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WO2007113575A2 - Réactifs et procédés de réticulation de molécules biologiques - Google Patents

Réactifs et procédés de réticulation de molécules biologiques Download PDF

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WO2007113575A2
WO2007113575A2 PCT/GB2007/001277 GB2007001277W WO2007113575A2 WO 2007113575 A2 WO2007113575 A2 WO 2007113575A2 GB 2007001277 W GB2007001277 W GB 2007001277W WO 2007113575 A2 WO2007113575 A2 WO 2007113575A2
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cross
linking reagent
reagent according
group
complex
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WO2007113575A3 (fr
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James Dowden
Melanie Joanne Welham
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University of Bath
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University of Bath
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution

Definitions

  • the present invention relates to the study of interactions between biological molecules, such as proteins and nucleic acids. It provides novel reagents which may be used to crosslink biological molecules which interact non-covalently and often transiently with one another, and so facilitate the identification and characterisation of such interactions.
  • Chemical cross -linking 1101 provides covalent capture of transient protein-protein interactions and can facilitate topological analysis using mass spectrometry.
  • 111 ' 121 A limitation of chemical cross-linkers is that tether length and reactivity must be optimized for individual protein-protein complexes. Additionally, reagents do not discriminate between inter- versus intra-molecular links or non-productive modifications. A modular synthetic route was recently reported that offered rapid access to a versatile arsenal of cross-linkers. 1131 However, individual compound evaluation is still necessary to select the optimum reagent for each system. Summary of the Invention
  • a chemical cross-linker that does not require specific optimization for use with different target molecules or complex mixtures, would be highly desirable.
  • a further desirable feature would be to increase the likelihood of forming inter-molecular cross-links over intra-molecular links, to increase the efficiency of such a reagent in cross- linking different members of non-covalently associated complexes .
  • Calixarenes are cylic oligomers derived from condensation reactions between an aldehyde and a phenol. Calixarenes typically contain either 6 phenyl units in a ring (referred to as calix[6] arenes) or 4 phenyl units in a ring (referred to as calix[4] arenes) .
  • the phenol component is typically phenol, resorcinol (1, 3-benzenediol) or pyrogallol (1,2,3- benzenetriol) but could also be catechol (1,2- benzenediol) , or a derivative of any of these.
  • a reaction between the aldehyde RCHO and resorcinol provides a resorcinarene (calix [4] arene) as follows :
  • Calixarenes have an lower (or inner) rim and an upper (or outer) rim.
  • the lower rim is shown as the interior of the macrocyclic ring, and the upper rim is that carrying the eight hydroxy1 groups.
  • the same nomenculature is applied to other calixarenes.
  • the molecule can be relatively flexible, with a degree of rotational freedom for the phenyl groups.
  • the R groups are larger, they restrict rotation and tend to lock the molecule into a particular configuration.
  • Such a rigid calixarene core would provide a well-defined geometrical structure for a cross-linking reagent, which could display multiple functional groups (e.g. groups reactive with various kinds of biological macromolecules) over a large surface area by tethering them to the hydroxy groups of the calixarene via spacer arms .
  • functional groups e.g. groups reactive with various kinds of biological macromolecules
  • spacer arms By offering multiple copies of reactive functionality in an organised arrangement, such a structure should bias cross-linking reactions toward inter-molecular links between proteins and thus lead to greater cross-linking efficiency.
  • formula I shows a calix [4] arene and formula II shows a calix [6] arene .
  • the invention further provides compounds having formula III
  • Calixarenes based on resorcinol have been found to be particularly suitable.
  • the invention provides compounds of formula V:
  • R groups are the same or different. They can be independently chosen according to the properties desired for the molecule, and will be described in more detail below.
  • the X groups are also the same or different and may independently be -H, -OH, or may have the formula -O-Y-Z, where Z is a terminal functional group and Y is a spacer arm.
  • the functional group is preferably a reactive functional group, capable of reacting with a group found on a target molecule.
  • one or more X groups may have the formula R or -0-R.
  • Preferably none, one or two X groups per molecule has the formula R- or -0-R.
  • the compounds described herein are used as cross-linking reagents. They may be used to form intra-molecular crosslinks (by linking groups found on the same target molecule) and/or inter-molecular links (by linking groups found on two or more non-covalently associated target molecules) .
  • the compounds of the invention must carry at least two such reactive functional groups .
  • R groups may carry reactive functional groups (see below) , preferably at least two X groups comprise reactive functional groups and therefore have the formula -O-Y-Z.
  • At least one X group on each phenyl group in the calixarene ring has the formula -O-Y-Z.
  • at least two X groups on each phenyl group in the calixarene ring have the formula -O-Y-Z.
  • all X groups have the formula -O-Y-Z.
  • X groups in formulae V and VI represent the formula -O-Y-Z.
  • Typical target molecules are biological macromolecules such as peptides, proteins and nucleic acids (DNA or RNA) .
  • peptide is used to refer to peptides of less than 100 amino acids, and "polypeptide” or “protein” for a molecule of 100 amino acids or more. However, reference to one should be taken to include the other unless the context specifically demands otherwise.
  • target molecules include anything capable of forming a non-covalent complex with a biological macromolecule such as a protein, peptide, or nucleic acid.
  • a biological macromolecule such as a protein, peptide, or nucleic acid.
  • organic molecules such as drug molecules, modulators of protein activity, isolated amino acids, nucleotides or nucleosides, protein cofactors such as heme groups, vitamins, etc ..
  • Proteins and peptides display various chemical groups which can be targeted by cross-linking reagents such as free amine groups (e.g. in lysine side chains and at the N-terminus) , free hydroxy groups (e.g. in serine and threonine side chains) , thiol groups (cysteine side chains) , carboxyl groups (aspartate and glutamate side chains, and the C-terminus of the molecule) .
  • Suitable reactive functional groups are well- known in the art; see e.g. Bioconjugate Techniques, by Greg T Hermanson; ISBN 0-12-342336-8.
  • amine-reactive functional groups which may be used in the cross -linking reagents described here include isothiocyanates, isocyanates, acyl azides, N-Hydroxysuccinimide (NHS) esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, carbonates, arylating agents, i ⁇ ddoesters, carbodimides and acid anhydrides.
  • isothiocyanates isocyanates
  • acyl azides N-Hydroxysuccinimide (NHS) esters
  • sulfonyl chlorides aldehydes, glyoxals, epoxides, carbonates, arylating agents, i ⁇ ddoesters, carbodimides and acid anhydrides.
  • NHS N-Hydroxysuccinimide
  • Thiol-reactive functional groups which may be used in the cross-linking reagents described here include haloa ⁇ etyl and alkyl halide derivatives, maleimides, aziridines, acryloyl derivatives, and disulfides, which can participate in thiol- disulfide exchange reactions.
  • Carboxylate-reactive functional groups which may be used in the cross -linking reagents described here include diazoalkanes and diazoacetyl compounds (diazoacetate esters and diazoacetamides) , carbonyldiimidazoles and carbodiimides .
  • Hydroxyl-reactive functional groups which may be used in the cross-linking reagents described here include carbonates.
  • Aldehyde- or ketone- reactive functional groups which may be used in the cross -linking reagents described here include hydrazines, and amines. These groups may be useful for targeting carbohydrates (e.g. those found in protein glycosylation) and non-naturally occurring amino acids.
  • Reactive functional groups which may be used to react with carbon atoms carrying "active hydrogen” atoms include diazonium derivatives. These may be used, inter alia, for reaction with carbon atoms carrying hydrogen in aromatic rings by electrophilic addition.
  • the reactive functional group may not be capable of reacting with a target molecule until a particular stimulus is applied.
  • certain groups known generally as photoreactive groups will only react on irradiation, typically with visible or UV irradiation. Lasers are often used to deliver the radiation because of their ability to deliver high-intensity monochromatic radiation at a very closely defined location.
  • Photoreactive groups include aryl azides, benzophenones and diazirine compounds . Photoactivation leads to formation of reactive free radical groups, which can react with various groups on the target molecule. They tend to be less specific in their reaction than the other reactive functional groups described above, but normally react with alkyl and aryl groups on the target molecule. They can be particularly useful for cross-linking nucleic acids, either to other nucleic acids or to other molecules such as proteins.
