched Ci-Cealkyl or Ci-Cealkenyl. As used herein the term ‘substituted’ is meant to encompass any kind of alkyl comprising one or more substituents. Said substituents may typically incorporate one or more hetero-atoms, such as nitrogen, oxygen, sulfur, phosphorus, boron, chlorine, bromine, or iodine. A moiety of formula -S-R4, creating a disulphide compound, as will be understood by the skilled person, is effective as a protecting group in general peptide synthesis procedures. Hence, since the minimal function of the -S-R4 moiety, if present, is only to block the thiol or selenol moiety, e.g. during protein or peptide synthesis, the exact structure of-R4 is not particularly critical. In a preferred embodiment -R4 represent a branched or linear Ci-C6 alkyl or alkenyl moiety, more preferably a branched or linear Ci-Cö alkyl, most preferably a group selected from methyl, ethyl, propyl, butyl and tert-butyl. In a particularly preferred embodiment, -R4 represents tert-butyl.
Furthermore, -R2 in the above formulae may represent a thiol protecting group. The term ‘thiol protecting group’ refers to any organic moiety which is readily attached to a thiol sulphur atom atom, and which, when bound to said atom, renders the resulting protected group inert to the reaction conditions to be conducted on other portions of the compound and which, at the appropriate time, can be removed to regenerate the thiol group. Suitable examples of such protecting groups are known to the person skilled in the art. Preferably the protecting group in accordance with the present invention is selected from the group consisting of benzyl; 4-methoxybenzyl; trityl; methoxytrityl; t-butyl; t-butylthiol; methylthiol; acetyl; 3-nitro-2-pyridinesulphenyl; acetamidomethyl; Cbz and 2-nitrobenzyl. The use of thiol-protecting groups is well known in the art, cf, for example, T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999). X represents a moiety selected from the group consisting of CH2; CH2-CH2; CH2-CH2-CH2; CHRa; CHRa-CH2 ; CH2-CHRa; CHRa-CH2- CH2; CH2-CHRa-CH2; or CH2-CH2- CHRa, preferably CH2 or CHRa, most preferably CHRa.
As mentioned above, Ra represents an amino acide side chain, typically an amino acid side chain of one of the naturally occurring amino acids, most preferably a side chain of an amino acid selected from the group consisting of Histidine; Alanine; Isoleucine; Arginine; Leucine; Asparagine; Lysine; Aspartic acid; Methionine; Cysteine; Phenylalanine; Glutamic acid; Threonine; Glutamine; Tryptophan; Glycine; Valine; Proline; Selenocysteine; Serine; and Tyrosine. As will be appreciated by those skilled in the art, based on the disclosure herein, the preferred meaning depends on the ABP or inhibitor to be produced with the building block. In particular, it will be preferred, in one embodiment, that Ra corresponds to the amino acid side chain of the amino acid residue in the natural protease substrate that is to be replaced by the building block. Hence, the invention is not particularly limited in this regard. However, in one embodiment of the invention it is preferred that X represents CH2. Building blocks wherein X represents CH2 will be relatively easy to synthesize. R3 is selected from the group of halogens and hydroxyl. Preferably R3 is a halogen, preferably chloro, bromo, fluor or Iodo, to provide a good leaving group. In a particularly preferred embodiment of the invention, R3 represents chloro.
In a particularly preferred embodiment of the invention a building block according to formula (I) is provided wherein -X- represents CHRa; -R1 represents an amine protecting group selected from Fmoc, Boc, trityl or methoxytrityl; and -R2 represents an amine protecting group selected from Fmoc, Boc, trityl or methoxytrityl; or a salt or ester thereof.
In another particularly preferred embodiment of the invention a building block according to formula (II) is provided wherein -X- represents CHRa; -R1 represents an amine protecting group selected from Fmoc, Boc, trityl or methoxytrityl; -R2 represents; and -R3 represents chloro; or a salt or ester thereof.
As will immediately be recognized by the skilled person, the building blocks of the invention contain at least one centre of chirality, such that four potential stereoisomers are possible. The present invention encompasses (the use of) any isomerically pure compound as well as any racemic or non-racemic mixture. An embodiment of the invention concerns a building block as defined herein above wherein the β-carbon atom of the building block has the L-configuration.
Esters, salts, and other derivatives of these thiobuilding blocks are also within the ambit of the invention. Any such derivative may thus be provided without departing from the scope of the invention, provided it is still suitable for use as a building block in protein or peptide synthesis to provide the the Michael Acceptor, without the need for major chemical modification prior to said use.
The building blocks of the present invention can be produced by a person skilled in the art by subjecting commercially available starting materials to reaction steps commonly known in the art.
An aspect of the invention, concerns an optimized method of synthesizing the building block according to formula (la) as defined herein before, said method comprising the steps of (i) treating commercially available 4-amino-3-hydroxybutanoic acid so as to result in esterification of the carboxylic acid and protection of the amine group (ii) tosylation of the esterified and amine protected compound obtained in step (i), (iii) converting the tosylated compound obtained in step (ii) into a thioester (iv) selective hydrolysis of the thioester to obtain a thiol group. Optionally, step (iv) may be followed by protecting the thiol group. Typically, step (i) comprises protecting the amine group with an amine protecting group as defined herein elsewhere. Step (i) also typically comprises converting the carboxylic acid group into an ester group, preferably a methyl, ethyl or allyl ester. In step (ii), the compound obtained in step (i) is typically tosylated by reacting it, in a suitable solvent, with a tosylate, preferably tosyl chloride. In step (iii), the tosylated compound is then reacted with thiocarboxylic acid or salt thereof in a suitable solvent to yield the corresponding thio-ester. In a preferred embodiment of the invention thioacetic acid or a salt thereof is used. Next, hydrolysis of the thio-ester, furnishes the thiol which, can be converted to any other compound of formula (I) by combining it, in a suitable solvent, with an agent that can transfer a group -R2 to said thiol. For example, a thioester may be converted to the corresponding thiol using hydroxylamine, which thiol, in solution, is subsequently combined, drop-wise, with a solution of S-methyl methanethiosulfonate yielding the compound of formula (I) wherein -R2 represents -S-S-CH3.
As will be understood by the skilled person, the corresponding unprotected compounds can be obtained by omitting this last step, and, if desired, by removing any other protecting groups.
Since all of the above reactions are per se known in the art, albeit in relation to the synthesis of different compounds, it is entirely within the skills of the trained professional to determine proper and optimal reaction conditions for each of the above steps as well as to apply any additional isolation or purification technique between the above steps in order to obtain the desired compound in the highest possible yield. A complete step by step description of the synthesis of certain compounds of the invention is given in the examples below.
The present invention also concerns any of the intermediate products obtained in the synthesis of the present building blocks. A third aspect of the invention concerns activity based probes or protease inhibitors, resulting from the modification of a protease substrate by ‘substitution’ of the amino acid residue involved in the peptide bond or isopeptide bond cleaved by the protease, with a building block of the invention and by subsequent transformation of the β-mercapto carbonyl or the β-seleno carbonyl group into an α-β unsaturated carbonyl group. The details of the relevant reaction pathways are given here below.
In the most generalized form, these peptides or proteins according to the invention have a structure according to one of the following formulas:
Figure NL2012026CD00121
wherein: η = O, 1, 2 or 3; X has the same meaning as in formulas (I) and (II) as defined above;
Ra and Ra represent independently selected amino acid side chains; [PEPTIDE], [PEPTIDE]’ and [-PEPTIDE]’ represent peptide chains having a length of at least 1 amino acid residue, preferably at least 2 amino acid residues, more preferably at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, or at least 15 amino acids.
In an embodiment, such peptides or proteins are provided having a total length of at least 4 amino acid residues, preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 12, more preferably at least 15, more preferably at least 20 more preferably at least 25 amino acid residues. In an embodiment, such peptides or proteins are provided having a length equal to at least 50%, at least 60 %, at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 92.5 %, at least 95 %, at least 97 %, at least 98 % or at least 99 % of the full length natural substrate for the protease of interest.
As will be understood by those of average skill in the art, the amino acid side chains Ra and Ra and the peptide chains represented by [PEPTIDE], [PEPTIDE]’ and [-PEPTIDE]’ in the above formulas are preferably chosen such that the resulting structure has the capability of being recognized by the target protease and/or to bind to the target protease. In one embodiment, this means that said amino acid side chains and said peptide chains are chosen such that they resemble the corresponding amino acid residue in the natural protease substrate.
The intermediate products, in particular those obtained before transformation of β-mercapto carbonyl or the β-seleno carbonyl group into an α-β unsaturated carbonyl group are also encompassed by the scope of this invention. For ease of reference, the ABP’s, protease inhibitors and intermediates are also collectively referred to herein as ‘the modified protease substrates’. A protease substrate may be a linear amino acid sequence or a non-linear protein conjugate comprising two (or more) linear amino acid sequences conjugated through an isopeptide bond, e.g. between a C-terminal carboxylic acid group and an epsilon amine of a lysine residue. As will be understood by those skilled in the art, a protein that is a substrate or target for a protease contains a specific sequence of amino acids that results in recognition and cleavage by the protease. Said sequence is referred to herein as ‘recognition sequence’ or ‘cleavage sequence’. A protease will cleave a specific amide bond within the substrate, which may be a linear amide bond or an isopeptide bond, resulting in two ‘fragments’. Within the context of this invention, this specific amide bond is referred to as the ‘cleavage site’ or the ‘scissile bond’ and the fragments resulting from cleavage by the protease are referred to as ‘cleavage fragments’.
