PHOSPHONATE-CONTAINING CROSSLINKERS

Abstract

Disclosed are phosphonate-containing trifunctional crosslinkers and methods for synthesizing same. The invention further pertains to using the trifunctional crosslinkers in methods for crosslinking, purifying, and characterizing biomolecules such as peptides, proteins and polynucleotides.

Claims

1. A compound according to Formula (1): ##STR00025## wherein m is 0 or 1, wherein each n is independently 0 or 1, wherein each R.sub.1 is independently selected from the group consisting of C(O)H, C(O)Cl, C(O)Br, C(O)F, C(O)O-pentafluorophenyl, NCS, NCO, S(O).sub.2Cl, S(O).sub.2F, phenylsulfonyl fluoride, phenylsulfonyl chloride, C(O)N.sub.3, C(O)OC(O)CH.sub.3, OC(O)O(CH.sub.2).sub.qCH.sub.3, C(NH)O(CH.sub.2).sub.qCH.sub.3, C.sub.2 epoxidyl, ##STR00026## wherein the wiggly lines indicate a bond to R.sub.2 or to R.sub.3, wherein R.sub.5 is selected from the group consisting of H, CH.sub.3, CH.sub.2CH.sub.3, OCH.sub.2OCH.sub.3, OCH.sub.2O(CH.sub.2).sub.2Si(CH.sub.3).sub.3, SO.sub.3H, and OCH.sub.2O(CH.sub.2).sub.2OCH.sub.3, wherein one Q is a nitrogen atom and the other Q are CH, wherein each R.sub.2 is independently selected from the group consisting of C.sub.1-4 alkylene, C.sub.2-4 alkenylene, C.sub.2-4 alkynylene, S(O)CH.sub.2(CH.sub.2).sub.q, (CH.sub.2).sub.qSS(CH.sub.2).sub.q, N((CH.sub.2).sub.qC(O)H)C(O)N((CH.sub.2).sub.qC(O)H), -aspartyl-prolyl, and -valyl-prolyl, wherein each q is independently an integer in a range of from 0 to 6, wherein when m is 0, then R.sub.3 is selected from the group consisting of CR.sub.6, benzenetriyl, 5-7 membered heteroarenetriyl, 3-7 membered (hetero)cycloalkanetriyl, 3-7 membered cycloalkenetriyl, and 5-7 membered heterocycloalkenetriyl, wherein when m is 1, then R.sub.3 is selected from the group consisting of CR.sub.6, P, N, benzenetriyl, 5-7 membered heteroarenetriyl, 3-7 membered (hetero)cycloalkanetriyl, 3-7 membered cycloalkenetriyl, and 5-7 membered heterocycloalkenetriyl, wherein the P(O)(OH).sub.2 group is attached to a carbon atom, wherein R.sub.4 is selected from the group consisting of linear or branched C.sub.1-4 alkylene, C.sub.2-4 alkenylene, and C.sub.2-4 alkynylene, wherein R.sub.6 is selected from the group consisting of hydrogen, linear or branched C.sub.1-4 alkyl, and C.sub.2-4 alkenyl, wherein said benzenetriyl, heteroarenetriyl, (hetero)cycloalkanetriyl, and (hetero)cyclocalkenetriyl groups are optionally substituted with at least one group selected from the group consisting of linear or branched C.sub.1-3 alkyl, OCH.sub.3, Cl, Br, F, and I, wherein said alkyl, alkenyl, alkylene, alkenylene, and alkynylene groups are optionally substituted with at least one group selected from the group consisting of ?O, ?NH, Cl, Br, F, and I, and optionally contain a heteroatom selected from the group consisting of N, O, and S.

2. A compound according to claim 1, wherein the compound has a molecular weight of at most 1500 Da.

3. A compound according to claim 1, wherein at least one atom selected from the group consisting of .sup.1H, .sup.12C, .sup.14N, and .sup.16O is replaced with its stable isotope analogue, wherein if .sup.1H is replaced, it is replaced with .sup.2H, wherein if .sup.12C is replaced, it is replaced with .sup.13C, wherein if .sup.14N is replaced, it is replaced with .sup.15N, and if .sup.16O is replaced, it is replaced with .sup.18O.

4. A compound according to claim 1, wherein both R.sub.1 are C(O)ON-succinimide.

5. A compound according to claim 1, wherein the compound has a Log P value in a range of from ?2 to 1.

6. A compound according to claim 1, wherein the compound is selected from the group consisting of ##STR00027## wherein each X is independently selected from the group consisting of CR.sub.6, and N.

7. A method for preparing a compound according to claim 1, said method comprising the step of subjecting a compound according to Formula (2): ##STR00028## to hydrogenation, wherein in Formula (2) R.sub.1, R.sub.2, R.sub.3, R.sub.4, n, and m are defined as for Formula (1).

8. A method for preparing a compound according to claim 1, wherein in said compound each R.sub.1 is independently selected from the group consisting of C(O)Cl, C(O)Br, C(O)F, C(O)O-pentafluorophenyl, ##STR00029## wherein said method comprises the subsequent steps of: a) subjecting a compound according to Formula (3) to reduction so as to obtain a compound according to Formula (4): ##STR00030## wherein R.sub.7 is selected from the group consisting of CH.sub.3 and CH.sub.2CH.sub.3; ##STR00031## b) subjecting the compound according to Formula (4) to oxidation so as to obtain a compound according to Formula (5): ##STR00032## c) activating the carboxylic acids of the compound according to Formula (5) so as to obtain a compound according to Formula (6): ##STR00033## d) subjecting the compound according to Formula (6) to hydrogenation, wherein for Formulae (3) to (6) R.sub.2, R.sub.3, R.sub.4, n, and m are defined as for Formula (1), wherein for Formula (6) each R.sub.1 is independently selected from the group consisting of C(O)Cl, C(O)Br, C(O)F, C(O)O-pentafluorophenyl, ##STR00034## wherein the wiggly lines, R.sub.5 and Q are as defined for Formula (1).

9. A method for crosslinking molecules, said method comprising the step of: a) bringing a compound according to claim 1 into contact with at least one molecule selected from the group consisting of proteins, peptides, and polynucleotides.

10. A method for purifying crosslinked molecules, said method comprising the step of: a) subjecting the mixture obtained in step a) of claim 9 to chromatography.

11. A method according to claim 10, wherein the chromatography is selected from the group consisting of immobilized-metal affinity chromatography and strong anion exchange chromatography.

12. A method for purifying peptides, said method comprising the subsequent steps of: a) bringing a compound according to claim 1 into contact with a solution comprising at least one protein; b) bringing a reducing agent into contact with the solution obtained in step a); c) bringing an alkylating agent into contact with the solution obtained in step b); d) bringing a digesting agent into contact with the solution obtained in step c); e) subjecting the solution obtained in step d) to chromatography.

13. A method according to claim 12, wherein the chromatography method in step e) is selected from the group consisting of immobilized-metal affinity chromatography and strong anion exchange chromatography.

14. A method for characterizing peptides, said method comprising the steps a)-e) according to claim 12, said method further comprising a step f) that is carried out after step e), wherein step f) comprises subjecting the purified compounds obtained under step e) to mass spectrometry.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0048] FIG. 1 depicts a possible explanation for how the phosphonate handle leads to excellent enrichment properties. In blue, the residue of the crosslinker according to the invention is shown, and in yellow a particle within a column with an iron complex bound to it. Panel A depicts how the phosphonate strongly binds to the column in case of a crosslinked peptide. As non-modified peptides do not carry the non-endogenous phosponate moieties these will not bind and simply flow-through during the binding step. Release can however easily be effected with an elution buffer containing 1 to 10% ammonia or other types of buffers like phosphate buffers ensuring an efficient and full release of the bound material. Combined this may explain the higher enrichment for inter- and intra-links when using molecules of the invention.