  • reactive functional groups include benzophenone and diazirine, aryl azides, hydrazides, alkyl halides and maleimides, epoxides, alkynes , phosphines, reactive esters, carbonates and anhydrides.
  • the spacer arms Y may be straight-chain or branched groups which are O- linked to the phenyl group at one end and linked to the reactive functional group at the other.
  • the spacer arms may consist of or comprise saturated or unsaturated chains of any desired length comprising, for example, one or more substituted or unsubstituted alkyl, alkene, aldehyde, ketone, alcohol or aryl groups, amino acids, or sugars, which may be linked by carbon or heteroatom (0, N, S, etc.) linkages such as ether, amine, amide, alkane, alkene, alkyne, thiol or ester linkages, or any other suitable linkage known to the skilled person.
  • linear chain of between 1 and 20 atoms between the reactive functional group and the respective phenol-derived oxygen of the calixarene ring to which it is linked.
  • the linear chain may be 2, 3, 4, 5., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 atoms in length, or more depending on the circumstances .
  • spacer arms Y may comprise or' consist of any one of the following:
  • Ci -10 alkyl optionally substituted with one or more substituents as defined herein, e.g. a group which is a substituted or unsubstituted Ci -I0 alkyl, C 1-10 haloalkyl, C 1 - I0 hydroxyalkyl , C 1-10 carboxyalkyl , C 1-10 aminoalkyl group;
  • Ci-io cycloalkyl-Ci-io alkyl optionally substituted with one or more substituents as defined herein,-
  • C 5-20 aryl optionally substituted with one or more substituents as defined herein, e.g. C 5-20 carboaryl or C 5-20 heteroaryl;
  • a spacer arm may comprise the formula -OCH 2 COO-
  • the spacer arm may carry two or more reactive functional groups.
  • Hyperbranched or dendritic architectures are possible.
  • Dendritic polymers are polymers derived from branched monomers attached to a central core . Thus each successive addition of monomer (referred to as a generation) results in an increase in the number of terminal groups, as the number of branches increases.
  • Commonly used dendrimers include polyamidoamines , polyamines, polyamides, poly(aryl ethers), polyesters and carbohydrates. (See Lee et al . 2005, Nature Biotech 23(12), 1517 for a review, and references cited therein) .
  • Dendrimeric spacer arms may therefore carry a plurality of reactive functional groups, to a maximum of one per chain terminus .
  • One Y group may also be connected to two or more phenyl groups within the calixarene ring, as shown in the following illustration:
  • Y alkyl, aryl or other briding atoms This may be achieved using Y groups such as alkyl, polyethylene glycol, polyalkyl amine, eneone derivatives • and polyamides, or by olefin polymerisation.
  • the spacer arms may contain selectively cleavable groups, which can be cleaved e.g. by a suitable chemical reaction, enzymatic reaction or irradiation. Such- groups facilitate the analysis of complexes cross-linked by the reagents described because the residues of reactive groups bound to target molecules can be detached from the calixarene core and separated from one another.
  • a spacer arm may contain a selectively cleavable group such as an alkene, disulfide, hydrazinobenzoic acid derivative (e.g.
  • ester amide or anhydride
  • diol or other group cleavable on contact with periodate, dithionite derivative, hydroxylamine-cleavable ester, base-labile sulfone derivative, hydrazide or photolabile group such as a nitrophenylethyl ether, ester, or amide.
  • these groups are exemplary only. The skilled person will be aware of other alternatives which may be used.
  • Any given spacer arm may contain two or more different types of selectively cleavable group if desired.
  • spacer arms may contain selectively cleavable groups as described above .
  • Branched spacer arms having two or more terminal reactive functional groups Z may contain one or more selectively cleavable groups for each reactive functional group Z, so that each can be cleaved from the calixarene core structure and separated from other groups carried by the same spacer arm. It is therefore possible to disrupt a cross- linking reagent so that each reactive functional group (or each residue of a reactive functional group bound to a target molecule) may be separated from all other reactive functional groups or residues thereof. That is to say, so that none of the reactive functional groups or residues thereof remain covalently linked to any other such group or residue, either via the calixarene core, or via a branched spacer arm or portion thereof .
  • Y and Z groups in any one calixarene molecule may be the same or different.
  • one molecule may contain spacer arms of two or more different lengths, and of different compositions. Different cross-linking applications may require different length spacer arms for optimal results.
  • any one cross-linking reagent may comprise more than one type of reactive functional group, e.g. groups capable of reacting with different target groups on target molecules.
  • a single resorcinarene-based reagent having 8 spacer arms may have 4 groups capable of reacting with amine groups (such as NHS groups) and four photoreactive groups .
  • the reagent may have an [(AB) n ] configuration in which spacer arms having different groups alternate around the calixarene ring. This could be achieved during initial synthesis, or by later modification of the upper rim.
  • [(A) 1n (B)J configuration might be used. This would provide different binding functionalities at the two opposite hemispheres of the calixarene molecule.
  • the R groups in any given molecule may also be the same or different. Using two aldehydes having different R groups in the original synthesis of the calixarene ring will result in a calixarene having both types of R group. Alternatively one or more R groups can be selectively modified after calixarene synthesis .
  • R groups may comprise one or more substituted or unsubstituted alkyl, alkene, aldehyde, ketone, alcohol, alkene or aryl groups, amino acids, sugars, etc. which may be linked by carbon or heteroatom (O, N, S, etc.) linkages such as ether, amine, amide, alkane, alkene, alkyne, thiol or ester linkages, or any other suitable linkage known to the skilled person.
  • each R group may comprise or consist of any. one of the following:
  • C 1-10 alkyl optionally substituted with one or more substituents as defined herein, e.g. a group which is a substituted or unsubstituted C 1-10 alkyl, C 1-10 haloalkyl, C 1-10 hydroxyalkyl , C 1-10 carboxyalkyl , C 1-10 aminoalkyl group;
  • Ci-io cycloalkyl-C 1-10 alkyl optionally substituted with one or more substituents as defined herein;
  • C 5-20 aryl optionally substituted with one or more substituents as defined herein, e.g. C 5-2O carboaryl or C 5-2O heteroaryl;
  • substituted means a parent group which bears one or more substituents.
  • substituted is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, appended to, or if appropriate, fused to, a parent group.
  • substituents are well known in the art, and methods for their formation and introduction into a variety of parent groups are also well known.
  • substituteduents as defined herein are independently selected from hydrogen, halo; hydroxy,- oxo; ether (e.g., Ci -7 alkoxy) ; formyl; acyl (e.g., C 1-7 alkylacyl ,
  • acylhalide carboxy,- ester,- acyloxy; amido; acylamido; thioamido,- tetrazolyl,- amino,- nitro; nitroso; azido; cyano; isocyano; cyanato,- isocyanato,- thiocyano,- isothiocyano,- sulfhydryl; thioether (e.g., C 1-7 alkylthio) ; sulfonic acid; sulfonate; sulfone; sulfonyloxy; sulfinyloxy; sulfamino; sulfonamino,- sulfinamino; sulfamyl; sulfonamido,-
  • Cx ⁇ alkyl including, e.g., unsubstituted Ci_ 7 alkyl , C 1-7 haloalkyl, C ⁇ hydroxyalkyl, Ci_ 7 carboxyalkyl , Ci -7 aminoalkyl,
  • C 5-6 heterocyclyl ,- or C s _ 20 aryl (including, e.g., C 5-20 carboaryl,
  • substituent (s) are independently selected from:
  • phenyl substituents if the phenyl group has less than the full complement of substituents, they may be arranged in any combination. For example, if the phenyl group has a single substituent other than hydrogen, it may be in the 2-, 3-, or 4-position. Similarly, if the phenyl group has two substituents other than hydrogen, they may be in the 2,3-,
  • phenyl group has three substituents other than hydrogen, they may be in, for example, the 2,3,4-, 2,3,5-, 2,3,6-, 2,4,5-, 2,5,6-, or 3 , 4, 5-positions . If the phenyl group has four substituents other than hydrogen, they may be in, for example, the
  • -CF 3 -CHF 2 , -CH 2 F, -CCl 3 , -CBr 3 , -CH 2 CH 2 F, -CH 2 CHF 2 , and -CH 2 CF 3 ; -CH 2 OH, -CH 2 CH 2 OH, and -CH(OH)CH 2 OH; and, -CH 2 NH 2 , -CH 2 CH 2 NH 2 and -CH 2 CH 2 NMe 2 .