The term ‘primed side’ refers to the fragment containing the primary amine group that contributes to the amide that is cleaved by the protease in the corresponding substrate protein. This may also be referred to as the N—>C cleavage fragment. In this designation the cleavage site is taken as the point of reference, meaning that the N-terminal site of the fragment contributes to the amide cleaved by the protease in the corresponding substrate protein. Similarly the term ‘non-primed side’ refers to the fragment containing a terminal carboxylic acid group that contributes to the amide that is cleaved by the protease in the corresponding substrate protein. This may also be referred to as the C^-N cleavage fragment. In this designation the cleavage site is taken as the point of reference, meaning that the C-terminal site of the fragment contributes to the amide cleaved by the protease in the corresponding substrate protein.
For ease of reference, the amino acid residues in the protease substrate and, hence, the corresponding modified protease substrate, are identified herein based on their position in the protein backbone relative to the cleavage site. In the context of the present invention the amino acid positions of the non-primed side fragment are designated 1, 2, 3, ...., co, wherein 1 denotes the position adjacent to the cleavage site and co denotes the N-terminal amino acid position. The amino acids at these positions are designated P1, P2, P3, ..., Ρω, wherein P1 is thus used to denote the amino acid containing the terminal amine group that contributed to the cleaved amide in the corresponding (natural) protease substrate.
Similarly, the amino acid positions of the primed side fragment are designated Γ, 2’, 3’, ..., ω’, wherein Γ denotes the position adjacent to the cleavage site and the amino acid residues are designated P1’, P2’, P3’, ..., Ρω’, wherein a'1 denotes the amino acid residue containing the carboxylic acid group that contributes to the cleaved amide in the corresponding (natural) protease substrate.
The protease substrate may contain an isopeptide bond as the cleavage site, typically between the C-terminal carboxyl group of one fragment and an amino acid side chain amine group of the other fragment, as is the case for e.g. diubiquitins, which are substrates for deubiquitinating proteases. In such embodiments, the amino acid positions of the peptide chain containing the carboxyl group involved in the isopeptide bond are designated 1, 2, 3, ...., ω, wherein 1 denotes the position adjacent to the cleavage site and ω denotes the N-terminal amino acid position. The amino acids at these positions are designated P1, P2, P3, ..., Ρω, wherein P1 is thus used to denote the amino acid containing the terminal amine group that contributed to the cleaved amide in the corresponding (natural) protease substrate. The amino acid positions of the peptide chain containing the lysine residue involved in the isopeptide bond are designated -ω’,......, -3’, -2’, -Γ, 0’, Γ, 2’, 3’, ...., ω’, wherein 0’ denotes the position of the lysine residue involved in the isopeptide bond, Γ and -Γ denote the position adjacent to said lysine residue and -co’ and co’ denotes the N-and C-terminal amino acid positions. The amino acids at these positions are designated P_t0’, p-35 p-2 > p-lj p05 pi 5 p2 5 p3> ρω?
As described above, the modified protease substrate is obtained by the introduction of a building block of this invention and, optionally, transformation of the β-mercaptocarbonyl or β-selenocarbonyl into the α,β-unsaturated carbonyl. The building block, typically, is introduced by substitution of the amino acid residue P1, following the denomination set out above. A structure is accordingly obtained having a Michael acceptor group exactly at the position of the cleavage site of the corresponding ‘natural’ protease substrate, i.e. when the capturing agent and the ‘natural’ modified protease substrate are projected over one another. For instance, the experimental part below, describes ‘substitution’ of the C-terminal glycine residue of ubiquitin with a building block of this invention and subsequent isopeptide linkage to a second ubiquitin (which was modified by replacing the lysine residue at position 0’ by a diaminobutyric acid residue).
Hence, in one embodiment, the invention concerns a modified protease substrate represented by any one of formulas (111)-(VI):
Figure NL2012026CD00151
Figure NL2012026CD00161
wherein: η = O, 1, 2 or 3; R2 and X have the same meaning as in formulas (I) and (II) as defined above;
Ra# represents an amino acid side chain identical to the amino acid side chain of the amino acid at the corresponding position in the non-modified protease substrate; [PEPTIDE] and [PEPTIDE]’ represent peptide chains having the amino acid sequences Ρ3-Ρω and P3 -Ρω respectively, wherein P# represents an amino acid residue identical to the amino acid residue in the corresponding position in the non-modified protease substrate, wherein the positions are defined relative to the site of hydrolysis, P1 and P1 representing the amino acid residues adjacent to the site of hydrolysis and Ρω and Ρω representing the N-terminal and C-terminal amino acid residues; or N- and/or C-terminally truncated variants of said modified protease substrate, wherein [PEPTIDE] and [PEPTIDE]’ may comprise a number of amino acid residues of equal to or higher than 0; or a homologue, conjugate or derivative thereof.
In another embodiment, the invention concerns a modified protease substrate represented by any one of formulas (VII)-(X):
Figure NL2012026CD00171
Figure NL2012026CD00181
wherein: R2 and X have the same meaning as in formulas (I) and (II) as defined above; n = 0, 1, 2 or 3;
Ra# represents an amino acid side chain identical to the amino acid side chain of the amino acid at the corresponding position in the non-modified protease substrate; [PEPTIDE] represents a peptide chain having the amino acid sequence Ρ2-Ρω; [PEPTIDE]’ and [-PEPTIDE]’ represent peptide chains having an amino acid sequence P1 -Ρω and P'1 -Ρ'ω, wherein P# represents an amino acid residue identical to the one in the corresponding position in the non-modified protease substrate, wherein the positions are defined relative to the site of hydrolysis, P1 representing the C-terminal amino acid residue of the isopeptide bond; P1 and P'1 representing the amino acid residues adjacent to the lysine residue of the isopeptide bond; and Ρω, Ρω and Ρ'ω representing the terminal amino acid residues of the respective peptide chains; or N- and/or C-terminally truncated variants of said modified protease substrate, wherein [PEPTIDE], [PEPTIDE]’ and/or [-PEPTIDE]’ may comprise a number of amino acid residues of equal to or higher than 0; or a homologue, conjugate or derivative thereof.
As will be clear from the definition and explanations in the foregoing, Ra# corresponds to the side chain of the amino acid residue at the respective position in the corresponding (natural) protease substrate.
As will be understood, based on the explanation provided herein, the variable n in formulas (VII)-(X) indicates that the lysine residue of the ‘natural’ protease substrate (i.e. amino acid residue P0’) may be replaced with a corresponding nonnatural amino acid, having a shorter side chain than lysine, such as diaminobutyric acid (DAB). This may compensate for the (minor) shift resulting from the introduction of the building block of this invention, so as to further ‘optimize’ the fit of the modified protease substrate at the protease active site. In an embodiment of the invention n = 1. Similarly, in formulas (III)-(VI) n indicates that the chain length may be varied to optimize the fit of the modified protease substrate at the recognition site. As will be recognized by those skilled in the art, when n = 1, the amino acid residues Ρ2’-Ρω (prime side) coincide exactly with the corresponding amino acid residues in the non-modified protease substrate, which represents the most preferred embodiment.
Furthermore, as will be clear from the explanation and definitions above, [PEPTIDE], [PEPTIDE]’ and [-PEPTIDE]’, in the above formulas, typically represent amino acid chains identical to the corresponding portion of the naturally occurring protease substrate. In this context ‘corresponding portion’ means the amino acid chain found in the naturally occurring protease substrate at the same position relative to the cleavage site. For example, if the protease is a deubiquitinating enzyme, ρΆρ1 in formula (VI) represent the entire naturally occurring ubiquitin sequence and [PEPTIDE] in formula (I) thus defines said entire ubiquitin sequence minus the two C-terminal amino acids.
Truncated versions of the modified protease substrates defined above, homologues of the modified protease substrates and/or conjugates comprising the modified protease substrates are also encompassed by the scope of the invention, provided that the resulting agent is still capable of being recognized by and interacting with the active site of the protease.
Specific embodiments of the invention concern truncated versions of the modified protease substrates of the invention. It will be understood by those skilled in the art that, for maintaining the capability of the (modified) protease substrate to be recognized by the protease active site, truncations of the terminal part(s) of the primed side(s) and/or non-primed side, distant from the cleavage site of the corresponding protease substrate, may not affect recognition and binding by the protease. The length of any truncation is not particularly limited provided that the remaining peptide is still capable of being recognized by the protease. In a preferred embodiment of the invention an N- and/or C-terminally truncated variants of the modified protease substrates are provided having a length of at least 4 amino acid residues, preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 12, more preferably at least 15, more preferably at least 20 more preferably at least 25; or a homologue, conjugate or derivative thereof. It will be understood by those skilled in the art that in determining the length of (truncated versions of) the modified protease substrate, the moiety containing the Michael Acceptor group counts as a single amino acid residue.
Hence, in an embodiment [PEPTIDE], [PEPTIDE]’ and/or [-PEPTIDE]’ represent truncated versions of the corresponding portions of the ‘wild-type’ protease substrate, with the proviso that the resulting agent is still capable of being recognized by and interacting with the active site of the protease. Preferably, in the above formulae (III)-(VI) [PEPTIDE] and [PEPTIDE]’ represent an amino acid sequence having a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, or at least 15. Preferably, in the above formulae (VII)-(X) [PEPTIDE] represents an amino acid sequence having a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, or at least 15 and [PEPTIDE]’ and/or [-PEPTIDE]’ represent an amino acid sequence having a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, or at least 15. Preferably, each of the peptides represented by [PEPTIDE], [PEPTIDE]’ and [-PEPTIDE]’ have a length ranging from 1-10 amino acid residues, preferably 2-8 amino acid residues, more preferable 2-6. In another preferred embodiment of the invention the fragment is the full length modified protease substrate. In another embodiment, the modified protease substrate is a truncated version containing at least 50%, at least 60 %, at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 92.5 %, at least 95 %, at least 97 %, at least 98 % or at least 99 % of the amino acid sequence of the full length modified protease substrate.