[0049] FIG. 2 depicts the enrichment efficiency on bovine serum albumin (XL: crosslinked peptides; Mono: mono-linked peptides; Peptide: linear peptides). (a) Not performing enrichment for crosslinked peptides complicates identification of crosslinked products, resulting in low identification rates for crosslinked peptides given their very low abundances compared to those of linear peptides. (b) Measuring the flow-through results in a single identified mono-linked peptide and furthermore only linear peptides, which are dominating the abundances plot. (c) Measuring the eluate results in a drastic decrease in identification and abundance levels for linear peptides. Conversely, the number of identified crosslinked peptides increases dramatically due their elevated abundance levels in the sample.

DETAILED DESCRIPTION OF THE INVENTION

[0050] The invention, in a broad sense, is based on the judicious insight that trifunctional crosslinkers according to Formula (1) possess excellent enrichment properties for inter- and intra-links after crosslinking. This is mainly due to the use of a phosphonate group as an enrichment handle. In compounds of the invention, the R.sub.1 groups are able to react with naturally occurring moieties, such as amines in lysine residues within proteins. As two R.sub.1 groups are present, the compounds of the invention function as a crosslinker. The two R.sub.1 groups combined with the phosphonate group ensure that the compounds of the invention bear three functional groups.

[0051] Without wishing to be bound by theory, it is believed that this improvement in enrichment of inter- and intralinks is achieved as depicted in FIG. 1. The phosphonate has high reactivity towards the IMAC material, while linear peptides do not and will flow-through. Release can easily and highly effectively be effected with a buffer containing 1 to 10% ammonia or other types of buffers like phosphate buffers. It is believed that in this way, a higher enrichment selectivity towards mono, inter- and intralinks is achieved.

[0052] In addition, after enrichment a sample of high purity has been generated from which in a single measurement a large degree of information can be obtained. If more information is required, the sample can be fractionated with techniques like SCX or SEC which are currently employed for crosslinked peptide enrichment due to their property of separating linear and mono-linked peptides from crosslinked peptides. As the linear peptides have been removed in the IMAC enrichment step, the mono-linked peptides can therefore largely be separated from the crosslinked peptides.

[0053] In another aspect compounds according to Formula (1) are small molecules that are able to reach the core of large biomolecules, for example proteins. It was found that the use of a small phosphonate handle is advantageous over other, often bulky affinity tags.

[0054] In another aspect, the molecules of the invention introduce the enrichment handle in one step (i.e. in the crosslinking step). An additional step (e.g. a click reaction) through which the enrichment handle is introduced is not required and a higher yield is achieved.

[0055] Moreover, the phosphonate group enables molecules contacted with a compound according to Formula (1) to be purified using standard chromatography techniques, such as strong anion exchange chromatography and in particular immobilized-metal affinity chromatography. Thus, the molecules and consequent sample handling steps according to the invention are easily implemented in laboratories where such chromatography setups are readily available.

[0056] Furthermore, the phosphonate groups are stable during mass spectrometry analysis, especially during fragmentation. This provides a further advantage over other affinity tags such as phosphates that are labile during that analysis (see P. J. Boersma, S. Mohammed, A. J. R. Heck, Phosphopeptide fragmentation and analysis by mass spectrometry, J. Mass Spect. 2009, volume 44, pages 861-878).

[0057] In another aspect, the phosphonate group is not cleaved off by phosphatases that are typically used to remove phosphate groups from phosphorylated peptides. This dramatically improves the performance for samples with a large degree of phosphorylated peptides. Typically, these heavily phosphorylated peptides are difficult to separate from crosslinked peptides, as the phosphate groups bind to the same column as the phosphonate tag. The entire peptide mixture, comprising phosphorylated peptides, can be treated with phosphatase, selectively removing the phosphate groups that might otherwise interfere with the phosphonate groups during chromatography steps. Hence, the background signal of non-crosslinked phosphorylated peptides is .heavily reduced or even completely removed.

[0058] As yet a further advantage, the molecules of the invention are readily synthesized as depicted in Scheme 1.

Definitions

[0059] The verb to comprise, and its conjugations, as used in this description and in the claims is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0060] 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 is one and only one of the elements. The indefinite article a or an thus usually means at least one.

[0061] The compounds disclosed in this description and in the claims may comprise one or more asymmetric centres, and different diastereomers and/or enantiomers may exist of the compounds. The description of any compound in this description and in the claims is meant to include all diastereomers, and mixtures thereof, unless stated otherwise. In addition, the description of any compound in this description and in the claims is meant to include both the individual enantiomers, as well as any mixture, racemic or otherwise, of the enantiomers, unless stated otherwise. When the structure of a compound is depicted as a specific enantiomer, it is to be understood that the invention of the present application is not limited to that specific enantiomer, unless stated otherwise.

[0062] The compounds may occur in different tautomeric forms. The compounds according to the invention are meant to include all tautomeric forms, unless stated otherwise. When the structure of a compound is depicted as a specific tautomer, it is to be understood that the invention of the present application is not limited to that specific tautomer, unless stated otherwise.

[0063] Unless stated otherwise, the compounds of the invention and/or groups thereof may be protonated or deprotonated. It will be understood that it is possible that a compound may bear multiple charges which may be of opposite sign. For example, in a compound containing an amine and a carboxylic acid, the amine may be protonated while simultaneously the carboxylic acid is deprotonated.

[0064] It is understood that a phosphonate group can be written as PO(OR.sub.a)(OR.sub.b) or PO(OH).sub.2, wherein in both cases the phosphor atom P is attached to a carbon atom that is part of the remainder of the molecule, and wherein R.sub.a and R.sub.b may be alkyl or aryl groups or combinations thereof (and R.sub.a may be the same as R.sub.b).

[0065] It is further understood that the PO(OH).sub.2 group, wherein the phosphor atom P is attached to a carbon atom that is part of the remainder of the molecule, may also be referred to as a phosphonic acid.

[0066] In several formulae, groups or substituents are indicated with reference to letters such as Q or X, and various (numbered) R groups. In addition, the number of repeating units may be referred to with a letter, e.g. q in (CH.sub.2).sub.q. The definitions of these letters are to be read with reference to each formula, i.e. in different formulae these letters, each independently, can have different meanings unless indicated otherwise.

[0067] Herein, cyclic moieties may be preceded by e.g. 3-7 membered, 5-7 membered, and so forth. These numbers refer to the total number of atoms within the cycle. For example, benzene is a 6 membered cycle as the ring consists of six carbon atoms. As another example, tetrahydrofuran is a 5 membered cycle as the ring consists of four carbon atoms and one oxygen atom. A further example is tetrazine, which is a 6 membered cycle as the ring consists of two carbon atoms and four nitrogen atoms.

[0068] Herein, in several chemical formulae and in text reference is made to alkyl, heteroalkyl, aryl, heteroaryl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, arylene, cycloalkyl, cycloalkenyl, cycloakynyl, arenetriyl, and the like. The number of carbon atoms that these groups have, excluding the carbon atoms comprised in any optional substituents as defined below, can be indicated by a designation preceding such terms (e.g. C.sub.1-C.sub.8 alkyl means that said alkyl may have from 1 to 8 carbon atoms). For the avoidance of doubt, a butyl group substituted with a OCH.sub.3 group is designated as a C.sub.4 alkyl, because the carbon atom in the substituent is not included in the carbon count.