  • the compounds of the invention may be derivatised in various ways.
  • “derivatives” of the compounds includes well known ionic, salt, solvate and protected forms of the compounds or their substituents mentioned herein.
  • a reference to carboxylic acid ( -COOH) also includes the anionic (carboxylate) form (-C00 " ) , a salt or solvate thereof, as well as conventional protected forms.
  • a reference to an amino group includes the protonated form (-N + HR 1 R 2 ) , a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group.
  • a reference to a hydroxyl group also includes the anionic form (-0 " ) , a salt or solvate thereof, as well as conventional protected forms.
  • Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and transforms,- E- and Z-forms; c-, t-, and r- forms; endo- and exo- forms; R-, S-, and meso-forms; D- and L-forms; d- and 1-forms ; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; ⁇ and ⁇ -forms; axial and equatorial forms,- boat-, chair-, twist-, envelope-, and halfchair-forms ,- and combinations thereof, collectively referred to as "isomers” (or "isomeric forms").
  • isomers are structural (or constitutional) isomers (i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space) .
  • a reference to a methoxy group, -OCH 3 is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH 2 OH.
  • a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta- chlorophenyl .
  • Ci_ 7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta- , and para-methoxyphenyl
  • Ci_ 7 alkyl includes n-propyl and iso-propyl
  • butyl includes n-, iso-, sec-, and tert-butyl
  • methoxyphenyl includes ortho-, meta- , and para-methoxyphenyl
  • keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
  • H may be in any isotopic form, including 1 H, 2 H (D) , and 3 H (T) ; C may be in any isotopic form, including 12 C, 13 C, and 14 C; 0 may be in any isotopic form, including 15 O and 18 O; and the like.
  • a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof.
  • Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
  • a salt may be formed with a suitable cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ .
  • Suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ) .
  • Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine .
  • An example of a common quaternary ammonium ion is N (CH 3 ) 4 + .
  • a salt may be formed with a suitable anion.
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous .
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric.
  • R groups are shown in the examples below, and include -C 6 H 4 (OCH 2 CH 2 ) 2 OCH 3 , -C 6 H 4 (OCH 2 CH 2 ) 3 OCH 3 , C 6 H 4 O (CH 2 ) 3 CH 3 , - (CH 2 ) 2 CHO, ⁇ (CH 2 ) 4 CH 3/ and -(CH 2 J 2 CH 2 OH.
  • Any R group may consist of or comprise a reactive functional group as described above.
  • one or more R groups e.g. 1, 2, 3, 4, 5, or 6 R groups
  • one or more R groups in a given molecule may consist of or comprise the formula -Y-Z as defined above in relation to the X groups.
  • the R groups will not normally carry reactive functional groups of the type carried by the X groups .
  • R groups may also carry other functional groups.
  • Examples include affinity tags, such as biotin, hexahistidine tags, or epitope peptides, which may be used to aid detection and/or purification of cross-linked complexes.
  • tags such as radiolabels and spectrophotometrically detectable moieties such as fluorescent dyes (BODIPY ⁇ ; fluorescein, rhodamine etc.) .
  • binding agents such as antibodies
  • Peptide sequences which are capable of targeting protein molecules to particular intracellular compartments may also be added, in order to target the cross-linking reagent to those compartments.
  • An example of a nuclear localisation sequence is PKKKRKV (derived from the SV40 large T antigen) .
  • An example of an ER retention sequence is KDEL, which will target proteins to the KDEL receptor on the endoplasmic reticulum.
  • Other moieties which direct ' molecules into cells or to particular sub-cellular compartments may also be used.
  • certain peptides are known which are capable of crossing the plasma membrane and can also transduce associated (e.g. covalently attached) molecules across the plasma membrane, such as the 16 amino acid peptide named "Penetratin" , which is derived from Antennapedia protein.
  • examples below utilise a monohydroxy resorcinarene in which 3 of the R groups are identical and the other contains a free OH group, which is readily derivatised by conventional chemistries.
  • the examples show incorporation of a biotin tag and a fluorescein tag.
  • R groups can also be derivatised as required by rapid and conventional techniques such as transamidation, "click” chemistry, and native Staudinger ligation.
  • Click chemistry is a set of reactions which are normally used for rapid modification of molecules, particularly biological molecules.
  • the 1,3 dipolar addition of azides to alkynes is one example of click chemistry:
  • Native Staudinger ligation involves the addition of phosphines to azides, then intramolecular transfer to form a stable amide; e.g.
  • R groups which consists of or comprises a peptide may be further derivatised by peptide ligation techniques, e.g. by intein mediated ligation.
  • R groups enables the compounds of the invention to cross the plasma membranes of living cells, which is highly desirable as it allows the trapping of interactions in a cellular environment without disrupting the cells. This may be particularly useful for trapping transient and/or low affinity interactions which may not survive cell lysis or may not take place in cell-free systems.
  • Amine functional groups such as morpholine-derivatives may be desirable, as these may be protonated in physiological milieu (which may improve solubility), but may also cross membranes.
  • solubilising groups particularly charged groups may be useful to increase compound solubility.
  • Arginine derivatives are particularly interesting because they are known to have cell penetrating properties.
  • R groups may have, or may comprise, the formula -Y- Z' , where Y is a spacer as defined above in relation to X groups, and Z' is a reactive functional group Z or is an alternative functional group such as an affinity tag, a spectrophotometrically-detectable moiety such as a fluorescent dye, a radiolabel, a peptide (e.g. of 2 to 50 amino acids, 2 to 30 amino acids, 2 to 20 amino acids or 2 to 10 amino acids) , etc ..
  • Y is a spacer as defined above in relation to X groups
  • Z' is a reactive functional group Z or is an alternative functional group such as an affinity tag, a spectrophotometrically-detectable moiety such as a fluorescent dye, a radiolabel, a peptide (e.g. of 2 to 50 amino acids, 2 to 30 amino acids, 2 to 20 amino acids or 2 to 10 amino acids) , etc ..
  • Radioactive or non-radioactive isotope tags may be incorporated into R groups (or elsewhere in the molecule) to assist in mass spectroscopic analysis of the resulting complexes. Unusual isotopes are readily identifiable by mass spectroscopy and can provide useful information about the number of cross-linking reagents attached to a given protein or fragment .
  • the molecule may comprise one or more atoms of, e.g., 13 C, 14 C, 18 O, 2 H or 3 H.
  • Calixarenes exist in different configurations, depending on the orientation of the R groups.
  • calix[4] arenes (such as the resorcinarenes described in the examples) exist in the two configurations:
  • the orientation of the spacer arms and hence the reactive functional groups also varies depending on the configuration. Particular configurations may be preferred for particular applications .
  • the compounds described in this specification find use as cross-linking agents, particularly for the study of structures of biological macromolecules, interactions between biological macromolecules, and also interactions between biological macromolecules and other molecules.
  • the invention provides a method of cross-linking two or more sites in a complex containing two or more components including at least one biological macromolecule, comprising contacting said complex with a cross-linking reagent as described above such that at least two reactive functional groups of said reagent react independently with components of said complex to form a cross-linked complex.
  • the complex consists of two or more molecules non-covalently associated with one another, e.g. by hydrophobic and/or electrostatic interactions. Each individual molecule present in the complex is referred to as a component .
  • the complex comprises at least one biological macromolecule, which is normally a peptide or protein, although it may also be a nucleic acid. It may be non-covalently complexed with one or more other peptides or proteins, or with other non-peptide molecules . Thus the complex may comprise two or more copies of said target protein.