In an embodiment [PEPTIDE], [PEPTIDE]’ and/or [-PEPTIDE]’ represent homologues of the corresponding portions of the ‘wild-type’ protease substrate, with the proviso that the resulting agent is still capable of being recognized by and interacting with the active site of the protease. The term ‘homologue’ is used herein in its common meaning, as referring to polypeptides which differ from the reference polypeptide, by minor modifications, but which maintain the basic polypeptide and side chain structure of the reference peptide. Such changes include, but are not limited to: changes in one or a few amino acid side chains; changes in one or a few amino acids, including deletions, insertions and/or substitutions; changes in stereochemistry of one or a few atoms; additional N- or C- terminal amino acids; and/or minor derivatizations, including but not limited to: methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol. Non-naturally occurring mutants of particular interest furthermore include mutants comprising certain insertions and/or substitutions that create ligation handles, especially the substitution of lysine with 6-thiolysine, δ-selenolysine, γ-thiolysine, γ-selenolysine (all as described in co-pending patent application no. PCT/NL2010/050277) or δ-azido ornithine or the substitution of leucine with photoleucine. As used herein, a homologue or analogue has either enhanced or substantially similar functionality as the naturally occurring polypeptide. A homologue herein is understood to comprise a polypeptide having at least 70 %, preferably at least 80 %, more preferably at least 90 %, still more preferably at least 95 %, still more preferably at least 98 % and most preferably at least 99% amino acid sequence identity with the reference polypeptide, when optimally aligned, such as by the programs GAP or BESTFIT using default parameters, and is still capable of eliciting at least the immune response obtainable thereby. Generally, the GAP default parameters are used, with a gap creation penalty = 8 and gap extension penalty = 2. For proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752, USA. Alternatively percent similarity or identity may be determined by searching against databases such as FASTA, BLAST, etc.
Porteases, in accordance with the present invention, include any protease having an active site nucleophile and a general base. In particular, the invention pertains to (the substrates of) serine proteases, threonine proteases and cysteine proteases, in particular (the substrates of) threonine and cysteine proteases. Hence, in a preferred embodiment of the invention, the protease is a threonine protease or a cysteine protease and the modified protease substrate is a modified threonine protease substrate or a modified cysteine protease substrate.
Threonine proteases, in accordance with this invention, in particular, include the proteasome and/or the proteasome catalytic subunits.
Cysteine proteases, in accordance with this invention, may for example be selected from the group consisting of cathepsin B; cathepsin C; cathepsin F; cathepsin H; cathepsin K; cathepsin L; cathepsin L2; cathepsin O; cathepsin S; cathepsin W; cathepsin Z; cathepsin J; cathepsin M; cathepsin Q; cathepsin Q2; cathepsin Q2-like; cathepsin R; cathepsin-1; cathepsin-2; cathepsin-3; cathepsin-6; cathepsin-7-like; tubulointerstitial nephritis antigen; TINAG related protein; testin; testin-2; testin-3; bleomycin hydrolase; calpain 1; calpain 2; calpain 3; calpain 5; calpain 6; calpain 7; calpain 7-like; calpain 8; calpain 9; calpain 10; calpain 11; calpain 12; calpain 13; calpain 14; calpain 15/Solh protein; ubiquitin C-terminal hydrolase 1; ubiquitin C-terminal hydrolase 3; ubiquitin C-term. hydrolase BAP1; ubiquitin C-terminal hydrolase 5; ubiquitin C-terminal hydrolase 4; legumain; hGPI8; caspase-1; caspase-2; caspase-3; caspase-4/11; caspase-5; caspase-6; caspase-7; caspase-8; caspase-9; caspase-10; caspase-12; caspase-14; paracaspase; homologue ICEY; casper/FLIP; caspase-14-like; pyroglutamyl-peptidase I; pyroglutamyl-peptidase II; USP1; USP2; USP3; USP4; USP5; USP6; USP7; USP8; USP9X; USP9Y; USP10; USP11; USP12; USP13; USP14; USP15; USP16; USP17; USP17-like; USP18; USP19; USP20; USP21; USP22; USP24; USP25; USP26; USP27; USP28; USP29; USP30; USP31; NY-REN-60; VDU1; USP34; USP35; USP36; USP37; HP43.8KD; SADI; USP40; USP41; USP42; USP43; USP44; USP45; USP46; USP47; USP48; USP49; USP50; USP51; USP52; USP53; USP54; DUB-1; DUB-2; DUB2a; DUB2a-like; DUB2a-like2; DUB6; BAP1; UCHL1; UCHL3; UCHL5; gamma-glutamyl hydrolase; Gln-PRPP amidotransferase; Gln-fructose-6-P transamidase 1; Gln-fructose-6-P transamidase 2; Gln-fructose-6-P transamidase 3; sonic hedgehog protein; indian hedgehog protein; desert hedgehog protein; sentrin/SUMO protease 1; sentrin/SUMO protease 2; sentrin/SUMO protease 3; sentrin/SUMO protease 5; sentrin/SUMO protease 5-like 1; sentrin/SUMO protease 6; sentrin/SUMO protease 7; sentrin/SUMO protease 8; sentrin/SUMO protease 9; sentrin/SUMO protease 11; sentrin/SUMO protease 12; sentrin/SUMO protease 13; sentrin/SUMO protease 14; sentrin/SUMO protease 15; sentrin/SUMO protease 16; sentrin/SUMO protease 17; sentrin/SUMO protease 18; sentrin/SUMO protease 19; separase;autophagin-l; autophagin-2; autophagin-3; autophagin-4; DJ-1; cezanne/OTU domain containing 7B; cezanne-2; A20, TNFa-induced protein 3; TRAF-binding protein domain; VCP(p97)/p47-interacting protein; Hin-l/OTU domain containing 4; asparagine-linked glycosylation 13 homolog; OTU domain containing-3; OTU domain containing-1; OTU domain containing-6A; OTUD2/YOD1; OTU domain containing 6B; CGI-77b; otubain-1; otubain-1 like; otubain-2; cylindromatosis protein; secernin-1; secemin-2; secemin-3; Ufm-1 specific protease 1; Ufm-1 specific protease 2; nasal embryonic LHRH factor; epithelial cell transforming sequence 2 oncogene-like; OTU domain containing-5; ataxin-3; ataxin-3 like; josephin-1; josephin-2; acid ceramidase; HetF-like; zinc finger CCCH-type containing 12A; zinc finger CCCH-type containing 12B; zinc finger CCCH-type containing 12C; zinc finger CCCH-type containing 12D; NYN domain and retroviral integrase containing; KHNYN KH and NYN domain containing; NEDD4 binding protein 1. In accordance with the present invention the cysteine protease is preferably selected from the group consisting of deubiquitinating proteases, cathepsins, calpains, caspases and SUMO proteases, preferably from the group of deubiquitinating proteases, SUMO protease, caspases and cathepsins, more preferably from the group of deubiquitinating proteases and SUMO proteases, and most preferably from the group of deubiquitinating proteases.
Accordingly, as will be understood by those skilled in the art, the protease substrate preferably is a protein targeted by any one of the above defined proteases.
In a preferred embodiment of the invention, a modified protease substrate represented by any of formulae (VII)-(X) is provided according to which [Ρ'-Ρω] represents the ubiquitin peptide sequence and wherein [P_t0’, ...., P'2’, P'1’, P0’, P1’, P2’, ...., Ρω’] also represents the ubiquitin peptide sequence.
In an embodiment, the modified protease substrate of the invention is conjugated with another peptide or protein, in a linear or non-linear fashion, with the proviso that the capability of the resulting peptide chain to be recognized by and interact with the active site of the protease is retained. Such conjugates may be used to introduce or affect chemical or biological functionality, e.g. cell permeability enhancement, proteasome targeting, introduction of sites for directed chemical modifications (introduction of a so-called ‘ligation handle’), affinity tagging, etc. Preferred examples include addition of cell penetration enhancing peptide sequences such as (D-Arg)8, Tat and penetratin; addition of affinity tag peptide sequences, such as HA and His6; addition of a proteasome targeting handle such as L4; and substitution of N- or C-terminal residues.
In addition to variations in the amino acid sequence, the here described invention also entails structures comprising a derivative of the modified above defined modified modified protease substrates, typically comprising a ligand coupled to an amino acid side chain thereof and/or the N-terminus and/or the C-terminus thereof. As used herein the terms ‘derivative’ thus refer to products comprising a modified modified protease substrate as defined herein before, further comprising one or more ligands derivatized to the C-terminal carboxyl group, the N-terminal amine group and/or an amino acid side chain. Such ligands may, in principle, be of any nature, including peptides or proteins, lipids, carbohydrates, polymers and organic or inorganic agents. The introduction of the ligand typically introduces or affects a particular biological or chemical function. Particularly interesting examples include the introduction of detectable labels and tags, introduction of electrophilic traps, introduction of chemical ligation moieties, etc. Hence, in a preferred embodiment, a method as defined herein before is provided, wherein said derivative comprises a ligand selected from the group of fluorophores, affinity labels, biophysical labels, chelating agents, complexing agents and epitope tags, such as fluorescein, TAMRA or DOTA. Those skilled in the art will be familiar with these types of ligands and their introduction at a desired site can be accomplished using reagents and conditions that are generally known.
Another aspect of the present invention concerns a method of producing a modified protease substrate comprising the steps of: i) identifying a substrate for the protease of interest; ii) chemical, biochemical or biological synthesis or production of the protease substrate wherein the amino acid residue at position 1, i.e. the amino acid residue designated P1 according to the definitions in the foregoing, is substituted by a building block of the invention; and iii) modifying the building block in situ by transforming the β-mercapto carbonyl or β-seleno carbonyl moiety into an α,β-unsaturated carbonyl moiety. As will be understood by those skilled in the art, the substrate for the protease typically will be a/the natural substrate for the protease, examples of which have been described herein elsewhere.
Furthermore, as will be clear from the foregoing, the method may comprise additional modifications of the protease substrate, e.g. by truncations, derivatizations, conjugations, amino acid deletions, insertions or substitutions, etc., with the proviso that the capability of the resulting structure to be recognized by and interact with the active site of the protease is retained.