[0069] Unsubstituted alkyl groups have the general formula C.sub.nH.sub.2n+1 and may be linear or branched. Optionally, the alkyl groups are substituted by one or more substituents further specified in this document. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl, etc. In preferred embodiments, up to two heteroatoms may be consecutive, such as in for example CH.sub.2NHOCH.sub.3 and CH.sub.2OSi(CH.sub.3).sub.3. In some preferred embodiments the heteroatoms are not directly bound to one another. Examples of heteroalkyls include CH.sub.2CH.sub.2OCH.sub.3, CH.sub.2CH.sub.2NHCH.sub.3, CH.sub.2CH.sub.2S(O)CH.sub.3, CH?CHOCH.sub.3, Si(CH.sub.3).sub.3. In preferred embodiments, a C.sub.1-C.sub.4 alkyl contains at most 2 heteroatoms.

[0070] An alkenyl group comprises one or more carbon-carbon double bonds, and may be linear or branched. Unsubstituted alkenyl groups comprising one CC double bond have the general formula C.sub.nH.sub.2n?1. Unsubstituted alkenyl groups comprising two CC double bonds have the general formula C.sub.nH.sub.2n?3. An alkenyl group may comprise a terminal carbon-carbon double bond and/or an internal carbon-carbon double bond. A terminal alkenyl group is an alkenyl group wherein a carbon-carbon double bond is located at a terminal position of a carbon chain. An alkenyl group may also comprise two or more carbon-carbon double bonds. Examples of an alkenyl group include ethenyl, propenyl, isopropenyl, t-butenyl, 1,3-butadienyl, 1,3-pentadienyl, etc.

[0071] An alkynyl group comprises one or more carbon-carbon triple bonds, and may be linear or branched. Unsubstituted alkynyl groups comprising one CC triple bond have the general formula C.sub.nH.sub.2n?3. An alkynyl group may comprise a terminal carbon-carbon triple bond and/or an internal carbon-carbon triple bond. A terminal alkynyl group is an alkynyl group wherein a carbon-carbon triple bond is located at a terminal position of a carbon chain. An alkynyl group may also comprise two or more carbon-carbon triple bonds. Unless stated otherwise, an alkynyl group may optionally be substituted with one or more, independently selected, substituents as defined below. Examples of an alkynyl group include ethynyl, propynyl, isopropynyl, t-butynyl, etc.

[0072] An aryl group refers to an aromatic hydrocarbon ring system that comprises six to twenty-four carbon atoms, more preferably six to twelve carbon atoms, and may include monocyclic and polycyclic structures. When the aryl group is a polycyclic structure, it is preferably a bicyclic structure. Optionally, the aryl group may be substituted by one or more substituents further specified in this document. Examples of aryl groups are phenyl and naphthyl.

[0073] Preferably, heteroaryl groups comprise five to sixteen carbon atoms and contain between one to five heteroatoms. Preferably, heteroaryl groups comprise at least two carbon atoms (i.e. at least C.sub.2) and one or more heteroatoms N, O, or S. A heteroaryl group may have a monocyclic or a bicyclic structure. Optionally, the heteroaryl group may be substituted by one or more substituents further specified in this document. Examples of suitable heteroaryl groups include pyridinyl, quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl, pyrrolyl, furanyl, triazolyl, benzofuranyl, indolyl, purinyl, benzoxazolyl, thienyl, phospholyl and oxazolyl.

[0074] Herein, the prefix hetero- denotes that the group contains one or more heteroatoms selected from the group consisting of O, N, and S. It will be understood that groups with the prefix hetero- by definition contain heteroatoms. Hence, it will be understood that if a group with the prefix hetero- is part of a list of groups that is defined as optionally containing heteroatoms, that for the groups with the prefix hetero- it is not optional to contain heteroatoms, but is the case by definition.

[0075] When referring to a (hetero)aryl group the notation is meant to include an aryl group and a heteroaryl group. In general, when (hetero) is placed before a group, it refers to both the variant of the group without the prefix hetero- as well as the group with the prefix hetero-.

[0076] Herein, the prefix cyclo- denotes that groups are cyclic. An example of a cyclic group is cyclopropyl.

[0077] Herein, the suffix-ene denotes divalent groups, i.e. that the group is linked to at least two other moieties. An example of an alkylene is propylene (CH.sub.2CH.sub.2CH.sub.2), which is linked to another moiety at both termini. It is understood that if a group with the suffix-ene is substituted at one position with H, then this group is identical to a group without the suffix. For example, an alkylene substituted with H is identical to an alkyl group. I.e. propylene, CH.sub.2CH.sub.2CH.sub.2, substituted with H at one terminus, CH.sub.2CH.sub.2CH.sub.2H, is logically identical to propyl, CH.sub.2CH.sub.2CH.sub.3.

[0078] Herein, the suffix-triyl denotes trivalent groups, i.e. that the group is linked to at least three other moieties. An example of a benzenetriyl is depicted below:

##STR00004##

wherein the wiggly lines denote bonds to different groups of the main compound.

[0079] It is understood that if a group, for example an alkyl group, contains a heteroatom, then this group is identical to a hetero-variant of this group. For example, if an alkyl group contains a heteroatom, this group is identical to a heteroalkyl group. Similarly, if an aryl group contains a heteroatom, this group is identical to a heteroaryl group. It is understood that contain and its conjugations mean herein that when a group contains a heteroatom, this heteroatom is part of the backbone of the group. For example, a C.sub.2 alkylene containing an N refers to NHCH.sub.2CH.sub.2, CH.sub.2NHCH.sub.2, and CH.sub.2CH.sub.2NH.

[0080] If indicated that a group (optionally) contains a heteroatom, the group may contain a heteroatom at non-terminal positions or at one or more terminal positions. In this case, terminal refers to the terminal position within the group, and not necessarily to the terminal position of the entire compound. For example, if an ethylene group contains a nitrogen atom, this may refer to NHCH.sub.2CH.sub.2, CH.sub.2NHCH.sub.2, and CH.sub.2CH.sub.2NH. For example, if an ethyl group contains a nitrogen atom, this may refer to NHCH.sub.2CH.sub.3, CH.sub.2NHCH.sub.3, and CH.sub.2CH.sub.2NH.sub.2.

[0081] Herein, it is understood that cyclic compounds (i.e. aryl, cycloalkyl, cycloalkenyl, etc.) are understood to be monocyclic, polycyclic or branched. It is understood that the number of carbon atoms for cyclic compounds not only refers to the number of carbon atoms in one ring, but that the carbon atoms may be comprised in multiple rings. These rings may be fused to the main ring or substituted onto the main ring.

[0082] Unless stated otherwise, any group disclosed herein that is not cyclic is understood to be linear or branched. In particular, (hetero)alkyl groups, (hetero) alkenyl groups, (hetero)alkylene groups, (hetero)alkenylene groups, (hetero)alkynylene groups, and the like are linear or branched, unless stated otherwise.

[0083] The term protein is herein used in its normal scientific meaning. Herein, polypeptides comprising about 10 or more amino acids are considered proteins. A protein may comprise natural, but also unnatural amino acids. The term protein herein is understood to comprise antibodies and antibody fragments.

[0084] The term peptide is herein used in its normal scientific meaning. Herein, peptides are considered to comprise a number of amino acids in a range of from 2 to 9.

[0085] The term polynucleotide is herein used in its normal scientific meaning. Polynucleotides may for example be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Unless indicated otherwise, polynucleotides as disclosed herein can be single-stranded or double-stranded. Herein, polynucleotides are understood to comprise at least 13 nucleotides.