  • the complex may further comprise at least one other protein, a nucleic acid, a protein cofactor, a modulator of protein activity or a drug molecule.
  • the complex may comprise a predetermined target biological macromolecule. That is to say, cross-linking may be performed with the specific intention of identifying molecules which associate non-covalently with a predetermined biological macromolecule. However, it is not always necessary to look for binding partners of a predetermined molecule. It is also possible to perform cross-linking in a system containing a plurality of biological macromolecules and subsequently identifying components of any cross-linked complexes obtained without prior knowledge of any components of the complex.
  • the complex to be cross-linked by the reagents described here may be present in an intact cell, as some of the cross-linking reagents described are capable of crossing a cell's plasma membrane. Alternatively, they may be present in a cell-free solution, such as a cell lysate (which may contain intact cellular organelles such as nuclei, endoplasmic reticulum or Golgi vesicles, mitochondria, etc.) or a reconstituted system containing only selected target molecules .
  • the method typically comprises contacting a solution containing said complex with said reagent.
  • cross-linker compounds described in this specification make them particularly suitable for cross-linking of complexes containing more than two non-covalently associated protein subunits.
  • the relatively large rigid core structure tends to prevent the compound from penetrating the interior of most proteins.
  • the compound is likely to be restricted to the exterior of a protein complex and so may be less prone to forming purely intramolecular links than many commercially available bifunctional cross-linking reagents.
  • the compounds preferably contain a large number of reactive functional groups, on tether arms spaced around the core, one molecule is well-adapted to cross-link more than two different components of a complex. This would require two or more molecules of a conventional bifunctional reagent having only two reactive functional groups. Therefore cross-linking studies can use lower concentrations of the compounds described herein than of many conventional reagents, which in turn is likely to improve the specificity of the cross-linking reaction, and reduce the chance of forming "false-positive" links between molecules which do not naturally associate with one another. Consequently it is believed that the results achieved with the cross-linking reagents described in this specification may be more physiologically relevant than those achieved with conventional cross-linkers .
  • the complex may comprise at least three non- covalently associated biological macromolecule components. Potentially it may contain even more, such as four, five, six, seven, eight, nine, ten, twenty, thirty or more components, such as a ribosome, viroid or virus.
  • the reagent may be necessary to provide an appropriate stimulus to induce reaction between the reagent and the complex.
  • the reagent may contain photoreactive groups it will normally be necessary to apply electromagnetic radiation of a suitable wavelength.
  • the method will further comprise the step of isolating the resulting cross-linked complex, e.g. from other components in the solution.
  • the cross-linked complex may be isolated from unreacted reagent and/or from un- cross-linked complex, and/or from target biological macromolecules not part of a complex.
  • the cross- linked complex will also be isolated from other biological macromolecules in the solution.
  • the purification may involve affinity purification. Typically, this will involve contacting the cross -linked complex with a binding partner capable of binding specifically to a component of the complex.
  • the affinity purification comprises contacting said cross-linked complex with a binding partner capable of binding specifically to the cross-linking reagent .
  • the reagent may possess an affinity tag to facilitate this.
  • Affinity tags include epitopes for known antibodies . Suitable epitopes include small molecules or short (approximately 6 or more amino acid) peptides .
  • the reagent may carry a tag which is capable of binding by coordination to a metal ion, such as a hexa-histidine peptide, which can bind to immobilised nickel ions.
  • the reagent may also carry a biotin • moiety, which can be bound by avidin or streptavidin.
  • a nucleic acid tag which could be bound by hybridisation to a complementary nucleic acid probe.
  • the affinity purification may use a binding partner specific for a component of the complex, such as the target biological macromolecule.
  • Antibodies may be used as binding partners for almost any complex component, but particularly for proteins.
  • Complementary nucleic acids may be used as binding partners for nucleic acids.
  • the component for which the binding partner is specific may have been specifically engineered or modified to facilitate isolation.
  • it may be genetically engineered to contain a specific nucleic acid or peptide sequence, for binding to a chosen binding partner such as a complementary nucleic acid probe, antibody or metal ion.
  • a chosen binding partner such as a complementary nucleic acid probe, antibody or metal ion.
  • it may be chemically modified, e.g. to incorporate an affinity tag such as a biotin moiety.
  • a modified component may be introduced into a system (e.g. into a cell) in order to isolate and identify other molecules capable of forming a complex with that component.
  • the binding partner is typically immobilised on a solid phase so that the bound cross -linked complex can easily be isolated from undesirable contaminants.
  • the purification may comprise one or more steps of purification on the basis of a physical property such as molecular mass, size (e.g. hydrodynamic radius) or charge.
  • Preferred separation methods include differential centrifugation/sedimentation analysis (e.g. analytical ultracentrifugation) , chromatography, particularly HPLC (high pressure liquid chromatography) , SDS-PAGE or isoelectric focussing. Chromatographic purification techniques such as HPLC are particularly convenient, as the resultant purified fractions can readily be analysed by mass spectroscopy (see below) . A combination of HPLC and mass spectroscopic analysis also lends itself readily to automation.
  • Such methods may also comprise the step of determining the molecular mass of the cross-linked complex. This may give an indication of the number or nature of the components of the complex.
  • the method may comprise the step of determining (or verifying) the identity of one or more components of the complex. This may be achieved by contacting the complex with a binding partner capable of binding specifically to a component suspected of being present in the complex.
  • Suitable binding partners for proteins include antibodies, which may be used in immunoblotting techniques, or immunosorbent assays such as an enzyme-linked immunosorbent assay (ELISA) .
  • Suitable binding partners for nucleic acids include hybridisation probes and polymerase chain reaction primers. Thus it may be possible to identify a particular nucleic acid in a complex by a hybridisation reaction or PCR.
  • Mass spectroscopy has become a common technique in proteomic analysis. It is often used in conjunction with chemical cross-linking studies to determine structures of macromolecules and macromolecular complexes (especially proteins) and to identify components of such complexes.
  • the methods described may further comprise the step of analysing the cross-linked complex by mass spectroscopy. It may follow a purification or separation step, or be used as an alternative to a purification or separation step.
  • the cross-linked complex Prior to mass spectroscopy, it may be desirable to disrupt the cross-linked complex, e.g. for reasons described below, to separate the various components of the complex from one another and/or from the cross-linking reagent. It may also be desirable to disrupt individual components of the complex into smaller fragments, e.g. by cleaving proteins into peptide fragments .
  • disruption may comprise chemical cleavage of the cross- linked complex:, e.g. with periodate, bases, acids, hydrazides, by photolysis, or by any other suitable technique known to the skilled person.
  • disruption may comprise contacting the cross-linked complex with a protease, such as trypsin. Trypsin cleaves proteins on the C-terminal side of lysine or arginine residues, except where they are followed by proline.
  • the cross -linking reagent may comprise a selectively cleavable group between two of the reactive functional groups bound to the complex.
  • the method will therefore comprise selectively cleaving said cleavable group.
  • cleavage may be performed by a chemical or enzymatic reaction, or irradiation.
  • After disruption of the cross-linked complex there may be further purification steps to isolate the various disrupted components or fragments thereof, to facilitate subsequent analysis. HPLC is a preferred technique.
  • the present invention also provides methods as described above (mutatis mutandis) for the formation of intra-molecular cross-links in a single target molecule, although this is less preferred than the formation of inter-molecular links in a molecular complex between two or more molecules .
  • Such a method comprises cross-linking two or more sites in a target biological macromolecule, by contacting said macromolecule with a cross-linking reagent as described above such that at least two reactive functional groups of said reagent react independently with sites of said macromolecule to form a cross-linked macromolecule.
  • MS typically involves measuring a mass/charge (m/z) ratio for an analyte molecule. Having determined the m/z ratio for a particular protein analyte, a database of protein sequences can be interrogated to find likely candidate sequences for the analyte, leading to its identification. These techniques can be expanded to identification of protein fragments. Thus, when a parent protein is cleaved at particular residues or sequences (e.g.