Chemical peptide synthesis methods are well known to the person skilled in the art. In accordance with the present invention the peptides are typically chemically synthesized, preferably using solid phase synthesis. In accordance with the present invention, it is not critical whether the entire sequence is synthesised through stepwise elongation only or whether the process involves ligation of two or more separately obtained fragments. Typically, it may be preferable to at least produce the respective fragments corresponding to the cleavage fragments separately and ligate the building block at position P1 with the N-terminal P1’ amino acid residue (in case of a linear protease substrate) or the P0’ amino acid residue (in case of an isopeptide protease substrate) through processes known as fragment condensation and/or chemical ligation.
Alternatively the buidling block presented herein can be incorporated into a protein using orthogonal tRNA/aminoacyl-tRNA synthetase pairs, which incorporates the unnatural building block in response to a nonsense or four-base codon in the gene of the protein of interest. To date this technology has allowed the incorporation of approximately 50 unnatural amino acids (cf. J. M. Xie, P. G. Schultz, Nat. Rev. Mol. Cell Biol. 2006, 7, 775 and/or P. R. Chen, D. Groff, J. Guo, W. Ou, S. Cellitti, B. H. Geierstanger, and P. G. Schultz, Angew. Chem. Int. Ed. 2009, 75, 4052). For this purpose, the building block of the invention is preferably provided in non-conjugated and, preferably, non-protected form at the α-amine and carboxylic acid.
As noted before, the synthesis of the peptide is preferably performed on a solid phase substrate, yielding a peptide that is covalently attached to said substrate. In one embodiment, the building bock of the invention is attached to the C-terminal P amino acid residue while the peptide (fragment) is still attached to the solid support. In one embodiment of the invention, the peptide chain is released from the solid support after addition of the P2 amino acid and the building block of the invention is attached after re-dissolving the protected peptide chain.
One or more of the subsequent steps of the present method may be performed before or after release of the peptide from said solid phase substrate. Transformation of the building block is performed on the solid phase as well. The method of the invention may concern solid phase synthesis without release from the solid phase substrate prior to or after the subsequent steps of the invention, e.g. in the case of protein (micro)arrays, where the protein can be synthesized directly on the microarray surface.
In a particularly preferred embodiment of the invention, the method comprises transformation of the β-mercapto carbonyl moiety into an α,β-unsaturated carbonyl moiety in situ. This entails the particular advantage that the Michael acceptor is introduced post-ligation, preventing any side reactions of this very reactive group.
Another aspect of the invention concerns the use of the building blocks as defined herein in the synthesis of a selected peptide or protein, typically a threonine or cysteine protease substrate, for the purpose of introducing a Michael Acceptor in said selected polypeptide or protein sequence. In an embodiment of the invention the use of the building blocks as defined herein is provided for converting a natural protease substrate into a protease ABP and/or protease inhibitor.
Another aspect of the invention concerns modified protease substrates obtainable by the afore-defined methods.
Another aspect of the present invention concerns the use of the modified protease substrates as defined in any of the foregoing as a protease inhibitor in therapy and/or as an activity based probe in biochemistry research and/or diagnostics.
As will be understood by one skilled in the art ABPs of the present invention can be used to bind their corresponding protease, e.g. from a highly complex biological matrix, which can be of particular use in both diagnostics and fundamental research. Hence, the invention, in one aspect, also provides a method of capturing (or ‘pulling down’) of a protease from a biological sample, said method comprising the steps of: a) providing said sample comprising a proteases; b) combining the sample with a corresponding ABP of this invention, wherein said ABP is conjugated to a chelating agent, a complexing agent, an epitope tag or a solid phase, which allows for or results in immobilization of the ABP; c) subjecting the sample to conditions that allow for selective binding of the ABP; d) separating the sample from the immobilized ABP. Immobilization of ABPs, which take the form of (conjugated/derivatized) peptides, can be achieved using various techniques familiar to those skilled in the art. Depending on the choice of immobilization technique the above-described method may comprise the additional step of combining the sample comprising the ABP with a solid phase capable of immobilizing the ABP, prior to any one of steps a), b), c) or d). If the immobilization step is done after step b), as will be understood, a technique is to be selected involving selective trapping under condition which do not affect other components of the biological sample. Hence, it will be appreciated that a preferred embodiment of the method comprises immobilization of the ABP prior to step b). As will be understood by those skilled in the art, immobilization of the ABP can be accomplished in various ways. In one embodiment of the invention, the modified protease substrate is immobilized using CNBr-activated sepharose. In an embodiment of the invention, the above method involves the use of an ABP that is conjugated/derivatized with a detection label as defined herein above, wherein the method comprises one or more additional steps of quantifying the binding of protease to the ABP. As explained herein before the present ABPs bind their corresponding protease in a highly selective and irreversible manner, allowing for stringent washing conditions, which makes the present method highly effective. The above method may be used in research concerning any biological process involving the action of a protease and/or in diagnosing any condition or disease involving the action of a protease.
Since the present modified protease substrates are capable of selective and highly irreversible binding of their corresponding protease, it is also envisaged that they have utility as (competitive) protease inhibitors or antagonistic agents in various therapeutic methods. Typically such therapeutic methods are aimed at the treatment or prevention of a condition or disease, involving the action of a protease, in particular cysteine proteases or threonine proteases.
Conditions or diseases involving the action of cysteine proteases may include auto immune diseases, cancer (metastatic and non-metastatic), infections and lysosomal storage diseases. The invention, in further aspects, provides the use of a modified cysteine protease substrate as defined in the foregoing as an inhibitor of a corresponding cysteine protease; a method of inhibiting cysteine protease activity by exposing the cysteine protease to a corresponding modified cysteine protease substrate of this invention; and the modified cysteine protease substrate for use in any such method. Said methods are, in particular, aimed at treatment or prevention of any condition or disease involving the action of cysteine proteases, as recited above.
The invention, in further aspects, provides the use of a modified proteasome substrate as defined in the foregoing as a proteasome inhibitor; a method of inhibiting proteasome activity by exposing proteasome to a corresponding modified substrate as defined herein before; and the modified proteasome substrate for use in any such method. As will be understood by those skilled in the art, these methods are aimed at the treatment or prevention of any disease or condition involving the action of the proteasome, in particular diseases or conditions caused or characterized by an increase in proteasome expression or activity or any disorder, disease or condition in which inhibition of proteasome activity is beneficial. For example, proteasome inhibitors of the invention can have utility in treatment of disorders mediated via proteins (e.g., NFkB, p2ΊΚιρ, p2lWAF/CIP1, p53) which are regulated by proteasome activity, e.g. inflammatory disorders (e.g., rheumatoid arthritis, inflammatory bowel disease, asthma, chronic obstructive pulmonary disease (COPD), osteoarthritis, dermatosis (e.g., atopic dermatitis, psoriasis)), vascular proliferative disorders (e.g., atherosclerosis, restenosis), proliferative ocular disorders (e.g., diabetic retinopathy), benign proliferative disorders (e.g., hemangiomas), autoimmune diseases (e.g., multiple sclerosis, tissue and organ rejection), as well as inflammation associated with infection (e.g., immune responses), neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, motor neurone disease, neuropathic pain, triplet repeat disorders, astrocytoma, and neurodegeneration as result of alcoholic liver disease), ischemic injury (e.g., stroke), cachexia (e.g., accelerated muscle protein breakdown that accompanies various physiological and pathological states, (e.g., nerve injury, fasting, fever, acidosis, HIV infection, cancer affliction, and certain endocrinopathies)), and cancer (e.g. pancreatic cancer; bladder cancer; colorectal cancer; breast cancer, including metastatic breast cancer; prostate cancer, including androgen-dependent and androgen-independent prostate cancer; renal cancer, including, e.g., metastatic renal cell carcinoma; hepatocellular cancer; lung cancer, including, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung; ovarian cancer, including, e.g., progressive epithelial or primary peritoneal cancer; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer, including, e.g., squamous cell carcinoma of the head and neck; melanoma; neuroendocrine cancer, including metastatic neuroendocrine tumors; brain tumors, including, e.g., glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma; bone cancer; and soft tissue sarcoma).
Thus, the invention has been described by reference to certain embodiments discussed above. It will be recognized that these embodiments are susceptible to various modifications and alternative forms well known to those of skill in the art.
Many modifications in addition to those described above may be made to the structures and techniques described herein without departing from the spirit and scope of the invention. Accordingly, although specific embodiments have been described, these are examples only and are not limiting upon the scope of the invention.
Furthermore, for a proper understanding of this document and in its claims, it is to be understood that the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Examples
Examples 1 : Synthesis of building block
In this example a protected building according to this invention, 4-((tert-butoxycarbonyl)amino)-3-(tert-butyldisulfanyl)butanoic acid, was synthesized according to the following reaction scheme.