[0086] The term activated carboxylic acid herein refers to a carboxylic acid moiety (i.e. COOH) that has been modified in such a way that the poor leaving group OH has been replaced with a good leaving group. In particular, activated carboxylic acids as used herein may refer to moieties selected from the group consisting of C(O)Cl, C(O)Br, C(O)F, C(O)O-pentafluorophenyl,

##STR00005## [0087] wherein R.sub.5 and Q are as defined for Formula (1).

[0088] The compounds of the invention may be present as a salt, unless explicitly stated otherwise.

[0089] The term salt thereof means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation and the like.

[0090] Salt refers to salts derived from a variety of organic and inorganic counter ions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.

[0091] The logarithm of the partition-coefficient, i.e. Log P, is herein used as a measure of the hydrophobicity of a compound. Typically, the Log P is defined as

[00001]$\log \left(\frac{{\left[\mathrm{Solute}\right]}_{\mathrm{octanol}}^{\mathrm{un}-\mathrm{ionized}}}{{\left[\mathrm{Solute}\right]}_{\mathrm{water}}^{\mathrm{un}-\mathrm{ionized}}}\right)$

The skilled person is aware of methods to determine the partition-coefficient of compounds without undue experimentation. Alternatively, the skilled person knows that software is available to reliably estimate the Log P value, for example as a function within ChemDraw? software or online available tools.

[0092] The unified atomic mass unit or Dalton is herein abbreviated to Da. The skilled person is aware that Dalton is a regular unit for molecular weight and that 1 Da is equivalent to 1 g/mol (grams per mole).

[0093] It will be understood that herein, the terms moiety and group are used interchangeably when referring to a part of a molecule.

[0094] It will be understood that when a heteroatom is denoted as X(R).sub.2, wherein X is the heteroatom and R is a certain moiety, then this denotes that two moieties R are attached to the heteroatom.

Embodiments

[0095] The compounds of the invention satisfy Formula (1):

##STR00006## [0096] wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, m, and n are defined as disclosed herein.

[0097] In Formula (1), m is 0 or 1.

[0098] In Formula (1), each n is independently 0 or 1.

[0099] In Formula (1), the P(O)(OH).sub.2 group is attached to a carbon atom.

[0100] In Formula (1), each R.sub.1 is independently selected from the group consisting of C(O)H, C(O)Cl, C(O)Br, C(O)F, C(O)O-pentafluorophenyl, NCS, NCO, S(O).sub.2Cl, S(O).sub.2F, phenylsulfonyl fluoride, phenylsulfonyl chloride, C(O)N.sub.3, C(O)OC(O)CH.sub.3, OC(O)O(CH.sub.2).sub.qCH.sub.3, C(NH)O(CH.sub.2).sub.qCH.sub.3, C.sub.2 epoxidyl,

##STR00007## [0101] wherein the wiggly lines indicate a bond to R.sub.2 or to R.sub.3. Preferably, both R.sub.1 are C(O)ON-succinimide.

[0102] In Formula (1), one Q is a nitrogen atom and the other Q are CH.

[0103] In Formula (1), each R.sub.2 is independently selected from the group consisting of C.sub.1-4 alkylene, C.sub.2-4 alkenylene, C.sub.2-4 alkynylene, S(O)CH.sub.2(CH.sub.2).sub.q, (CH.sub.2).sub.qSS(CH.sub.2).sub.q, N((CH.sub.2).sub.qC(O)H)C(O)N((CH.sub.2).sub.qC(O)H), -aspartyl-prolyl, and -valyl-prolyl-.

[0104] In preferred embodiments, R.sub.2 is a C.sub.1-4 alkylene, preferably methylene, ethylene, propylene, or butylene.

[0105] In Formula (1), each q is independently an integer in a range of from 0 to 6. In some embodiments, each q is independently an integer in a range of from 0 to 5, more preferably in a range of from 0 to 4, even more preferably in a range of from 0 to 3, more preferably still in a range of from 0 to 2, most preferably each q is independently 0 or 1.

[0106] In preferred embodiments, R.sub.2 is a gas-phase cleavable linker selected from the group consisting of S(O)CH.sub.2(CH.sub.2).sub.q, (CH.sub.2).sub.qSS(CH.sub.2).sub.q, N((CH.sub.2).sub.qC(O)H)C(O)N((CH.sub.2).sub.qC(O)H), -aspartyl-prolyl, and -valyl-prolyl-. Gas-phase cleavable linkers are advantageous as they provide additional tools to obtain distinguishing ions when different types of gas phase fragmentation methods are used, and/or different levels of tandem mass spectrometry (such as MS2 and MS3). Thus, gas-phase cleavable linkers are able to improve the characterization of crosslinked proteins and peptides. Examples of such linkers are for instance described in A. Kao, et al, Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes, Molecular and Cellular Proteomics, 2011; M Q M?ller, F. Dreiocker, C. H. Ihling, M. Sch?fer;, A. Sinz, Cleavable cross-linker for protein structure analysis: reliable identification of cross-linking products by tandem MS, Analytical Chemistry, 2010; and A. S. Argo, C. Shi, F. Liu, M. B. Goshe, Performing protein crosslinking using gas-phase cleavable chemical crosslinkers and liquid chromatography-tandem mass spectrometry, Methods 2015, volume 89, pages 64-73.

[0107] In Formula (1), when m is 0, then R.sub.3 is selected from the group consisting of CR.sub.6, benzenetriyl, 5-7 membered heteroarenetriyl, 3-7 membered (hetero)cycloalkanetriyl, 3-7 membered cycloalkenetriyl, and 5-7 membered heterocycloalkenetriyl.

[0108] When m is 1, then R.sub.3 is selected from the group consisting of CR.sub.6, P, N, benzenetriyl, 5-7 membered heteroarenetriyl, 3-7 membered (hetero)cycloalkanetriyl, and 3-7 membered cycloalkenetriyl, and 5-7 membered heterocycloalkenetriyl.

[0109] Preferred 5-7 membered heteroarenetriyl groups for R.sub.3 are pyridinetriyl, pyrimidinetriyl, triazinetriyl, pyrazinetriyl, furantriyl, pyrroletriyl, imidazoletriyl, thiophenetriyl, oxazoletriyl, thiazoletriyl, and triazoletriyl groups. Preferably, heteroarenetriyl groups for R.sub.3 are 5-6 membered.

[0110] Preferred 3-7 membered cycloalkanetriyl groups for R.sub.3 are cyclopropanetriyl, cyclobutanetriyl, cyclopentanetriyl, cyclohexanetriyl, and cycloheptanetriyl groups.

[0111] Preferred 3-7 membered heterocycloalkanetriyl groups for R.sub.3 are aziridinetriyl, azetidinetriyl, oxetanetriyl, 1,3-oxazetidinetriyl, tetrahydrofurantriyl, pyrrolidinetriyl, imidazolidinetriyl, piperidinetriyl, piperazinetriyl, morpholinetriyl, thiomorpholinetriyl, oxathianetriyl, oxanetriyl, dioxanetriyl, thianetriyl, dithianetriyl, thiepanetriyl, oxepanetriyl, azepanetriyl, oxazepanetriyl, oxathiepanetriyl, thiazepanetriyl, dithiepanetriyl, dioxepanetriyl, and diazepanetriyl groups.

[0112] Preferred 3-7 membered cycloalkenetriyl groups for R.sub.3 are cyclopropenetriyl, cyclobutenetriyl, cyclopentenetriyl, cyclopentadienetriyl, cyclohexenetriyl, cyclohexadienetriyl, cycloheptenetriyl, cycloheptadienetriyl, and cycloheptatrienetriyl groups.