  • the m/z ratios of the resulting peptide fragments can be determined and the database then interrogated to identify peptide sequences which match both the protease' s cleavage preferences and the appropriate m/z ratio.
  • a number of different reactions may occur.
  • the cross-linking reagent may react at only one site on the protein or complex (i.e. not form any cross-links) yielding a residue carrying a tag.
  • an intra-molecular link may form, where the reagent links two residues within the same protein chain.
  • an inter-molecular link may form, between residues in different protein chains .
  • Digestion of the cross-linked assembly can therefore yield free peptides unconjugated to any linker, single peptides conjugated to one or more cross-linker molecules which are not linked to any other peptides, and pairs of peptides derived from the same or different protein chain connected by one or more cross-linker molecules.
  • a peptide bound to cross-linker will not have the same molecular mass or m/z ratio as an identical unconjugated peptide.
  • knowing the molecular mass of the cross- linking reagent and the chemistry by which it reacts with its target groups on a protein it will be possible to predict the effect of the cross-linker- on the m/z ratio of any given peptide. Therefore, if the algorithm used to interrogate the protein sequence database is able to take account of the molecular mass of the cross-linker when conjugated to one or more peptides (or the database itself contains suitable data) , it remains possible to identify the peptide.
  • cross- linking reagent may suggest the footprint of the reagent in relation to the cross-linked molecule (s) and thus lead to information about the topology of protein-protein interactions within a cross-linked complex.
  • incorporation of isotope tags into that part of the cross-linking reagent which remains associated with a target molecule or fragment thereof after any disruption step can be used for comparison of fragment populations.
  • cross -linking reagents which include groups which are cleavable (e.g. by a suitable chemical or enzymatic cleavage reaction) between reactive functional groups. This allows the peptides linked by a single cross-linking reagent to be separated when desired and analysed independently.
  • Cross-linking and MS can also be used to obtain structural information about proteins and protein complexes .
  • Proteins have complicated three-dimensional structures. A number of factors restricts the number and relative spatial location of groups on a protein which can be cross-linked by any given reagent. These include the conformation of the protein, the length and flexibity of the spacer between two reactive functional groups, whether the target groups in the protein are available on the surface of the molecule or buried internally, and whether the linker can access the interior and exterior of the protein or only the exterior. This can restricted by steric and electrostatic effects, as the interior of many proteins is hydrophobic, while parts of cross-linking reagents (particularly the reactive functional groups) are often hydrophilic.
  • identifying groups within a single protein or a protein complex which can be linked by a given cross-linking reagent provides information about the relative location of those residues. This may be used to deduce information about the structure of a molecule, the relative topological location of subunits within a complex, etc..
  • MS analysis involves acquiring data concerning molecular weight and/or charge of a candidate molecule, such as a protein, or protein fragment.
  • a candidate molecule such as a protein, or protein fragment.
  • the candidate molecule will be a component of a cross-linked complex or a fragment thereof.
  • the candidate molecule may be covalently linked to a cross- linking reagent as described in this specification or to a fragment of such a reagent .
  • a suitable database is interrogated in order to identify possible matches for the candidate compound, e.g.
  • Nd and Cd have mass distributions of 62 and 13 kDa, corresponding to a tetramer and monomer, respectively.
  • Lanes 1 (9 ⁇ M DnaD) and 2 (4.5 ⁇ M DnaD) indicate control reactions in the absence of SOXL whereas lanes 3 ⁇ protein (18 ⁇ M) /SOXL (180 ⁇ M) ⁇ and 4 ⁇ protein (18 ⁇ M) /SOXL (504 ⁇ M) ⁇ indicate reactions with increasing relative ratios of SOXL to DnaD or Nd.
  • Monomeric and dimeric species of DnaD are shown as (D)I and (D) 2 whereas monomeric, dimeric and tetrameric species of Nd as (Nd)I, (Nd) 2 and (Nd) 4, respectively.
  • Molecular weight markers (kDa) are shown in lanes M.
  • FIG. 4 The gel filtration profile of a mixture of Nd and Cd. The two domains elute at different positions through a superdex S200 analytical column. The individual proteins alone elute at exactly the same positions (data not shown) . Nd elutes earlier indicating an oligomer. No interaction between the domains was observed. Samples from the peaks were analysed by SDS-PAGE. The presence of Nd and Cd in the early and late peaks, respectively was verified by SDS-PAGE. The MW of Nd (16,056 kDa) is slightly bigger than that of Cd (13,730 kDa) and therefore it appears at a slightly higher position in the gel . B. Cross -linking of Nd at high concentration using SOXL.
  • Nd (30 ⁇ M) was cross-linked with SOXL (lane 2; 840 ⁇ M, lane 3; 1680 ⁇ M) .
  • Lane M and lane 1 show molecular weight markers (kDa) and unlinked Nd, respectively. Internal cross linking is apparent in the monomeric (Nd)I and dimeric (Nd) 2 species. Higher species corresponding to trimers (Nd) 3, tetramers (Nd) 4 and multimers (Nd)X are also apparent.
  • FIG. 5 SOXL cross-linking of Cd in the presence and absence of a ssl9mer oligonucleotide.
  • Panel A shows SOXL cross linking of Cd (18 ⁇ M) in the absence of oligonucleotide and control Cd in the absence of cross-linker, as indicated.
  • Panel B shows control Cd (18 ⁇ M) in the absence of cross-linker and also cross -linking reactions with increasing concentrations of SOXL (0.5, 1.0 and 1.5 mM) , at two different oligonucleotide concentrations (0.9 and 0.19 ⁇ M) , as indicated.
  • Internal cross-links as well as large crosslinked species are indicated as Cd and (Cd)X, respectively.
  • FIG. 6 Comparison of SOXLl cross-linking with cross- linking achieved by divalent reagents having equivalent span between reactive groups.
  • Cross-linking of GST was performed with 1, 2 and 4 molar equivalents of either SOXL 1 (lanes 1, 5 and 9), DSS 4 (lanes 2,6 and 10), DSG 5 (lanes 3, 7 and 11), or SAB 6 (lanes 4, 8 and 12), as indicated. After 30 mins cross- linking, proteins were denatured and separated by SDS-PAGE prior to immunoblotting with anti-GST antibodies. Position of GST complexes are indicated.
  • FIG. 7 Crosslinking of phosphoinositde 3 -kinase heteromers performed with SOXL 1, SOXL 2, SOXL 3 and DSS.
  • Figure 8 Comparison of the time-course of SOXLl and DSS-mediated crosslinking.
  • Cross-linking of GST was performed with 4 molar equivalents of either SOXL 1 (S) or DSS (D) , for the times indicated (in minutes) prior to the reaction being terminated. Proteins were denatured and separated by SDS-PAGE prior to immunoblotting with anti-GST antibodies.
  • Figure 9 SDS-PAGE gel showing cross-linking of DnaD Nd using cross-linking reagent 26 with cleavable groups in the linker arms.
  • x Nd' contains protein (N domain of DnaD) only.
  • the other lanes contain cross-linked protein and are labelled to reflect the molar ratio of protein: reagent in the cross- linking reaction.
  • Resorcinarene (3) (2.09 g, 1.45 tnmol) and Cs 2 CO 3 (4.72 g, 14.5 mmol) were combined in dry DMP (15 mL) and stirred at 50° C for 1 h.
  • ethyl bromoacetate (7.26 g, 43.5 mmol) was added and the mixture stirred at 50° C for 18 h.
  • the residue was partitioned between CH 2 Cl 2 and water.
  • the organic phase was washed with water and dried over MgSO 4 .
  • Octaester (4) (1.8 g, 0.85 mmol) was dissolved in THF (50 raL) . To this was added a KOH solution (5.7 g KOH in 30 mL water) and the reaction mixture was stirred vigorously at room temperature for 1 h. The mixture was washed with ether and the aqueous phase adjusted to pH 2. The mixture was left at - 20 °C for 18 h and the resultant precipitate was collected by suction filtration. The solid was dried (0.1 tnmHg) to give the octaacid (5) as a white solid (1.30 g, 80 %) ; m.p. 168- 172°C; v ⁇ /cra "1 (KBr) 3398s, 2931vs, 1754vs, 1610m, 1584m,
  • N- hydroxysuccinimide was co-evaporated with dry toluene (3 x 1 mL) , dissolved in dry THF (1 mL) , and cooled to -10 °C. Then the solution of the octaacid chloride and piperidinomethyl polystyrene HL (200 - 400 mesh) (413.5 mg, 1.65 mmol) was added, and the mixture was stirred for 2 h at room temperature.