Figure NL2012026CD00291
Chemical reagents were obtained from Sigma-Aldrich, Fluka and Acros of the highest available grade and used without further purification. 4-amino-3-hydroxybutanoic acid was purchased. Solvents were purchased from Biosolve or Aldrich. Peptide synthesis reagents were purchased from Novabiochem. Analytical thin layer chromatography was performed on aluminium sheets precoated with silica gel 60 F254. Column chromatography was carried out on silica gel (0.035-0.070 mm, 90A, Acros). Nuclear magnetic resonance spectra ('H-NMR, 13C-NMR) were determined in DMSO (¾ δ 2.50 ppm; 13C δ 39.5 ppm) or CDCI3 (3H δ 7.26 ppm; 13C δ 77.2 ppm) using a Bruker ARX 400 Spectrometer ^H-NMR: 400 MHz, 13C-NMR: 100 MHz) at 298 K. Peak shapes in NMR spectra are indicated with the symbols ‘d’ (doublet), ‘dd’ (double doublet), ‘s’ (singlet), ‘F (triplet) and ‘m’ (multiplet). Chemical shifts (δ) are given in ppm and coupling constants J in Hz. LC-MS measurements were performed on a system equipped with a Waters 2795 Separation Module (Alliance HT), Waters 2996 Photodiode Array Detector (190-750nm), Phenomenex Kinetex C18 (2.1x50, 2.6 pm) and LCT™ Orthogonal Acceleration Time of Flight Mass Spectrometer. Samples were run using 2 mobile phases: A = 1% CH3CN, 0.1% formic acid in water and B = 1% water and 0.1% formic acid in CH3CN. Data processing was performed using Waters MassLynx Mass Spectrometry Software 4.1 (deconvolution with MaxEnt I function). LC-MS program: Phenomenex Kinetex Cl8, (2.1x50 mm), 2.6 pM); flow rate= 0.8 mL/min, runtime= 6 min, column T= 40°C. Gradient: 0 - 0.5 min: 5% B; 0.5 - 4 min: 5 -> 95% B; 4 - 5.5 min: 95% B. methyl 4-amino-3-hydroxybutanoate (2) A suspension of 4-amino-3-hydroxybutanoic acid 1 (10.0 g, 83.96 mmol) in MeOH (300 mL), was cooled to 0°C. Then, thionyl chloride (61 mL, 839.6 mmol) was added dropwise turning the suspension into a yellowish solution. The mixture was refluxed for 3 h. and then allowed to cool to r.t. All volatiles were removed in vacuo, followed by co-evaporation with DCM (5x) to remove any residual thionyl chloride. The residue was lyophilized, yielding 12.3 g of crude compound 2. 3H NMR (400 MHz, DMSO): δ 8.05 (s, 1H, OH), 5.58-5.56 (d, 2H, NH2), 4.09 (s, 1H, pCH), 3.60 (s, 3H, OCH3), 2.93-2.69 (m, 2H, yCH2), 2.61-2.40 (m, 2H, aCH2). 13C APT NMR (100MHz, DMSO): δ 171.0 (COOMe), 64.2 (pCH), 51.5 (OCH3), 43.9 (yCH2), 36.2 (aCH2). m/z (ESI+): calculated: 133.07; measured: 134.05 [M+H]+. methyl 4-((tert-hutoxycarhonyl)amino)-3-hydroxyhutanoate (3)
To a solution of compound 2 (1.12 g, 8.40 mmol) and Et2N (4.7 mL, 33.58 mmol) in MeCN (75 mL), a solution of Boc anhydride (2.75 g, 12.59 mmol) in MeCN (5 mL) was added. The reaction mixture was stirred overnight. Solvents and volatiles were evaporated and the resulting residue was dissolved in EtOAc. The organic solution was extracted using 1M KHSO4 (50 mL, 3x) followed by saturated NaHCCri (50 mL, 3x). The organic layer was dried using anhydrous MgSCL and filtrated. The filtrate was concentrated in vacuo and the resulting residue was purified using column chromatography (EtOAc/hexanes, 1:2 -> 1:1, v:v), yielding 70% (over two steps) of compound 3 (1.23 g, 4.00 mmol) as colorless oil. 'HNMR (400 MHz, CDCI3): δ 5.01 (s, 1H, OH), 4.14-4.06 (m, 1H, βΟΗ), 3.70 (s, 3H, OCH3), 3.34-3.29 (m, 2H, yCH2), 2.50-2.47 (m, 2H, aCH2), 1.41 (s, 9H, Boc). 13C APT NMR (100 MHz, CDC13): δ 172.8 (COOMe), 156.7 (BocC=0), 79.6 (BocC), 67.8 (βΟΗ), 51.9 (OCH3), 45.7 (yCH2), 38.7 (aCH2), 28.4 (BocCH3). m/z (ESI+): calculated: 233.13; measured: 133.99 [M-Boc]+, 255.94 [M+Na]+, 489.93 [2M+Na]+. methyl 4-((tert-hutoxycarhonyl)amino)-3-(tosyloxy)hutanoate (4)
Compound 3 (1.00 g, 4.29 mmol) was dissolved in pyridine (6 mL) and cooled to 0°C. Tosyl chloride (2.45 g, 12.86 mmol) was added to the mixture, which was then allowed to warm-up to r.t. during overnight stirring. The reaction mixture was concentrated in vacuo and co-evaporated with toluene and DCM consecutively. The residue was dissolved in DCM and extracted using 1M KHSO4 (15 mL, 2x), followed by saturated NaHCCL (15 mL, 2x). The organic layer was dried using anhydrous MgSCL and filtrated. The filtrate was concentrated in vacuo and the resulting residue was purified using column chromatography (EtOAc/hexanes, 1:3 -> 1:1, v:v), yielding compound 4 (1.55 g, 4.00 mmol) as brown oil. ^NMR (400 MHz, CDCI3): δ 7.80-7.77 (d, 2H, J = 8.2 Hz, PhCH), 7.35-7.32 (d, 2H, J= 8.0 Hz, PhCH), 4.94-4.90 (t, 1H, J= 4.8 Hz, βΟΗ), 3.67 (s, 3H, OCH3) 3.42-3.40 (t, 2H, J= 4.9 Hz, yCH2), 2.65-2.63 (d, 2H, J= 6.3 Hz, aCH2), 2.44 (s, 3H, PhCH3), 1.41 (s, 9H, Boc). 13C APT NMR (100 MHz, CDC13): δ 169.5 (COOMe), 155.8 (BocC=0), 145.1 (PhCl), 133.5 (PhC4), 129.9 (PhC2, PhC6), 127.9 (PhC3, PhC5), 79.8 (BocC), 78.0 (βΟΗ), 51.9 (OCH3), 43.6 (yCH2), 36.9 (aCH2), 28.3 (BocCH3). m/z (ESI+): calculated: 387.14; measured: 287.87 [M-Boc]+, 409.85 [M+Na]+, 796.79 [2M+Na]+. methyl 3-(acetylthio)-4-((tert-hutoxycarhonyl)amino)hutanoate (5)
Thioacetic acid (7.0 mL, 98.21 mmol) and DBU (10.3 mL, 68.74 mmol) were dissolved in DMF (30 mL) and stirred for 5 minutes. A solution of compound 4 (7.61 g, 19.64 mmol) in DMF (60 mL) was added to the reaction mixture, which was then heated to 60°C and stirred overnight. DMF was removed in vacuo and the resulting residue dissolved in EtOAc. The solution was washed with saturated NH4C1, FLO (2x) and brine and the organic layer was dried using MgS04. After filtration and removal of the solvent, the resulting residue was purified using column chromatography (EtOAc/hexanes, 1:6 -> 1:1, v:v), yielding 83% of compound 5 (4.77 g, 16.38 mmol). 'HNMR (400 MHz, CDCL): δ 4.78 (s, 1H, NH), 3.97-3.93 (q, 1H, J= 6.5 Hz, βΟΗ), 3.69 (s, 3H, OCH3) 3.42-3.40 (t, 2H, J= 6.5 Hz, yCH2), 2.69-2.65 (m, 2H, J= 6.7 Hz, aCH2), 2.33 (s, 3H, SAcCH3), 1.41 (s, 9H, Boc). 13C APT NMR (100 MHz, CDC13): δ 197.7 (SAcC=0), 171.2 (COOMe), 155.9 (BocC=0), 79.6 (BocC), 51.9 (OCH3), 43.8 (γΟΗ2), 40.8 (βΟΗ), 36.5 (aCH2), 30.7 (SAcCH3), 28.4 (Boc-CH3). m/z (ESI+): calculated: 291.11; measured: 191.87 [M-Boc]+, 313.82 [M+Na]+, 604.68 [2M+Na]+. methyl 4-((tert-butoxycarbonyl)amino)-3-(tert-butyldisulfanyl) butanoate (6)
To a solution of compound 5 (4.77 g, 16.4 mmol), Et3N (9.12 mL, 65.5 mmol) and hydroxylamine HC1 (4.55 g, 65.5 mmol) in MeOH (160 mL) S-Zc/V-butyl methanesulfonothiate (13.8 g, 81.9 mmol) was added. The reaction mixture was stirred overnight at r.t. All volatiles were evaporated in vacuo and the resulting residue was dissolved in EtOAc. The solution was extracted with H20, 1M KHS04 and saturated NaHC03. The organic layer was dried using MgS04. After filtration and removal of the solvent, the resulting residue was purified using column chromatography (EtOAc/hexanes, 1:9 -> 2:1, v:v), yielding crude compound 6 as a red, oily suspension. 3H NMR (400 MHz, CDC13): δ 4.90 (s, 1H, NH), 3.67 (s, 3H, OCH3) 3.42-3.39 (t, 2H, J= 6.0 Hz, γΟΗ2), 3.31-3.25 (t, 1H, J= 6.0 Hz, βΟΗ), 2.60-2.58 (d, 2H, J= 6.0 Hz, aCH2), 1.40 (s, 9H, Boc), 1.30 (s, 9H, StBu). 13C APT NMR (100 MHz, CDCL): δ 171.6 (COOMe), 155.9 (NC=0), 79.6 (BocC), 51.9 (OCH3), 48.3 (βΟί), 48.1 (StBuC), 43.2 (γΟί2), 36.8 (aCH2), 30.0 (StBuCH3), 28.4 (BocCH3). m/z (ESI+): calculated: 337.14; measured: 237.87 [M-Boc]+, 359.81 [M+Na]+, 696.70 [2M+Na]+. 4-((tert-butoxycarbonyl)amino)-3-(tert-butyldisulfanyl)butanoic acid (7)
After dissolving crude compound 6 (6.65 g, 19.7 mmol) and trimethyltin hydroxide (10.96 g, 60.6 mmol) in DCE (60 mL), the reaction mixture was heated to 80°C and stirred for 60 hrs. The solvent was evaporated in vacuo and the resulting residue was dissolved in EtOAc. The solution was extracted with 0.01 M KHSO4, H20, and brine. The organic layer was dried using MgSCE. After filtration and removal of the solvent, the resulting residue was purified using column chromatography (MeOH/DCM, 1:99 -> 1:9). After concentrating the product under reduced pressure, lyophilization afforded compound 7 (2.3 g, 36% yield over two steps) as an off-white solid. *H NMR (400 MHz, CDCI3): δ 4.97 (s, 1H, NH), 3.56-3.48 (m, 2H, yCH2), 3.33-3.27 (q, 1H, J= 6.0 Hz, pCH), 2.65 (m, 2H, aCH2), 1.44 (s, 9H, Boc), 1.33 (s, 9H, StBu). 13C APT NMR (100 MHz, CDC13): δ 175.6 (COOH), 48.3 (StBuC), 48.1 (pCH), 42.9 (yCH2), 36.8 (aCH2), 30.0 (StBuCH3), 28.5 (BocCH3) m/z (ESI+): calculated: 323.12; measured: 223.95 [M-Boc]+, 345.90 [M+Na]+.