[0113] Preferred 5-7 membered heterocycloalkenetriyl groups for R.sub.3 are dihydrofurantriyl, dihydrothiophenetriyl, pyrrolinetriyl, tetrahydropyridinetriyl, dihydropyridinetriyl, tetrahydropyrimidinetriyl, dihydrooxazinetriyl, dihydrothiazinetriyl, dihydrothiopyrantriyl, thiopyrantriyl, dithiinetriyl, oxathiinetriyl, dihydropyrantriyl, pyrantriyl, dioxinetriyl, tetrahydroazepinetriyl, tetrahydrooxepinetriyl, tetrahydrooxazepinetriyl, tetrahydrothiazepinetriyl, tetrahydrothiepinetriyl, dihydrodioxepinetriyl, dihydrodithiepinetriyl, tetrahydrodiazepinetriyl, dihydrooxathiepinetriyl, dihydroazepinetriyl, dihydrodiazepinetriyl, dihydrotriazepinetriyl, dihydrooxazepinetriyl, dihydrothiazepinetriyl, dioxepinetriyl, dihydrooxepinetriyl, azepinetriyl, thiazepinetriyl, thiepinetriyl, dihydrothiepinetriyl, dithiepinetriyl, oxathiazepinetriyl, and oxazepinetriyl.

[0114] Regardless of whether m is 0 or 1, R.sub.3 is preferably benzenetriyl or pyrroletriyl.

[0115] In Formula (1), R.sub.4 is selected from the group consisting of linear or branched C.sub.1-4 alkylene, C.sub.2-4 alkenylene, and C.sub.2-4 alkynylene. Preferably, R.sub.4 is C.sub.1-4 alkylene, and more preferably R.sub.4 is C.sub.1-2 alkylene (i.e. methylene or ethylene).

[0116] In Formula (1), R.sub.5 is selected from the group consisting of H, CH.sub.3, CH.sub.2CH.sub.3, OCH.sub.2OCH.sub.3, OCH.sub.2O(CH.sub.2).sub.2Si(CH.sub.3).sub.3, SO.sub.3H, and OCH.sub.2O(CH.sub.2).sub.2OCH.sub.3. Preferably, R.sub.5 is H.

[0117] In Formula (1), R.sub.6 is selected from the group consisting of hydrogen, linear or branched C.sub.1-4 alkyl, and C.sub.2-4 alkenyl. Preferably, R.sub.6 is selected from the group consisting of hydrogen, methyl, and ethyl.

[0118] In Formula (1), the benzenetriyl, heteroarenetriyl, (hetero)cycloalkanetriyl, and (hetero)cyclocalkenetriyl groups are optionally substituted with at least one group selected from the group consisting of linear or branched C.sub.1-3 alkyl, OCH.sub.3, Cl, Br, F, and I.

[0119] Preferably, the benzenetriyl, heteroarenetriyl, (hetero)cycloalkanetriyl, and (hetero)cyclocalkenetriyl groups are optionally substituted with at most three, more preferably at most two, most preferably at most one group selected from the group consisting of linear or branched C.sub.1-3 alkyl, OCH.sub.3, Cl, Br, F, and I.

[0120] In Formula (1), the alkyl, alkenyl, alkylene, alkenylene, and alkynylene groups are optionally substituted with at least one group selected from the group consisting of ?O, ?NH, Cl, Br, F, and I, and optionally contain a heteroatom selected from the group consisting of N, O, and S.

[0121] Preferably, the alkyl, alkenyl, alkylene, alkenylene, and alkynylene groups are optionally substituted with at most three, preferably at most two, more preferably at most one group selected from the group consisting of ?O, ?NH, Cl, Br, F, and I.

[0122] Preferably, the alkyl, alkenyl, alkylene, alkenylene, and alkynylene groups optionally contain at most two, more preferably at most one heteroatom selected from the group consisting of N, O, and S.

[0123] The compounds according to Formula (1) preferably have a molecular weight of at most 1500 Da. In preferred embodiments, the compounds according to Formula (1) have a molecular weight of at most 1300 Da, more preferably at most 1100 Da, even more preferably at most 900 Da.

[0124] In the compounds according to Formula (1), at least one atom selected from the group consisting of .sup.1H, .sup.12C, .sup.14N, and .sup.16O may be replaced with its stable isotope analogue. If .sup.1H is replaced, it is replaced with .sup.2H. If .sup.12C is replaced, it is replaced with .sup.13C. If .sup.14N is replaced, it is replaced with .sup.15N. If .sup.16O is replaced, it is replaced with .sup.18O.

[0125] Compounds according to Formula (1) preferably have a Log P value in a range of from ?2 to 1. A compound with a Log P value in this range has the advantage that it is well-soluble in aqueous solutions and has improved chance to reach the hydrophobic core of the protein.

[0126] In preferred embodiments, compounds according to Formula (1) are selected from the group consisting of

##STR00008##

wherein each X is independently selected from the group consisting of CR.sub.6, and N. Preferably, at most two X are N, more preferably at most one X is N. In a preferred embodiment, at least two X are CH. More preferably, all X are CR.sub.6, and even more preferably all X are CH.

[0127] In other preferred embodiments, the compound according to Formula (1) is selected from the group consisting of

##STR00009##

[0128] The invention further relates to methods for preparing compounds according to Formula (1). In a broad sense, these methods comprising the step of subjecting a compound according to Formula (2):

##STR00010##

to hydrogenation, wherein in Formula (2) R.sub.1, R.sub.2, R.sub.3, R.sub.4, n, and m are defined as for Formula (1).

[0129] In one embodiment, the method for preparing a compound according to Formula (1) relates to the preparation of compounds wherein each R.sub.1 is independently selected from the group consisting of C(O)Cl, C(O)Br, C(O)F, C(O)O-pentafluorophenyl,

##STR00011##

In this embodiment, the method comprises the subsequent steps of: [0130] a) subjecting a compound according to Formula (3) to reduction so as to obtain a compound according to Formula (4):

##STR00012## [0131] wherein R.sub.7 is selected from the group consisting of CH.sub.3 and CH.sub.2CH.sub.3;

##STR00013## [0132] b) subjecting the compound according to Formula (4) to oxidation so as to obtain a compound according to Formula (5):

##STR00014## [0133] c) activating the carboxylic acids of the compound according to Formula (5) so as to obtain a compound according to Formula (6):

##STR00015## [0134] d) subjecting the compound according to Formula (6) to hydrogenation. For Formulae (3) to (6) R.sub.2, R.sub.3, R.sub.4, n, and m are defined as for Formula (1). For Formula (6) each R.sub.1 is independently selected from the group consisting of C(O)Cl, C(O)Br, C(O)F, C(O)O-pentafluorophenyl,

##STR00016## [0135] wherein the wiggly lines, R.sub.5 and Q are as defined for Formula (1).

[0136] The methods for synthesizing compounds according to Formula (1) are based on the judicious insight to use benzyl (Bn in Formulae (2) to (6)) groups as protecting groups for the phosphonate moiety. This is advantageous, as benzyl groups are compatible with protecting groups for other moieties. Especially in the synthesis of compounds according to the invention wherein R.sub.1 is an activated carboxylic acid, this strategy is particularly advantageous. It was found that in contrast with other phosphonate protecting strategies, the benzyl protection is fully compatible with the reduction/oxidation/activation strategy as described herein to synthesize the activated carboxylic acids.