  • Resorcinarene 2 R 1 C 6 H 4 (OCH 2 CH2) 3 ⁇ Me SOXL 1' wherein the correct formula of the molecule designated SOXLl is shown as SOXLl' .
  • the compounds described above should be named as follows: [(2,8,14,20-Tetra ⁇ 4- ⁇ 2- [2-(2- methoxy ethoxy) ethoxy] ethoxy ⁇ phenyl Jpentacyclo [19.3.1.1 3 ' 7 .1 9 ' 13 .1 15 ⁇ 19 ]tetracosa-l(25) , 3 , 5, 7 (28) , 9, 11, 13 (27) , 15, 17, 19 (26) , 21,23 - dodecaene-4,6,10,12,16, 18, 22, 24-octol;
  • Resorcinarene (7) (1.49 g, 1.94 mmol) and K 2 CO 3 (5.37 g, 38.9 mmol) were combined in dry DMF (5 mL) and stirred at 50 °C for 1 h.
  • Ethyl bromoacetate (6.49 g, 38.9 mmol) was added and the mixture stirred at 50° C for 18 h.
  • the residue was partitioned between CH 2 Cl 2 and water .
  • the organic phase was washed with water and dried over MgSO 4 .
  • Octaester (8) (0.94 g, 0.65 mmol) was dissolved in THF (30 mli) . To this was added a KOH solution (4.34 g KOH in 23 mL water) and the reaction mixture was stirred vigorously at room temperature for 1 h. The mixture was washed with ether and the aqueous phase adjusted to pH 2. The mixture was left at - 20 0 C for 18 h and the resultant precipitate was collected by- suction filtration.
  • Resorcinarene (11) (1.14 g, 0.80 mmol) and Cs 2 CO 3 (3.71 g, 11.4 mmol) were combined in dry DMF (15 mL) and stirred at 50 "C for I h.
  • Ethyl bromoacetate (2.15 g, 12.9 mmol) was added and the mixture stirred at 50 0 C for 18 h.
  • the residue was partitioned between CH 2 Cl 2 and water.
  • the organic phase was washed with water and dried over MgSO 4 .
  • Octaester (12) (0.81 g, 0.39 mmol) was dissolved in THF (50 mL) . To this was added a KOH solution (5.7 g KOH in 30 mL water) and the reaction mixture was stirred vigorously at room temperature for 1 h. The mixture was washed with ether and the aqueous phase adjusted to pH 2. The mixture was left at - 20 °C for 18 h and the resultant precipitate was collected by suction filtration.
  • a KOH solution 5.7 g KOH in 30 mL water
  • N- hydroxysuccinimide (59.1 mg, 0.51 mmol) was co-evaporated with dry toluene (3 x 1 mL) , dissolved in dry THF (1 mL) , and cooled to -10 0 C. Then the solution of the octaacid chloride and piperidinomethyl polystyrene HL (200 - 400 mesh) (467 mg, 1.91 mmol) was added, and the mixture was stirred for 2 h at room temperature.
  • Oxalyl chloride (26 ⁇ L, 0.28 mmol) was dissolved in CH 2 Cl 2 (200 ⁇ L) under an argon atmosphere and cooled to -78 0 C.
  • DMSO 60 ⁇ L, 0.84 mmol was added and the reaction mixture was stirred for 15 mins at -78 0 C.
  • Alcohol (15) (109.1 mg, 0.076 mmol) dissolved in CH 2 Cl 2 (100 ⁇ L) was added dropwise over 2 mins and the reaction was stirred for 2 hr at -78 0 C.
  • Et 3 N (53.9 ⁇ L, 0.38 mmol was added and the reaction was allowed to warm to 0 0 C using an ice bath over 1 hr.
  • Octaester (17) (35 mg, 0.019 mmol) was dissolved in THF (5 mli) . To this was added a KOH solution (0.57 g KOH in 3 mL water) and the reaction mixture was stirred vigorously at room ⁇ temperature for 1 h. The mixture was washed with ether and the aqueous phase adjusted to pH 2. The mixture was left at - 20 °C for 18 h and the resultant precipitate was collected by suction filtration. The solid was dried (0.1 mmHg) to give the octaacid (18) as a white solid (24 mg, 78 %) ;
  • NHS groups may be added to the ends of the spacer arms as described above.
  • Octaester (19) (53.3 mg, 0.032 mmol) was dissolved in THF (5 mL ⁇ . To this was added a KOH solution (0.57 g KOH in 3 mL water) and the reaction mixture was stirred vigorously at room temperature for 1 h. The mixture was washed with ether and the aqueous phase adjusted to pH 2. The mixture was left at - 20 °C for 18 h and the resultant precipitate was collected by- suction filtration. The solid was dried (0.1 mmHg) to give the octaacid (20) as a white solid (24 mg, 70 %) ;
  • NHS groups may be added to the ends of the spacer arms as described above .
  • iV-hydroxysuccinimide (9.2 rag, 0.08 mmol) was co- evaporated with dry toluene (3 x 1 mL) , dissolved in dry THF (1 mL) , and cooled to -10 0 C. Then the solution of the octaacid chloride and piperidinoraethyl polystyrene HL (200 - 400 mesh) (41.4 mg, 0.17 mmol) was added, and the mixture was stirred for 2 h at room temperature.
  • a sulfone group in the cross-linking reagent can allow for cleavage of a conjugate through hydrolysis of the linkage under basic conditions.
  • 0.1 M sodium phosphate adjusted to pH 11.6 by addition of Tris base, containing 6 M urea, 0.1 % SDS, and 2 mM DTT, sulfone groups have been successfully cleaved after incubation at 37 0 C for 2 h.
  • XL-lBlue E.coli (Stratagene, CA, USA) were transformed with the pGEX2T plasmid (AmerhsamBioscience) .
  • This plasmid drives ⁇ bacterial expression of the Schistosoma japonicum form of GST.
  • XL-I Blue harbouring pGEX2T were cultured in 2xYT containing 100 ⁇ g/ml ampicillin at 37 0 C, to an optical density between 0.6-0.8 OD 600 before being transferred to 28 0 C, at which time isopropyl- ⁇ -D-thiogalactoside was added to a final concentration of 0.1 mM, as previously described (1) .
  • Octavalent cross-linker, SOXL 1 was rapidly synthesized using acid-catalyzed Friedel-Crafts alkylation of resorcinol with 4- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) benzaldehyde in absolute ethanol at 80 0 C to provide resorcinarene 2 in 70 % yield after recrystallization from hot ethanol (scheme 1) .
  • t141 Subsequent alkylation of all eight resorcin[4] arene phenols proceeded in good overall yield of the octaethyl ester 3 using a two molar excess of ethyl 2-bromoacetate for each phenolate.
  • Glutathione S-transferases [E . C.2.5.1.18] tl ⁇ ) exist as homodimers , although higher order oligomers have also been reported. 1171 Experiments were performed on the Schistosoma japonicum form of GST purified from E.coli transformed with the pGEX2T expression vector . [18] Aliquots of between 1 and 8 molar equivalents of SOXL 1 or DSS 5 were added to GST solution in phosphate buffer to a constant final volume of DMSO. All solutions remained clear suggesting complete dissolution of either cross-linker.
  • SOXL 1 treated GST-dimer has a slightly higher molecular weight than that treated with DSS 5, most likely due to the additional molecular weight contributed by SOXL 1.
  • the nature of the higher molecular weight band in the DSS 5 treated lanes is currently not clear.
  • incorporation of photoactivatable diazirine functionality may offer a more reactive, but shortlived species upon irradiation and the timing of cross-linking to be controlled more precisely.