Example 2: Preparation pf diUb based DUB ABPs
The building block 7 prepared in example 1, was used for the total chemical synthesis of activity based deubiquitinating enzyme (DEIB) probes that resemble the native isopeptide linked diubiquitin structure. Probes based on Kll and K48 linked diUb were constructed. In order to preserve the native isopeptide Lys(Gly) length, Lysll or Lys48 of Ub is replaced by a diaminobutryic acid residue. The DUB ABPs were produced according to the following scheme.
Figure NL2012026CD00331
Using earlier reported total chemical synthesis of Ub by Fmoc based SPPS (cf WO2012/036551), Ub Lysl lDab(Alloc) and Ub Lys48Dab(Alloc) mutants were first constructed on solid phase. Next, the orthogonal protected Dab(Alloc) residue was selectively deprotected on-resin and coupled with building block 7. Global deprotection with TFA and purification by cation exchange and HPLC gave the desired Ub Lys-to-Dab(9) mutants in 7% overall yield. The required Ub(l-75)SEt thioester, representing the proximal part of the diUb probe design, was synthesised in a similar fashion by Fmoc SPPS. Native chemical ligation of the (distal) ubiquitin Ub Lys-to-Dab(9) mutant (20 mg/mL) and Ub(l-75)SEt (1.5 eq, 30 mg/mL) thioester was performed in 6M Gdn-HCl, 0.15M sodium phosphate and 250 mM MPAA (250 mM). Judged by LC-MS, overnight incubation at 37°C resulted in full consumption of the Ub Lys-to-Dab(9) mutant and formation of the ligation product as a MPAA disulfide. A short treatment with TCEP followed by preparative HPLC gave the Kll and K48 diUb conjugate 10 in 31% and 48% yield, respectively. Transformation of the β-mercepto carbonyl into an α-β unsaturated carbonyl (‘arming of the warhead’) was achieved by overnight incubation of the diUb conjugate (0.5 mg/mL) with 100 eq of 2,5-dibromohexanediamide in 50 mM sodium phosphate pH 8 at 37°C. LC-MS analysis (Figure 1; A and B: DiUb precursor at t = 0; C and D: Forming of sulfonium intermediate t= 3h; E and F: DiUb probe at t= 18h.) showed formation of the expected sulfonium intermediate (Figure IB) and the armed diUb probe 11 by basic elimination of the sulfonium group. After HPLC purification, both the Kll and K48 diUb probe (10) were isolated in 50% yield.
General Fmoc SPPS Strategy SPPS was performed on a Syro II MultiSyntech Automated Peptide synthesizer using standard 9-fluorenylmethoxycarbonyl (Fmoc) based solid phase peptide chemistry at 50 pmol scale, using fourfold excess of amino acids relative to pre-loaded Fmoc amino acid Wang type resin (0.2 mmol/g, Applied Biosystems®) or pre-loaded Fmoc amino acid trityl resin (0.2 mmol/g, Rapp Polymere GmbH). The Ub (mutant) peptide sequences were synthesized on resin following the procedures as described before. Optimization of this procedure led to discarding the capping step. Single couplings were performed for 45 min. After the first 30 cycles the coupling time was extended to 60 min. Dipeptides at position 9, 17, 20, 25, 57 and 61 are coupled for 2h. Double couplings are preformed in cycle 62-69 (35 min each).
Cation purification of ubiquitin mutants
The crude Ub mutant was taken up in a minimal amount of warm DMSO and diluted with 50 mM NaOAc pH 4.5 while the final DMSO concentration was kept as low as possible (2 - 10%). Next, the peptide was purified by cation chromatography using a MonoS column and a 0 - 1 M NaCl gradient in 50 mM NaOAc pH 4.5. Fractions that contained product were pooled and further purified by prep-HPLC using 2 mobile phases: A=0.05% TFA in MQ and B: 0.05% TFA in CH3CN. Prep-HPLC program: Waters C18-XBridge 5 μΜ (30 x 150 mm); flowrate: 30 mL/min. Gradient: 0-6 min: 5 -> 25% B; 6 - 21 min: 25 -> 75% B; 21 - 23 min: 75 -> 95% B. Pure fractions were pooled and lyophilized.
Ub Lys to Dab mutant
The Ub(l-76) peptide sequence was synthesized on a wang resin following the general procedure, with a Lys to Dab(Alloc) mutation. The resin bound polypeptide was treated with Pd(PPh3)4 (0.35 eq) and PhSiH (20 eq) in DCM (2x 20 min). Then, the free N-terminus was reacted with building block 1 (3 eq), PyBOP (3 eq) and DiPEA (6 eq). The resin bound peptide was shaken overnight at room temperature. After extensive washings (NMP (3x), DCM (3x), Et20 (3x)) the resin was treated with TFA/H20/TiS (95:2.5:2.5 v/v/v) for 3 h followed by precipitation with cold Et20/pentane 3:1 v/v. The crude peptide was lyophilized and purified according to the general procedure Further workup (i.e. lyophilization and purification by cation chromatography) was performed according to the general procedure.
Ub(l-76)K11: LC-MS: Rt = 3.25, ES MS+ (amu) calcd: 8742, found 8729 Ub(l-76)K48: LC-MS: Rt = 3.25, ES MS+ (amu) calcd: 8742, found 8729 Ub76 Ref: LC-MS: Rt = 3.43, ES MS+ (amu) calcd: 8565, found 8553
Synthesis of diUb probes - Native Chemical Ligation
To a solution of the Ub(l-76) Lys to Dab mutant in 0.15 M sodium phosphate buffer (pH 7) containing 6M Gdn-HCl and MPAA (250mM), a solution of UbSEt (1.5 equiv) in 0.2 M sodium phosphate buffer (pH 7) containing 6M Gdn-HCl and MPAA (250 mM) was added and the mixture (cone: 50 mg/mL) was incubated at 37°C. After incubation at 37°C overnight, TCEP was added to reduce the MPAA disulfide. The obtained mixture was purified by prep-HPLC using 2 mobile phases: A=0.05% TFA in MQ and B: 0.05% TFA in CH3CN. Prep-HPLC program: Waters Atlantis C18 10 μΜ (10x150 mm); flowrate: 6.6 mL/min. Gradient: 0-7 min: 5%B; 7-9 min 5 -> 30% B; 9 - 43 min: 30 -> 60% B; 43 - 44 min: 60 -> 95% B. Pure fractions were pooled and lyophilized.
Kll-linked diUb precursor: 5.8 mg (31%), LC-MS: Rt= 3.65, ES MS+ (amu) ealed 17143, found 17111 K48-linked diUb precursor: 8.3 mg (48%), LC-MS: Rt = 3.56, ES MS+ (amu) ealed 17143, found 17112
Ub76 Ref: LC-MS: Rt = 3.45, ES MS+ (amu) ealed: 8565, found 8550
Synthesis of diUb probes - Arming of warhead
To a solution of the Kll-linked precursor (4.6 mg, 0.27 Dmol) in 50 mM sodium phosphate buffer (pH 8, 0.5 mg/mL) 2,5-dibromohexandiamide (100 eq.) was added. The reaction mixture was incubated at 37°C overnight and spun down to remove the insoluble dibromide. The obtained mixture was purified by prep-HPLC using 2 mobile phases: A=0.05% TFA in MQ and B: 0.05% TFA in CH3CN. Prep-HPLC program: Waters Atlantis C18 10 pM (10x150 mm); flowrate: 6.6 mL/min. Gradient: 0-7 min: 5%B; 7-9 min 5 -> 30% B; 9 - 43 min: 30 -> 60% B; 43 - 44 min: 60 95% B. Pure fractions were pooled and lyophilized.
Kll-linked diUb probe: 2.5 mg (52%), LC-MS: Rt = 3.58, ES MS+ (amu) ealed 17111, found 17078 K48-linked diUb probe: 3.5 mg (50%), LC-MS: Rt = 3.54, ES MS+ (amu) ealed 17111, found 17078
Ub76 Ref: LC-MS: Rt = 3.45, ES MS+ (amu) ealed: 8565, found 8550
Example 3: Reactivity of diUb ABPs towards DUBs
To investigate the reactivity of the Kll and K48 diUb probes of example 2 toward DUBs, they were treated with USP8 (Figure 2), which is known to react with all seven isopeptide linked diUb conjugates. Incubation with the diUb probes showed efficient covalent labelling of USP8. Next, the OTU DUB Cezanne was treated which exhibits selectivity toward KI 1 diUb linkages. Incubation of Cezanne 1 with the KI 1 diUb probe indeed resulted in labelling, while incubation with the K48 diUb probe showed no labelling at all (Figure 3). This demonstrates the ability of these probes to selectively capture DUBs with distinct target preference. When Cezanne 1 was treated with the (unarmed) thiol precursor of the Kll diUb (10), no labeling was observed, confirming the requirement for a Michael acceptor at that position for covalent DUB labelling (Figure 4).