[0137] An alternative synthesis route, wherein the esters in Formula (3) are first hydrolyzed proved unsuccessful. The benzyl group, like other phosphonate protecting groups, turned out to be unstable under these hydrolysis conditions. Therefore, the judicious insight to successfully synthesize compounds according to the invention by using a benzyl protecting group and the reduction/oxidation/activation strategy as described herein was surprisingly advantageous.

[0138] The invention also pertains to methods for crosslinking molecules. Methods for crosslinking molecules according to the invention comprise the step of bringing a compound according to Formula (1) into contact with at least one molecule selected from the group consisting of proteins, peptides, and polynucleotides.

[0139] Without wishing to be bound by theory, it is believed that the compounds according to Formula (1) mainly react with nucleophilic groups such as amines, thiols, and hydroxyl groups. Depending on the nature of the R.sub.1 group of the compound according to Formula (1), reactivity towards specific groups can be achieved.

[0140] Furthermore, the invention is related to methods for purifying molecules crosslinked by a method of the invention. This method for purifying crosslinked molecules comprises the step of subjecting the mixture obtained by crosslinking molecules by a method of the invention to chromatography.

[0141] The invention also relates to methods for purifying peptides. These methods comprise the subsequent steps of: [0142] a) bringing a compound according to Formula (1) into contact with a solution comprising at least one protein; [0143] b) bringing a reducing agent into contact with the solution obtained in step a); [0144] c) bringing an alkylating agent into contact with the solution obtained in step b); [0145] d) bringing a digesting agent into contact with the solution obtained in step c); [0146] e) subjecting the solution obtained in step d) to chromatography.

[0147] The skilled person is aware of suitable reducing agents, alkylating agents, and digesting agents for use in methods of the invention. Similarly, the skilled person is able to use his general knowledge about the art to select suitable buffers for the different steps of the procedures. Likewise, if required, the skilled person is aware of methods known in the art to change buffer compositions such as centrifugation, dialysis, exchange chromatography, and the like.

[0148] In preferred embodiments, any one of the steps a)-d) in the method for purifying peptides further comprises contacting the solution with a phosphatase. Most preferably, the solution obtained in step d) is contacted with a phosphatase in a separate step d2 before proceeding to step e), amounting to a method comprising the steps of [0149] a) bringing a compound according to Formula (1) into contact with a solution comprising at least one protein; [0150] b) bringing a reducing agent into contact with the solution obtained in step a); [0151] c) bringing an alkylating agent into contact with the solution obtained in step b); [0152] d) bringing a digesting agent into contact with the solution obtained in step c); [0153] d2) bringing a phosphatase into contact with the solution obtained in step d) [0154] e) subjecting the solution obtained in step d2) to chromatography.

[0155] Preferably, the reducing agent is selected from the group consisting of tris-(2-carboxyethyl)phosphine, thiol-containing compounds, and combinations thereof. Preferred thiol-containing compounds are selected from the group consisting of dithiothreitol, 2-mercaptoethanol, 2-mercaptoethylamine, and cysteine.

[0156] Preferably, the alkylating agent is selected from the group consisting of iodoacetamide, chloroacetamide, C.sub.2-11 alkanal, and combinations thereof. Preferably, the alkylating agent is iodoacetamide.

[0157] Digestion of peptides and proteins can be performed using proteases or chemical agents known to the skilled person.

[0158] Preferably, digesting agents are proteases selected from the group consisting of ArgC proteinase, AspN endopeptidase, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, chymotrypsin, clostripain, enterokinase, coagulation factor Xa, glutamyl endopeptidase, granzyme B, lysyl endopeptidase, peptidyl-Lys metalloendopeptidase, neutrophil elastase, pepsin, proline endopeptidase, proteinase K, staphylococcal peptidase I, tobacco etch virus protease, thermolysin, thrombin, trypsin, and combinations thereof. A preferred protease is trypsin. The skilled person is aware of the conditions under which these proteases function.

[0159] For the digestion of polynucleotides, preferably deoxyribonucleases (DNases) or ribonucleases (RNases) are employed. These can be either exonucleases (e.g. any one of exonucleases I, II, III, IV, V, VI, VII, or VIII) or endonucleases (e.g. DNase I).

[0160] Alternatively, digestion is performed by contacting the mixture with a chemical agent. Preferred chemical agents for digestion are selected from the group consisting of formic acid, 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole), cyanogen bromide, hydroxylamine, iodosobenzoic acid, and 2-nitro-5-thiocyanatobenzoic acid. The preferred chemical agent for digestion is formic acid. When applying the chemical agent for digestion, in particular formic acid, the mixture is held at elevated temperatures, such as 80? C., or the mixture is heated up in a microwave.

[0161] The invention also pertains to methods for characterizing peptides. These methods comprise the steps a)-e) as described above for the method for purifying peptides, and further comprise a step f) that is carried out after step e). Step f) comprises subjecting the purified compounds obtained under step e) to mass spectrometry.

[0162] Preferred mass spectrometry methods are those selected from the group consisting of those using high resolution mass spectrometry like, but not restricted to Orbitrap or high resolution time-of-flight (TOF) instruments. Preferably, one mass selection device like, but not restricted to quadrupole or ion trap is incorporated. Furthermore, a high efficiency peptide fragmentation device is preferably incorporated like, but not limited to collisional activation like collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), electron activation like electron-transfer dissociation (ETD) and electron-induced dissociation (EID), ultraviolet (UV) or CO.sub.2 photodissociation (PD) needs to be incorporated.

[0163] During data acquisition, two types of methods are distinguished. For non gas-phase cleavable crosslinking reagents each precursor can be isolated and fragmented prior to recording in the mass spectrometer. In the case of collisional fragmentation it is advantageous to have the option for the isolated crosslinked peptide pair to undergo multiple rounds of activation at different energies to ensure optimal sequence coverage for both peptides. For gas-phase cleavable crosslinking reagents it is advantageous to do a tandem mass spectrometry, more specifically an MS3 experiment, where the crosslinked peptide pairs are separated from each other by a fragmentation step followed by isolation and fragmentation of each of the peptides. Alternatively, the peptide sequence information can also be read out in a single or several fragmentation steps on the crosslinked peptide pair ions.

[0164] In any of the methods of the invention comprising a chromatography step, the chromatography is preferably selected from the group consisting of immobilized-metal affinity chromatography and strong anion exchange chromatography.

[0165] In a preferred embodiment, the chromatography method chosen in any method of the invention comprising a chromatography step is immobilized-metal affinity chromatography.

[0166] In preferred embodiments, immobilized-metal affinity chromatography is performed using particles comprising a metal selected from the group consisting of TiO.sub.2, Ti.sup.4+, Fe.sup.3+, Ga.sup.3+, Al.sup.3+, Zr.sup.4+, and combinations thereof. In a particularly favorable embodiment, particles comprising Ti.sup.4+ are used for immobilized-metal affinity chromatography.

[0167] In preferred embodiments, immobilized-metal affinity chromatography is performed using particles with a size in a range of from 4 ?m to 20 ?m. In a particularly favorable embodiment, particles with a size in a range of from 14 ?m are used for strong anion exchange chromatrography.

[0168] In another embodiment, the chromatography method chosen in any method of the invention comprising a chromatography step is strong anion exchange chromatography.

[0169] In preferred embodiments, strong anion exchange chromatography is performed using particles comprising a moiety selected from the group consisting of trialkyl ammonium and trialkylbenzyl ammonium. In a particularly favorable embodiment, strong anion exchange chromatography is performed using particles comprising a moiety selected from the group consisting of trimethylbenzyl ammonium, dimethyl-2-hydroxyethylbenzyl ammonium, and dimethyl-2-hydroxyethylbenzyl ammonium particles are used.