  • adjustment of the aldehyde derived component may achieve modulation of solubility, or incorporation of biotin for affinity purification.
  • the defined geometry offered by SOXL 1 may offer insight into the topology of the interacting protein complex by analysis of its footprint, perhaps after partial digestion of the entrapped protein and mass spectrometry.
  • the SOXL molecule has also been used in a study of the domain organisation of the DnaD protein of Bacillus subtilxs.
  • the DnaD protein participates in an essential primosomal cascade to recruit the replicative helicase and primase proteins to the oriC or to a restart site (Sonenshein et al, 2002) .
  • DnaD consists of N-terminal (Nd) and C-terminal (Cd) domains.
  • Cd is tetrameric, binds DNA and DNA-binding induces its oligomerisation. Although Cd binds to the plasmid pBR322 it fails to open it up either alone or in the presence of Nd.
  • Nd oligomerisation is DNA independent whereas Cd oligomerisation is DNA dependent.
  • Nd and Cd must be linked in the same polypeptide to exhibit
  • Nd and Cd C-terminally His -tagged Nd and Cd were constructed from a pET22b-DnaD vector (Turner et al , 2004) by amplifying Ndel- Xhol gene fragments using PCR and cloning into the same sites of pET22b. Both domains were tagged with the ELH6 sequence at their C-termini. They were over-expressed in E. coli BL21(DE3) and purified using a HiTrap-Ni2+-chelating column and gel filtration through superdex S75. Both proteins were purified in 50 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT and made up to 10%v/v glycerol before snap-freezing in liquid nitrogen for storage at -80 0 C.
  • SOXL cross linking Proteins (DnaD, Nd) were incubated with SOXL at different protein: SOXL molar ratios (1:14 and 1:28) in 50 mM Tris pH7.5, 2 mM EDTA, 1 mM DTT, 350 mM NaCl (for DnaD) or 100 mM NaCl (for Nd and Cd) for 30 minutes at 37 0 C. Samples were then resolved by SDS-PAGE through a 12% gel.
  • a 25 mM stock of SOXL in DMSO was prepared for long storage and prior to a linking experiment SOXL was diluted further in 10% v/v DMSO and added to the protein solution to maintain the DMSO at a maximum of 2% v/v in the reaction mixture.
  • Cross-linking of Cd (18 ⁇ M) in the presence or absence of DNA was carried out with increasing concentrations of SOXL (0.5, 1.0, 1.5 mM) in the presence or absence of the same ssl9mer oligonucleotide (0.9 and 0.18 ⁇ M) that was used in the gel shift assays.
  • Analytical ultrac ⁇ ntrifugation was carried out in an Optima XL-A ultracentrifuge (Beckman Coulter, Palo Alto, CA, USA) . Sedimentation velocity was carried out at three loading concentrations in two channel centrepieces. All experiments were at 40,000 rpm, data were taken at 5 minute intervals, and analysed using SEDFIT (www.analyticalultracentrifugation.com). Sedimentation equilibrium was carried out in six channel centre pieces and samples were centrifuged at 12,000, 16,000, 22,000 and 28,000 rpm. Data were fitted globally using SEDPHAT. Errors were estimated by 1,000 runs of a Monte Carlo simulation.
  • Solvent density was determined using an Anton Paar DMA 5,000 density meter (Anton Paar, Hertford, UK). Solution viscosity was determined using a Schott Gerate viscometry unit attached to an Oswald viscometer (Schott Gerate, Germany) .
  • Nd residues 1-128
  • Cd residues 129-232
  • the Cd monomer binds DNA and exhibits a DNA-induced oligomerisation activity Cd bound to small oligonucleotides in gel shift assays (not shown) .
  • Nd did not exhibit any DNA binding activity.
  • the Cd monomer is small the observed shifts were large and comparable to these obtained with the full length D ⁇ aD.
  • the sizes of the complexes appear to be very large as they fail to enter into the gel. Similar shifts were observed with longer 54mer and shorter 19mer ss oligonucleotides, as well as with an intermediate 30mer ds oligonucleotide.
  • the large complexes are not the result of individual Cd molecules binding side by side to the oligo substrates, but instead there is an inherent oligomerisation activity that is induced by binding to DNA.
  • a Cd molecule binds to DNA it acts as a ⁇ seed' to induce the binding of ⁇ more Cd molecules, forming large complexes.
  • SOXL cross-linking in the presence and absence of DNA (Fig. 5) .
  • a ssl9mer oligonucleotide large complexes of Cd molecules with variable stoichiometries were cross-linked appearing as a smear higher up the gel (Fig. 5) .
  • Cross-linkers with C 4v symmetry (derived from alkyl aldehydes, S0XL2) were compared with C 2h symmetric analogues (derived from 4-alkoxyphenyl aldehydes, SOXL3) . While each compound has the same number of reactive esters, distinct cross-linking efficiency was apparent.
  • the crown shaped octavalent linker SOXL2 displays all eight esters on the same face, but is less efficient, and also displays lower cross-linking when compared directly to the bivalent linker DSS or to SOXLl.
  • the 320 kDa species is consistent with a complex of two pllO ⁇ subunits and one p85 ⁇ subunit, the 282 kDa specific is consistent with a complex of one pllO subunit and two p85 ⁇ subunits. Comparison of the time-course of SOXLl and DSS-mediated crosslinking.
  • Cross-linking of GST was performed with 4 molar equivalents of either SOXLl (S) or DSS (D) , for 1, 5, 10 and 30 minutes before the reactions were terminated. Proteins were denatured and separated by SDS-PAGE prior to immunoblotting with anti- GST antibodies. The position of molecular weight standards (in kDa) and GST complexes are indicated. The results (Fig. 8) indicate that SOXL-I initiates cross-linking within 1 minute and the reaction plateaus at around 30 minutes.
  • Protein (DnaD, Nd) was incubated with the chemically cleavable linker 26 at different protein: linker molar ratios (1:14 and 1:28) in Tris pH 7.5, 2 mM EDTA, ImM DTT, 100 mM NaCl for 30mins at 37 0 C. Samples were then resolved by SDS-PAGE through a 12% gel. Prior to cross-linking the chemically cleavable linker was diluted further to 5mM in 10% v/v DMSO and added to the protein solution to maintain the DMSO at a maximum of 2% v/v in the reaction mixture.
  • band 1 contains Nd protein alone
  • band 2 contains Nd monomers + linker 26
  • band 3 contains Nd dimers + linker 26
  • bands 4 to 6 contain higher oligomers of Nd + linker 26.
  • Gel bands were excised, dessicated with acetonitrile, then treated with buffer containing 3mM DTT, 6M urea, 10OmM tris buffer and 10OmM phosphoric acid adjusted to pH11.6, by addition of 1OM NaOH, for 2 hours at 37 0 C. This treatment cleaves the sulfone groups in the linker arms.
  • Crosslinked proteins will therefore be separated into monomers, but those monomers will still carry residual fragments of the cross- linking reagent (extending from the sulfone group to the reactive NHS group) covalently bound to those residues with which they reacted in the cross-linking reaction.
  • the chemically cleaved material was reduced with DTT and alkylated with iodoacetamide before trypsin digestion in situ.
  • the extracted material was then digested with trypsin, to yield a mixture of peptides.
  • Those peptides containing residues with which the cross-linking reagent bound will have greater molecular weight than expected simply from their amino acid sequence because of the residual fragments of the cleaved reagent which remain bound to them.
  • Mass spectrometry of the cleaved and digested material was performed at the Biopolymer Synthesis and Analysis Unit, University of Nottingham. Tryptic peptides were desalted by binding and then elution from a C18 Zip-tip (Millipore) , and then analyzed by electrospray mass spectrometry (Waters QTOF2 hybrid quadrupole mass spectrometer) .
  • CAM(B) CAM Cysteine
  • Fusion proteins encoding growth factor receptor binder 2 (Grb2, 28 kDa) , grb2-associated binder 2 (Gab2, 90 kDa) , src homology phosphatase 2 (Shp2, 68 kDa) and the 85kDa regulatory subunit of class IA phosphoinositide 3 -kinases (p85, 85 kDa) have all been expressed in XL-lBlue E.coli, purified and the fusion partner removed by thrombin cleavage. Further purification of each protein has been conducted to yield preparations of greater than 95% purity.