Next probes holding a fluorophore were generated to allow direct in-gel fluorescence scanning. The fluorophore Cy5 was introduced at the N-terminus of the distal Ub(l-75), followed by transthioesterification. Then ligation with the proximal Ub(l-76) Lys to Dab mutant and arming of the thiol gave access to the Cy5 labelled KI 1 and K48 diUb probes in 11% overall yield after isolation with preparative HPLC.
The diUb probes were compared to the classical Ub VME probe in profiling DUB activity, for that the El-4 cell lysate was incubated with increasing probe concentrations (Figure 5). Labeled DUBs were visualized by in gel-fluorescence measurements. The diUb probes showed to be me more selective in capturing DUBs in lysate than the classical Ub VME probe
Iln conclusion, it has been shown that the new building block of this invention enables the synthesis of diUb based probes. This synthetic approach offers significant advantages over the established methods, placing the warhead in the correct position compared to the native isopeptide bond. The straightforward synthesis of the peptides, combined with the efficient reaction and an ‘in situ’ arming reaction, now allows a convenient preparation of diUb probe conjugates. Having routine strategies for the synthesis of these probes, virtually any cysteine, serine or threonine protease activity-based probe is now within practical reach. DUB labeling assays - USP8 USP8 (2.5 pg) was incubated with different concentrations of Kll and K48-diUb probe (0.63 pg, 2.5 pg, 5 pg, 10 pg) and UbVME (2.5 pg ) at 37°C for 30 min in a reaction buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl and 10 mM DTT. The reactions were quenched by the addition of reducing sample buffer and heating (70°C, 10 min). Samples were separated by SDS-PAGE and then stained with Coomassie brilliant blue for detection. DUB labeling assays - Cezanne
Cezanne (1 μΜ) was incubated with 5 μΜ different probes, Kll precursor and Kll diUb at 37°C for 30 min in a reaction buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl and 10 mM DTT. To evaluate the selectivity of the probes to cezanne, samples were taken of the incubation with the Kll and K48 diUb probe at different time points (5, 30, 60 min). The reactions were quenched by the addition of reducing sample buffer and heating (70°C, 10 min). Samples were separated by SDS-PAGE and then stained with Silver stain for detection. DUB labeling assays - Preparation of EL-4 cell extracts and DUB activity based profiling EL4 cells were grown in Gibco RPMI 1640 medium (Life technologies) supplemented with fetal calf serum (FCS, 10% v/v) at 37°C in a 5% CO2 atmosphere, overnight and then harvested. Cells were lysed by sonication in HR lysis buffer: 50 mM Tris (pH=7.4), 250 mM sucrose, 5 mM MgCk, 1 mM DTT, 2 mM DTT, and clarified by spinning (16000 g, 10 min, 4°C). 25 pg cell lysates were then incubated with 0.1, 0.2 and 0.5 μΜ Cy5-labelled Kll-diUb probe and Cy5 labelled K48-diUb probe respectively and 0.5 μΜ Cy5-Ub-VME as a control. Labeling experiments were performed in buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl and 1 mM DTT at 37°C for 30 min. The reactions were terminated by addition of reducing sample buffer and heating (70°C, 10 min). Samples were separated by SDS-PAGE and signals were detected by in-gel fluorescence scanning.
Examples 4 : Synthesis of building block
In this example, the synthesis of a protected building according to this invention, is described. The synthesis is performed according to the following reaction scheme. Synthesis of the starting comound(s) is based on the prior art method (cf. Harbut et al. Proc Natl Acad Sci, 2011, 108(34), E526-34). Nuclear magnetic resonance spectroscopy and LC-MS measurements are performed following the procedures described in example 1, confirming the identity of the intermediate and end products.
Figure NL2012026CD00391
PG= protective group

Claims (15)

1. Bouwsteen gekozen uit de groep bestaande uit de verbindingen volgens formules (Ia), (Ib), (Ha) of (Ilb) en zouten en esters daarvan:A building block selected from the group consisting of the compounds according to formulas (Ia), (Ib), (Ha) or (Ilb) and salts and esters thereof: <img img-format="tif" img-content="drawing" file="NL2012026CC00401.tif" id="icf0001" /><img img-format = "tif" img-content = "drawing" file = "NL2012026CC00401.tif" id = "icf0001" /> waarin: X een groep voorstelt gekozen uit CH2; CH2-CH2; CH2-CH2-CH2; CHRa; CHRa-CH2; CH2-CHRa; CHRa-CH2- CH2; CH2-CHRa-CH2; en CH2-CH2- CHRa , waarin Ra een aminozuurzij keten voorstelt; R1 (i) waterstof of (ii) een amine-beschermende groep voorstelt; R2 (i) waterstof; (ii) een groep volgens formule -S-R4 voorstelt, waarin R4 een eventueel gesubstitueerde vertakte of lineaire Ci-Cö alkyl of Ci-Cealkenyl voorstelt; of (iii) een thiol-beschermende groep; en R3 halogeen of hydroxyl voorstelt; met uitzondering van de verbinding 4-amino-3-mercaptobutaanzuur.wherein: X represents a group selected from CH 2; CH 2 CH 2; CH 2 CH 2 CH 2; CHRa; CHR a-CH 2; CH 2 CHRa; CHR a - CH 2 - CH 2; CH 2 -CH 3 -CH 2; and CH 2 CH 2 CHR a, wherein R a represents an amino acid side chain; R 1 represents (i) hydrogen or (ii) an amine protecting group; R2 (i) hydrogen; (ii) represents a group of formula -S-R 4, wherein R 4 represents an optionally substituted C 1 -C 6 branched or linear alkyl or C 1 -C 6 alkenyl; or (iii) a thiol protecting group; and R 3 represents halogen or hydroxyl; with the exception of the compound 4-amino-3-mercaptobutanoic acid. 2. Bouwsteen volgens conclusie 1, waarin -X- de groep -CHRa voorstelt.Building block according to claim 1, wherein -X- represents the group -CHRa. 3. Bouwsteen volgens conclusie 1 of 2, waarin de aminebeschermende groep wordt gekozen uit de groep bestaande uit carbobenzyloxy; /;-methoxybenzyl carbonyl; tert-butyloxycarbonyl (Boe); 9-fluorenylmethyloxycarbonyl (Fmoc); benzyl; p-methoxybenzyl; 3,4-dimethoxybenzyl; /;-methoxyphenyl; tosyl; sulfonamides; allyloxycarbonyltrityl en methoxytrityl.The device of claim 1 or 2, wherein the amine protecting group is selected from the group consisting of carbobenzyloxy; methoxybenzyl carbonyl; tert-butyloxycarbonyl (Boe); 9-fluorenylmethyloxycarbonyl (Fmoc); benzyl; p-methoxybenzyl; 3,4-dimethoxybenzyl; methoxyphenyl; tosyl; sulfonamides; allyloxycarbonyltrityl and methoxytrityl. 4. Bouwsteen volgens een van de voorgaande conclusies, waarin de thiolbeschermende groep wordt gekozen uit de groep bestaande uit benzyl; 4-methoxybenzyl; trityl; methoxytrityl; t-butyl; t-butylthiol; acetyl; 3-nitro-2-pyridinesulphenyl; acetamidomethyl; methaanthiol en 2-nitrobenzyl.A building block according to any one of the preceding claims, wherein the thiol protecting group is selected from the group consisting of benzyl; 4-methoxybenzyl; trityl; methoxytrityl; t-butyl; t-butyl thiol; acetyl; 3-nitro-2-pyridine sulphenyl; acetamidomethyl; methanethiol and 2-nitrobenzyl. 5. Werkwijze voor het produceren van een gemodificeerd protease sub straat omvattende: i) het identificeren van een substraat voor het protease; ii) het chemisch, biochemisch of biologisch synthetiseren of produceren van het proteasesubstraat waarbij het aminozuurresidue op de cleavage site van het non-primed side fragment, gesubstitueerd wordt door een bouwsteen zoals gedefinieerd in een van de voorgaande conclusies; en iii) het modificeren van de bouwsteen door omzetting van de β-mercapto of β-selenocarbonylgroep naar een α,β-onverzadigde carbonylgroep.A method for producing a modified protease substrate comprising: i) identifying a substrate for the protease; ii) chemically, biochemically or biologically synthesizing or producing the protease substrate wherein the amino acid residue at the cleavage site of the non-primed side fragment is substituted by a building block as defined in any one of the preceding claims; and iii) modifying the building block by converting the β-mercapto or β-selenocarbonyl group to a α, β-unsaturated carbonyl group. 6. Werkwijze volgens conclusie 5, waarin de productie van de gekozen polypeptide- of eiwitsequentie het inbouwen van de bouwsteen omvat met behulp van orthogonale tRNA/aminoacyl-tRNA synthetase paren, waarin de bouwsteen wordt ingebouwd in respons op een nonsense of een vier-base codon dat is ingebouwd in het gen dat codeert voor de gekozen sequentie.The method of claim 5, wherein the production of the selected polypeptide or protein sequence comprises incorporating the building block using orthogonal tRNA / aminoacyl-tRNA synthetase pairs, wherein the building block is incorporated in response to a nonsense or a four-base codon built into the gene encoding the chosen sequence. 7. Werkwijze volgens conclusie 5 of 6, waarin het gekozen proteasesub straat een in de natuuur voorkomend polypeptide of eiwit is of een functionele variant of fragment daarvan.The method of claim 5 or 6, wherein the selected protease substrate is a naturally occurring polypeptide or protein or a functional variant or fragment thereof. 8. Werkwijze volgens conclusie 5-7, waarin het protease wordt gekozen uit cysteineproteases en threonineproteases.The method of claims 5-7, wherein the protease is selected from cystein proteases and threonine proteases. 