[0170] More preferably, strong anion exchange chromatography is performed using particles comprising polystyrene. In another preferred embodiment, strong anion exchange chromatography is performed using particles comprising Amberlite?. In yet another preferred embodiment, strong anion exchange chromatography is performed using particles comprising Dowex?. Herafter, the invention is further illustrated with non-limiting examples.

EXAMPLES

Example 1: Synthesis of a Compound According to Formula (1)

[0171] Scheme 1 depicts the general synthetic route towards compound (6), which is a compound according to the invention. It also provides a crystal structure of compound (6).

##STR00017## ##STR00018##

[0172] Below, the experimental details of each step of the synthesis in Scheme 1 are provided.

Example 1.1: Synthesis of dimethyl 5-(diphenoxyphosphoryl)isophthalate (2)

##STR00019##

[0173] TsOH.Math.H.sub.2O (4.4 g, 22.6 mmol, 1.2 eq) was dissolved in 200 mL of MeCN (HPLC grade). A slurry of Arylamine (1) (4 g, 18.9 mmol, 1 eq) in 50 mL MeCN was added and a white precipitate formed. The turbid solution was cooled to 0? C., tBuONO (3.4 mL, 28.6 mmol, 1.5 eq.) was added, and the reaction mixture was stirred at 0? C. for 30 min, followed by the addition of P(OPh).sub.3 (12.6 g, 57.2 mmol, 3 eq). The resulting reaction solution was stirred for 8 h at room temperature. During this time the reaction mixture became a clear orange-reddish solution. The solution was then concentrated under reduced pressure, and the crude residue was purified by silica gel column chromatography (PE/EtOAc 6:1) to afford product (2) (5.6 g, 13.2 mmol, 70%) as a red to pink solid.

[0174] HRMS (m/z): [M+H].sup.+ calcd. for C.sub.22H.sub.20O.sub.7P, 427.0941; found, 427.0967

[0175] .sup.1H NMR (400 MHz, CDCl.sub.3) ?8.89 (d, J=1.3 Hz, 1H, ArH), 8.83 (d, J=1.6 Hz, 1H, ArH), 8.79 (d, J=1.6 Hz, 1H, ArH), 7.37-7.27 (m, J=18.1, 9.8 Hz, 5H, PhH), 7.23-7.13 (m, 5H, PhH), 3.97 (s, 6H, 2?CH.sub.3).

[0176] .sup.31P NMR (162 MHz, CDCl.sub.3) ?8.48 (s).

Example 1.2: Synthesis of dimethyl 5-(bis(benzyloxy)phosphoryl)isophthalate (3)

##STR00020##

[0177] Compound (2) (899 mg, 2.10 mmol) was dissolved in dry THF and the solution was cooled to 0? C. BnOH (479 ?L, 4.66 mmol, 2.2 eq) and NaH (199 mg, 8.32 mmol, 4 eq.) was added and the reaction mixture was stirred for 3 h at 0? C. After stopping the reaction by the addition on MeOH, the reaction mixture was diluted with DCM and washed with brine (2?50 mL). The organic layer was dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo. The crude product was purified by column chromatography (PE/EtOAc 4:1 to 2:1) to obtain (3) as a colorless oil (400 mg, 0.880 mmol, 42%).

[0178] HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.24H.sub.23O.sub.7PNa, 477.1097; found, 477.1103.

[0179] .sup.1H NMR (600 MHz, CDCl.sub.3) ?8.80 (s, 2H, ArH), 8.59 (d, J=3.4 Hz, 1H, ArH), 8.57 (d, J=1.4 Hz, 1H, ArH), 7.35-7.26 (m, 10H, BnH), 5.17-5.04 (m, 4H, 2?CH.sub.2), 3.94 (s, 6H, CH.sub.3).

[0180] .sup.13C NMR (151 MHz, CDCl.sub.3) ?165.27 (C?O), 136.84, 136.77 (CP), 135.70 (CAr), 135.66 (CAr), 134.29 (CAr), 134.3 (CAr), 131.14 (CAr), 131.04 (CAr), 128.67 (CAr), 128.23 (CAr), 68.28, 68.24 (CH.sub.2Ar), 52.7 (CH.sub.3)

[0181] .sup.31P NMR (162 MHz, CDCl.sub.3) ?16.65 (s).

Example 1.3: Synthesis of 5-(Bis(benzyloxy)phosphoryl)isophthalic acid (4a)

##STR00021##

[0182] Compound (3) (390 mg, 0.859 mmol) was dissolved in DCM and the solution was cooled to 0? C. DIBALH (1M in THF, 3.43 mL, 3.43 mmol, 4 eq.) was added dropwise and the reaction mixture was stirred at 0? C. until TLC showed complete conversion of the starting material. The reaction was stopped by addition of MeOH. The crude reaction mixture was diluted by DCM (50 mL) and washed with brine (2?50 mL). After purification by column chromatography compound (4a) was obtained as a colourless oil (171 mg, 0.43 mmol, 50%).

[0183] HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.22H.sub.23O.sub.5PNa, 421.1181; found, 421.1200.

[0184] .sup.1H NMR (600 MHz, CDCl.sub.3) ?7.68 (s, 1H, ArH), 7.64 (s, 1H, ArH), 7.56 (s, 1H, ArH), 7.32 (s, 10H, BnH), 5.12-5.01 (m, 4H, 2?CH.sub.2), 4.69 (s, 4H, 2?CH.sub.2).

[0185] .sup.31P NMR (162 MHz, CDCl.sub.3) ?19.38(s).

Example 1.4: Synthesis of 5-(Bis(benzyloxy)phosphoryl)isophthalic acid (4b)

##STR00022##

[0186] For the preparation of Jones reagent, CrO.sub.3 (0.8 g, 8 mmol) was mixed with H.sub.2SO.sub.4 (0.85 mL, 16 mmol) at 0? C. H.sub.2O (2.5 mL) was carefully added and the mixture stirred for 15 min until a dark red color remained. Alcohol (4a) (20 mg, 0.05 mmol) was dissolved in 4 mL acetone (technical grade) and Jones reagent was added at 0? C. until the color remained red. After stirring for 3 h, TLC analysis showed complete conversion of the starting material. Isopropanol was added and the reaction color turned from green to blue. H.sub.2O was added and the aqueous phase was extracted with EtOAc, washed with brine and the combined organic phases were dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Compound (4b) was obtained without further purification was a colorless oil (21 mg, 0.05 mmol, quantitative).

[0187] HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.22H.sub.19O.sub.7PNa, 449.0786; found, 449.0766.

[0188] .sup.1H NMR (600 MHz, MeOD) ?8.75 (s, 1H, ArH), 8.49 (s, 1H, ArH), 8.45 (s, 1H, ArH), 7.33-7.30 (m, 10H, BnH), 5.12-5.01 (m, 4H, 2?CH.sub.2).

[0189] .sup.31P NMR (162 MHz, MeOD) ?19.94 (s).

Example 1.5: Synthesis of Bis(2,5-dioxopyrrolidin-1-yl) 5-(bis(benzyloxy)phosphoryl)isophthalate (5)

##STR00023##

[0190] Compound (4b) (27 mg, 0.063 mmol, 1 eq) was dissolved in pyridine (technical grade) and NHS (20 mg, 0.173 mmol, 2.5 eq) was added. POCl.sub.3 (15 ?L, 0.160 mmol, 2.7 eq) was added dropwise at 0? C. and immediately a precipitate formed. The reaction mixture was stirred for 30 mins more at 0? C. and the reaction stopped by the dropwise addition of ice-cold H.sub.2O. The reaction mixture was extracted with EtOAc (2?), the combined organic phases were dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo. The crude product was purified by column chromatography (DCM/MeOH 50:1 to 30:1) to yield NHS-ester (5) as a colourless oil (25 mg, 0.040 mmol, 63%).