  • Gab2 has been expressed in XL-I Blue E.coli expressing an active tyrosine kinase, resulting in constitutive tyrosine phosphorylation of Gab2. It is envisaged that these purified proteins will be used in a series of in vitro experiments. In some cases, the uncleaved fusion proteins may be used instead of the cleaved protein product, especially if undesirably small amounts of pure protein are obtained following the cleavage and re-purification steps.
  • Grb2 binds via its SH3 domains to proline-rich motifs of Gab2. This represents a hetero-dimeric interaction.
  • Shp2 and p85 can bind to Gab2 when it is tyrosine phosphorylated, via SH2 domains present within Shp2 and p85. This represents a hetero-trimeric interaction.
  • the compounds described may be used to capture heteromeric protein interactions in an incremental manner as described below.
  • Purified Grb2 and non-phosphorylated Gab2 proteins will each be used at a final concentration between 0.1 ⁇ M and 3 ⁇ M and mixed together in 30% (v/v) PBS, pH 7.2, 70% (v/v) water, or used separately as controls.
  • BSA will be added at equimolar concentrations.
  • Stock solutions of the cross-linkers (SOXL and derivatives) will be dissolved in DMSO at identical concentrations and between 1 and 8 molar equivalents added to the protein solutions in a final reaction volume of 50 ⁇ l . Reactions will be incubated for 60 minutes at 21°C and quenched by addition of 1 ⁇ l of IM tris-HCl pH7.5.
  • Purified Shp2, p85, tyrosine phosphorylated and non- phosphorylated Gab2 proteins will each be used at a final concentration between 0.1 ⁇ M and 3 ⁇ M and incubated either individually or in combination in 30% (v/v) PBS, pH 7.2, 70% (v/v) water.
  • BSA will be added at equimolar concentrations.
  • Stock solutions of the cross- linkers (SOXL and derivatives) will be dissolved in DMSO at identical concentrations and between 1 and 8 molar equivalents added to the protein solutions in a final reaction volume of 50 ⁇ l . Reactions will be incubated for 60 minutes at 21°C and quenched by addition of 1 ⁇ l of IM Tris-HCl pH7.5.
  • Multimeric complexes containing Grb2 , Gab2 , Shp2 and p85 Multimeric complexes containing Grb2 , Gab2 , Shp2 and p85.
  • Purified Shp2 , p85 ; tyrosine phosphorylated and non- phosphorylated Gab2 and Grb2 proteins will each be used at a final concentration between 0.1 ⁇ M and 3 ⁇ M and incubated either individually or in combination in 30% (v/v) PBS, pH 7.2, 70% (v/v) water.
  • BSA will be added at equimolar concentrations.
  • Stock solutions of the cross-linkers SOXL and derivatives
  • DMSO DMSO at identical concentrations and between 1 and 8 molar equivalents added to the protein solutions in a final reaction volume of 50 ⁇ l .
  • Reactions will be incubated for 60 minutes at 21°C and quenched by addition of 1 ⁇ l of IM Tris-HCl pH7.5. 5 x SDS sample buffer will then be added to each sample, prior to boiling to denature the proteins. Samples will then be subjected to SDS-PAGE and immunoblotting using standard techniques. A multimeric complex containing all four proteins would yield a protein complex of approximately 271 kDa, which is close to the limits of resolution by SDS-PAGE. However, we should be able to also detect hetero-trimers of varying composition and most likely also hetero-dimers of varying composition. The composition of such complexes will be distinguished through the use of protein selective antibodies .
  • BaF/3 cells will be expanded in IL-3 -containing media. Prior to experimentation, the cells will be washed free of cytokine and incubated in serum-free and cytokine-free medium for Ih at 37°C, after which time SOXL or another compound of the invention will be added to the cells at concentrations between 0.5 and 20 ⁇ M and incubated for a further Ih at 37 0 C. A proportion of cells would be lysed directly and a further portion treated with lOng/ml rmIL-3 for 2-30 minutes to induce protein phosphorylation and subsequent protein-protein interactions. Control cells, to which no cross-linker is added, would be treated in an identical manner for comparison.
  • protein concentrations of the lysates would be determined and equivalent amounts of protein (at least 500 ⁇ g per sample) would be immunoprecipitated using standard techniques with antibodies specific to Shp2, Gab2, Grb2 or p85. After extensive washing, the immunoprecipitates would be denatured prior to separation by SDS-PAGE and immunoblotting . Immunoblotting would be performed as described above, using antibodies specific for the different protein anticipated to participate in protein- protein interactions.
  • CCDC 297190 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www. cede . cam.ac .uk/data_request/cif . " . [16] J. D. Hayes, J. U. Flanagan, I. R. Jowsey, Ann. Rev. Pharmacol. Toxicol. 2005, 45, 51-88.

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Abstract

L'invention concerne de nouveaux réactifs à base de calixarène et particulièrement des structures de résorcinarène. Les réactifs sont utiles en tant que réactifs de réticulation permettant l'étude des interactions non covalentes entre des macromolécules biologiques. La présente invention concerne également des procédés d'utilisation des nouveau réactifs.
PCT/GB2007/001277 2006-04-05 2007-04-05 Réactifs et procédés de réticulation de molécules biologiques Ceased WO2007113575A2 (fr)

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GBGB0606947.0A GB0606947D0 (en) 2006-04-05 2006-04-05 Reagents and methods for cross-linking biological molecules
GB0606947.0 2006-04-05

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010067621A1 (fr) * 2008-12-11 2010-06-17 出光興産株式会社 Composés cycliques et compositions de résine photosensible les utilisant
WO2010067622A1 (fr) * 2008-12-11 2010-06-17 出光興産株式会社 Composés cycliques stéréoisomères, leur procédé de fabrication, compositions comprenant les composés cycliques stéréoisomères et leur procédé de fabrication
CN107033032A (zh) * 2017-04-27 2017-08-11 同济大学 一种含氮杯[4]芳烃衍生物及其制备方法
CN109160995A (zh) * 2018-08-19 2019-01-08 南京理工大学 一种柱[5]芳烃自组装弹性体材料及其制备方法
CN119899947A (zh) * 2025-04-02 2025-04-29 长春黄金研究院有限公司 环保无氰浸金药剂及其提取电子废弃物中金的工艺

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Publication number Priority date Publication date Assignee Title
FR2797442B1 (fr) * 1999-08-13 2003-10-03 Commissariat Energie Atomique Derives acetamido de calixarenes, leur preparation et leur utilisation pour l'extraction du strontium
JP2002220556A (ja) * 2001-01-25 2002-08-09 Fuji Photo Film Co Ltd インク組成物
DE10352466A1 (de) * 2003-11-07 2005-06-16 Henkel Kgaa Verwendung von Calixaren-Verbindungen als geruchsbindendes Mittel
ATE536177T1 (de) * 2004-10-04 2011-12-15 Univ Minnesota Calixaren-basierte peptidkonformationsmimetika, verfahren zu ihrer verwendung und verfahren zu ihrer herstellung

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010067621A1 (fr) * 2008-12-11 2010-06-17 出光興産株式会社 Composés cycliques et compositions de résine photosensible les utilisant
WO2010067622A1 (fr) * 2008-12-11 2010-06-17 出光興産株式会社 Composés cycliques stéréoisomères, leur procédé de fabrication, compositions comprenant les composés cycliques stéréoisomères et leur procédé de fabrication
CN107033032A (zh) * 2017-04-27 2017-08-11 同济大学 一种含氮杯[4]芳烃衍生物及其制备方法
CN109160995A (zh) * 2018-08-19 2019-01-08 南京理工大学 一种柱[5]芳烃自组装弹性体材料及其制备方法
CN119899947A (zh) * 2025-04-02 2025-04-29 长春黄金研究院有限公司 环保无氰浸金药剂及其提取电子废弃物中金的工艺

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