9. Werkwijze volgens een van de conclusies 5-8, waarin het protease proteasome en/of een katalytische proteasome-subunit is.The method of any one of claims 5-8, wherein the protease is proteasome and / or a catalytic proteasome subunit. 10. Peptide dat een structuur heeft volgens een van de volgende formules:A peptide having a structure according to one of the following formulas: <img img-format="tif" img-content="drawing" file="NL2012026CC00421.tif" id="icf0002" /><img img-format = "tif" img-content = "drawing" file = "NL2012026CC00421.tif" id = "icf0002" /> waarin: η = O, 1,2 of 3; X dezelfde betekenis heeft als in formules (I) en (II) volgens conclusie 1; Raen Ra onafhankelijk van elkaar gekozen aminozuurzijketens voorstellen; en [PEPTIDE], [PEPTIDE]’ en [-PEPTIDE]’ peptideketens voorstellen met een lengte van tenminste 1 aminozuurresidu, bij voorkeur tenminste 2 aminozuurresiduen, met meer voorkeur tenminste 3 aminozuurresiduen.wherein: η = O, 1.2 or 3; X has the same meaning as in formulas (I) and (II) according to claim 1; Ra and Ra represent independently selected amino acid side chains; and [PEPTIDE], [PEPTIDE] "and [-PEPTIDE]" represent peptide chains with a length of at least 1 amino acid residue, preferably at least 2 amino acid residues, more preferably at least 3 amino acid residues. 11. Proteasesubstraat dat gemodificeerd is op de cleavage site en een structuur heeft volgens een van de formules (III)-(VI):11. Protease substrate modified at the cleavage site and having a structure according to one of the formulas (III) - (VI): <img img-format="tif" img-content="drawing" file="NL2012026CC00431.tif" id="icf0003" /><img img-format = "tif" img-content = "drawing" file = "NL2012026CC00431.tif" id = "icf0003" /> <img img-format="tif" img-content="drawing" file="NL2012026CC00441.tif" id="icf0004" /><img img-format = "tif" img-content = "drawing" file = "NL2012026CC00441.tif" id = "icf0004" /> waarin: η = O, 1,2 of 3; R2 en X dezelfde betekenis hebben als in formules (I) en (II) volgens conclusie 1; Ra# een aminozuurzij keten voorsteelt overeenkomend met de aminozuurzij keten in het correponderende niet-gemodificeerdeprotease substraat; [PEPTIDE] en [PEPTIDE]’ peptideketens voorstellen met de aminozuursequenties Ρ3-Ρω respectievelijk P3 -Ρω, waarin P# een aminozuurresidu voorstelt identiek aan het aminozuurresidu op de overeenkomstige positie in het niet-gemodificeerde proteasesubstraat, waarin de posities genummerd zijn ten opzichte van de cleavage site, waarbij P1 en P1 de aminozuurresiduen aan weerszijden van de cleavage site voorstellen en Ρω en Ρω de N-terminale en C-terminale aminozuurresiduen voorstellen; of N- en/or C-terminaal getrunceerde varianten van deze gemodificeerde proteasesub straten, waarin [PEPTIDE] en [PEPTIDE]’ een aantal aminozuurresiduen bevatten gelijk aan of groter dan 0; of een homoloog, conjugaat of derivaat daarvan.wherein: η = O, 1.2 or 3; R2 and X have the same meaning as in formulas (I) and (II) according to claim 1; R a # represents an amino acid side chain corresponding to the amino acid side chain in the corresponding non-modified protease substrate; [PEPTIDE] and [PEPTIDE] 'represent peptide chains with the amino acid sequences Ρ3-Ρω and P3 -Ρω, respectively, in which P # represents an amino acid residue identical to the amino acid residue at the corresponding position in the unmodified protease substrate, in which the positions are numbered relative to the cleavage site, where P1 and P1 represent the amino acid residues on either side of the cleavage site and Ρω and Ρω represent the N-terminal and C-terminal amino acid residues; or N- and / or C-terminally truncated variants of these modified protease substrates, wherein [PEPTIDE] and [PEPTIDE] "contain a number of amino acid residues equal to or greater than 0; or a homologue, conjugate or derivative thereof. 12. Proteasesub straat dat gemodificeerd is op de cleavage site en een structuur heeft volgens een van de formules (VII)-(X):12. Protease substrate modified at the cleavage site and having a structure according to one of the formulas (VII) - (X): <img img-format="tif" img-content="drawing" file="NL2012026CC00442.tif" id="icf0005" /><img img-format = "tif" img-content = "drawing" file = "NL2012026CC00442.tif" id = "icf0005" /> <img img-format="tif" img-content="drawing" file="NL2012026CC00451.tif" id="icf0006" /><img img-format = "tif" img-content = "drawing" file = "NL2012026CC00451.tif" id = "icf0006" /> waarin: η = O, 1,2 of 3; R2 en X dezelfde betekenis hebben als in formules (I) en (II) volgens conclusie 1; Ra# een aminozuurzij keten voorstelt overeenkomend met de aminozuurzij keten in het correponderende niet-gemodificeerde proteasesubstraat; [PEPTIDE] een peptideketen voorstelt met de aminozuursequentie Ρ2-Ρω; [PEPTIDE]’ en [-PEPTIDE]’ peptideketens voorstellen met de aminozuursequenties P1 -Ρω respectievelijk P'1 -Ρ'ω, waarin P# een aminozuurresidu voorstelt identiek aan het aminozuurresidu op de overeenkomstige positie in het niet-gemodificeerde proteasesub straat, waarin de posities genummerd zijn ten opzichte van de cleavage site, waarbij P1 en P'1 de aminozuurresiduen aan weerszijden van het lysineresidu van de isopeptidebinding; en Ρω, Ρω en Ρ'ω de N-terminale en C-terminale aminozuurresiduen voorstellen van de respectievelijke aminozuurketens; of N- en/or C-terminaal getrunceerde varianten van deze gemodificeerde proteasesub straten, waarin [PEPTIDE], [PEPTIDE]’ en/of [-PEPTIDE]’ een aantal aminozuurresiduen bevatten gelijk aan of groter dan 0; of een homoloog, conjugaat of derivaat daarvan.wherein: η = O, 1.2 or 3; R2 and X have the same meaning as in formulas (I) and (II) according to claim 1; Ra # represents an amino acid side chain corresponding to the amino acid side chain in the corresponding unmodified protease substrate; [PEPTIDE] represents a peptide chain with the amino acid sequence Ρ2-Ρω; [PEPTIDE] 'and [-PEPTIDE]' represent peptide chains with the amino acid sequences P1 -Ρω and P'1 -Ρ'ω, respectively, where P # represents an amino acid residue identical to the amino acid residue at the corresponding position in the unmodified protease substrate, wherein the positions are numbered relative to the cleavage site, wherein P1 and P'1 are the amino acid residues on either side of the lysine residue of the isopeptide bond; and Ρω, Ρω and Ρ'ω represent the N-terminal and C-terminal amino acid residues of the respective amino acid chains; or N- and / or C-terminally truncated variants of these modified protease substrates, wherein [PEPTIDE], [PEPTIDE] "and / or [-PEPTIDE]" contain a number of amino acid residues equal to or greater than 0; or a homologue, conjugate or derivative thereof. 13. Proteasesub straat volgens conclusie 11 of 12, waarin het protease een cysteineprotease of een threonineprotease is.The protease substrate of claim 11 or 12, wherein the protease is a cystein protease or a threonine protease. 14. Werkwijze voor het synthetiseren van een bouwsteen volgens formule (Ia) zoals gedefinieerd in conclusie 1, waarin de werkwijze de stappen omvat van: (i) het behandelen van 4-amino-3-hydroxybutaanzuur om de carbonzuurgroep te veresteren en de aminegroep te beschermen; (ii) het tosyleren van de veresterde en amine-beschermde verbinding verkregen in stap (i); (iii) het omzetten van de getosyleerde verbinding verkregen in stap (ii) tot een thioester; en (iv) het selectief hydrolyseren van de thioester om een thiolgroep te verkrijgen.A method for synthesizing a building block of formula (Ia) as defined in claim 1, wherein the method comprises the steps of: (i) treating 4-amino-3-hydroxybutanoic acid to esterify the carboxylic acid group and the amine group to protect; (ii) tosylating the esterified and amine-protected compound obtained in step (i); (iii) converting the tosylated compound obtained in step (ii) to a thioester; and (iv) selectively hydrolyzing the thioester to obtain a thiol group. 15. Gebruik van een bouwsteen volgens een van de conclusies 1-2 als een bouwsteen bij de synthese van een gemodificeerd proteasesub straat om een Michael Acceptor in het protease substraa in te bouwen.Use of a building block according to any of claims 1-2 as a building block in the synthesis of a modified protease substrate to incorporate a Michael Acceptor into the protease substrate.
NL2012026A 2013-12-24 2013-12-24 Building blocks for introducing michael acceptors in selected peptide sequences. NL2012026C2 (en)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
US5378691A (en) * 1988-04-14 1995-01-03 Merck Patent Gesellschaft Mit Beschrankter Haftung Amino acid derivatives

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Publication number Priority date Publication date Assignee Title
US5378691A (en) * 1988-04-14 1995-01-03 Merck Patent Gesellschaft Mit Beschrankter Haftung Amino acid derivatives

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Title
JEONG KYU BANG ET AL: "Solid-Phase Syntheses of Olefin-Containing Inhibitors for HTLV-1 Protease Using the Horner-Emmons Reaction", THE JOURNAL OF ORGANIC CHEMISTRY, vol. 70, no. 25, 1 December 2005 (2005-12-01), pages 10596 - 10599, XP055134846, ISSN: 0022-3263, DOI: 10.1021/jo051872s *
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