[0191] .sup.1H NMR (600 MHz, CDCl.sub.3) ?8.90 (s, 1H, ArH), 8.66 (dd, J=13.4, 1.3 Hz, 2H, ArH), 7.32 (s, 11H, ArH), 5.19-5.07 (m, 5H, 2?CH.sub.2), 2.93 (s, 8H, 4?CH.sub.2).

[0192] .sup.31P NMR (162 MHz, CDCl.sub.3) ?14.36 (s).

Example 1.6: (3,5-Bis(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)phosphonic acid (6)

##STR00024##

[0193] Compound (5) (11 mg, 0.017 mmol) was dissolved in dry THF. The solution was degassed for 30 min by bubbling H.sub.2 through the solution. Pd/C (5 mol %) was added and the reaction was set under an H.sub.2-atmosphere using a balloon filled with H.sub.2-gas. After stirring for 40 min at room temperature, the reaction mixture was filtered over celite and the solvent was removed in vacuo. The crosslinker (6) could thereby be obtained without further purification as a colorless oil (7.4 mg, 0.017 mmol, quantitative).

[0194] HRMS (m/z): [M+H].sup.+ calcd. for C.sub.16H.sub.14N.sub.2O.sub.11PNa, 441.0330; found, 441.0390.

[0195] .sup.1H NMR (600 MHz, MeOD) ?8.92 (s, 1H, ArH), 8.83 (d, J=12.7 Hz, 2H, 2?ArH), 2.93 (s, 8H, 4?CH.sub.2).

[0196] .sup.31P NMR (162 MHz, CDCl.sub.3) ?14.36 (s).

Example 2: Crosslinking Protein Using a Compound According to Formula (1) and Digestion of the Crosslinked Protein

[0197] In Scheme 2, a workflow for the crosslinking, processing and characterization of proteins is depicted. Examples 2-5 describe one or several steps of these processes.

[0198] A crosslinking reagent according to formula (1) (i.e. compound (6)) was freshly dissolved at a concentration of 20 mM in anhydrous DMSO. The protein mixture was dissolved at a concentration of 1 mg/mL in an amine-free buffer-system. Compound (6) was added to the protein mixture at a final concentration of 2 mM and the mixture was incubated at room temperature for 45 minutes. The crosslinking reaction was quenched by addition of Tris.Math.HCl (100 mM, pH 8) to a final concentration of 10 mM. Residual crosslinking reagent was removed by size-cut-off filters (Vivaspin 500K 10 kDa MWCO centrifugal filter units) with 3 volumes of Tris HCl (50 mM, pH 8). The crosslinked protein mixture (in 50 mM Tris HCl, pH 8) was reduced with DTT (final concentration of 2 mM) for 30 min at 37? C., followed by alkylation with IAA (final concentration of 4 mM) for 30 min at 37? C. This reaction was quenched by addition of DTT (final concentration of 2 mM). Then the sample was digested by incubation with LysC (1:75 enzyme to protein) and Trypsin (1:50 enzyme to protein) for 10 h at 37? C., after which formic acid (1%) was added to quench the digestion. Finally, peptides were desalted by C.sub.18 Seppak prior to both IMAC enrichment as well as LC-MS analysis.

Example 3: Purifying Peptides Derived from a Protein Crosslinked with a Compound According to Formula (1)

[0199] Crosslinked peptides were enriched with Fe(III)-NTA 5 ?L in an automated fashion using the AssayMAP Bravo Platform (Agilent Technologies; Santa Clara, Ca). Fe(III)-NTA cartridges were primed with 250 ?L of 0.1% TFA in ACN and equilibrated with 250 ?L of loading buffer (80% ACN/0.1% TFA). Samples were dissolved in 200 ?L of loading buffer and loaded onto the cartridge. The columns were washed with 250 ?L of loading buffer, and the crosslinked peptides were eluted with 25 ?L of 10% ammonia directly into 25 ?L of 10% formic acid. Samples were dried down and stored in 4? C. until subjected to LC-MS/MS. For LC-MS/MS analysis the samples were resuspended in 10% formic acid.

Example 4: Mass Spectrometry Analysis of Purified Peptides Crosslinked with a Compound According to Formula (1)

[0200] Data were acquired using an UHPLC 1290 system (Agilent Technologies; Santa Clara, Ca) coupled on-line to an Orbitrap Fusion mass spectrometer (Thermo Scientific; San Jose, Ca). Peptides were first trapped (Dr. Maisch Reprosil C18, 3 ?m, 2 cm?100 ?m) prior to separation on an analytical column (Agilent Poroshell EC-C18, 2.7 ?m, 50 cm?75 ?m). Trapping was performed for 10 min in solvent A (0.1 M formic acid in water), and the gradient was as follows: 0-10% solvent B (0.1 M formic acid in 80% ACN) in 5 min, 10-44% in 20 min, 44-100% in 3 min, and finally 100% for 2 min (flow was passively split to approximately 200 nL/min). The mass spectrometer was operated in data-dependent mode. Full-scan MS spectra from m/z 350-1300 Th were acquired in the Orbitrap at a resolution of 60,000 after accumulation to a target value of 1?10.sup.6 with a maximum injection time of 20 ms. In-source fragmentation was activated and set to 15 eV. The cycle time for the acquisition of MS/MS fragmentation scans was 3 s. Charge states included for MS/MS fragmentation were set to 3-8. Dynamic exclusion properties were set to n=1 and to an exclusion duration of 15 s. HCD fragmentation (MS/MS) with stepped collision energy at 20, 30 and 40 NCE was performed in the Ion Trap and the spectrum acquired in the Orbitrap at a resolution of 30,000 after accumulation to a target value of 1?10.sup.5 with an isolation window m/z 1.4 Th and maximum injection time 120 ms.

Example 5: Data Analysis of Peptides Crosslinked with a Compound According to Formula (1)

[0201] The acquired raw data were processed using Proteome Discoverer (version 2.2.0.388) with the XlinkX nodes integrated. For linear peptides a database search was performed against a FASTA file containing only bovine serum albumin (BSA) using the standard Sequest node as the search engine. Cysteine carbamidomethylation was set as fixed modification. Methionine oxidation and protein N-term acetylation as dynamic modification. To support the search for potential monolinks, water-quenched (C.sub.8H.sub.5O.sub.6P) and Tris-quenched (C.sub.12H.sub.14O.sub.8PN) versions of the crosslinker according to formula (1) were additionally set as dynamic modifications. Trypsin was specified as the cleavage enzyme with a minimal peptide length of six and up to two miss cleavages were allowed. Filtering at 1% false discovery rate (FDR) at the peptide level was applied through the Percolator node. For crosslinked peptides, a database search was performed against the same FASTA file using the XlinkX nodes for crosslink analysis. The crosslinker according to formula (1) (C.sub.8H.sub.3O.sub.5P) was set as the crosslink modification. Cysteine carbamidomethylation was set as a fixed modification and methionine oxidation and protein N-term acetylation was set as dynamic modifications. Trypsin was specified as enzyme and up to two miss cleavages were allowed. Furthermore, identifications were only accepted with a minimal score of 40 and a minimal delta score of 4. Otherwise, standard settings were applied. Filtering at 1% FDR at peptide level was applied through the XlinkX Validator node. The standard proteome discoverer node Minora Feature Detector was used for precursor ion quantification for all identifications.