Description

The present invention relates to compounds, reagent combinations and methods useful in isolating proteins from complex mixtures such as cell lysates or living cells.

Tri-functional compounds for selective reaction with proteins are known from

WO2004/064972A2. The capture compounds described therein comprise three essential functionalities X, Y and Q linked through a central core Z. These functions impart the selective affinity targeting of the capture compound for certain target proteins (selectivity function or moiety Y), the light-mediated covalent linkage of the capture compound to the target protein (reactivity function or moiety X), and the subsequent removal or isolation of the capture-compound-protein complex from the mixture by a sorting function Q.

Different methods for analysing or identifying the isolated ("captured") proteins can be employed. For high-throughput analysis, mass spectrometry has proven effective. The combination of capture compound isolation and mass spectrometric analysis is referred to as CCMS (Capture Compound Mass Spectrometry).

The isolation step in CCMS involves the equilibration of the capture compound with a complex mixture of proteins, for example a cell homogenate, an isolated organ or cell with intact cellular architecture, or an organelle. This first contacting step is performed in the absence of light that is able to activate the reactivity function X. The capture compound selectively associates (non-covalently) with certain proteins to which the selectivity function has an affinity. One example is a capture compound having a pharmaceutical drug as Y, which will then specifically interact with drug target proteins and, in addition, with any protein that the drug does also interact specifically but is not designed to do so ("off-targets").

Once equilibration has occurred, the sample is irradiated with light of a wavelength sufficient to activate the reactivity function X, rendering a highly (non-specifically reactive) species, e.g. a carbene, that immediately reacts with any suitable group within its neighbourhood, effectively linking the target or off-target protein to the capture compound. Subsequently, the capture compound, with any attached protein, is removed from the mixture by contacting the mixture with a ligand for the sorting function that allows for removal of Q-containing molecules by attachment to a surface. One prominent example of Q is biotin, which can mediate removal of biotinylated molecules via highly stable association to streptavidin bound to magnetic particles or surfaces ("matrices"). One area of useful application of CCMS is the analysis of drug molecule interactions with proteins to understand toxicity and side effects of pharmaceutical drugs and drug

development candidates.

During CCMS, matrices to which captured proteins are attached, bind proteins non- specifically to a certain extent. These non-specifically bound proteins form a background "noise" appearing also in control experiments, potentially drowning out signals from proteins that have bound specifically in small quantity.

The objective of the present invention is to provide methods and reagents to overcome the stated problems of the state of the art and to extend the applicability of CCMS to whole cell systems using membrane-permable capture compounds. This objective is attained by the subject-matter of the independent claims.

Summary of the Invention

The central feature of the invention is the introduction of a light-cleavable function or moiety as part of the capture compound linked to the captured protein. Light-induced cleavage allows for the captured proteins' selective and careful disassociation from the matrix used in removing the captured proteins from the mixture.

A further central aspect of the invention is the separation of the selectivity (Y) and reactivity functions into one compound, and the sorting function (Q) into another compound, both of which compounds can be selectively rejoined upon selection of suitable reaction conditions. Thereby, the capture compound having all three essential functions X, Y and Q is assembled stepwise, and can -by means of the light-cleavable function- be disassembled after the protein has reacted and has been isolated.

According to a first aspect of the invention, a method for isolating protein molecules is provided, comprising the following steps:

In a first binding step, a mixture comprising protein molecules is contacted with a first compound I

(I) wherein

- X is a reactivity function moiety activated at a first wavelength λ-ι, forming a highly reactive molecular species upon activation by light,

- Y is a selectivity function comprising a moiety of a molecular mass of less than 3500 u, - Z is a carbon, nitrogen, phosphorus or silicon atom;

D is a first connecting moiety, which is able to selectively engage with a second connecting moiety E at reaction conditions at which D and E do not react with polypeptides;

- each S independently from any other S is a covalent linking moiety comprising

carbon, nitrogen, oxygen, sulphur and/or silicon atoms bridging Z with X, Y, and D.

Subsequently the reactivity function moiety X is activated by exposure to light of λ-ι, whereby protein molecules in close proximity react with the highly reactive species formed by activation of X to form a covalent bond between the protein and compound (I).

Subsequent to said first binding step, said mixture is contacted with a second compound (II) described by a general formula E-S-F-S-Q in a second binding step, wherein S bridges F and D and Q; respectively, and S has the meaning indicated above. Furthermore, E is the second connecting moiety, cited above, which is able to selectively engage with the first connecting moiety D at reaction conditions at which D and E do not react with polypeptides; F is a moiety cleavable by light of a wavelength λ ₂ , and Q is a sorting function. Reaction conditions are set so that D selectively engages with E, thereby forming a complex in which the first compound (I) is now both connected to a protein by virtue of having reacted through its X moiety, and to the second compound (II), thereby connecting the protein to the sorting function Q.

In a subsequent first isolation step, molecules comprising said sorting function Q are separated from the mixture as a reaction fraction. This reaction fraction concentrates the isolated proteins, as they now bear the sorting function Q attached as part of the capture compound.

In a subsequent cleavage step, F is cleaved by exposure to light of λ ₂ , thereby severing the covalent link between the (target/off-target) protein and Q, and in a second isolation step, removal of molecules comprising Q from the reaction fraction renders isolated proteins.

According to a second aspect of the invention, a compound for use in a method of the invention is provided, which is described by a general formula E-S-F-S-Q, wherein

- each S independently is a covalent linking moiety comprising carbon, nitrogen, oxygen, sulphur and/or silicon atoms bridging F, E and Q;

E is a second connecting moiety, which is able to selectively engage with a first connecting moiety D at reaction conditions at which D and E do not react with polypeptides;

F is a moiety cleavable by light of a wavelength λ ₂ , and - Q is a sorting function.

According to a third aspect of the invention, a reagent combination for use in a method of the invention is provided, comprising a first compound described by a general formula I

(I) wherein X, Y, Q, Z, S and D have the meanings indicated above, and a second compound described by a general formula E-S-F-S-Q as specified above.

Detailed description of the invention

The method of the invention for isolating protein molecules comprises the steps of

in a first binding step, contacting a mixture comprising protein molecules with a first compound I

(I) wherein

- X is a reactivity function activated at a first wavelength λ-ι, forming a highly reactive species upon activation that will react with any protein being in close proximity,

- Y is a selectivity function, in certain embodiments a pharmaceutical drug molecule or drug development candidate, or a metabolite of a drug or drug candidate, which is able to bind to at least some proteins with high affinity, typically in the micro- or nanomolar range, and which is also capable of binding to its target or off-target protein highly selectively;

- Z is central moiety linking the X, Y and D function, such as a carbon, nitrogen

phosphorus or silicon atom;

D is a first connecting moiety able to selectively engage with a second connecting moiety E at reaction conditions at which D and E do not react with polypeptides;

- each S independently is a covalent spacer comprising carbon, nitrogen, oxygen, sulphur and/or silicon atoms bridging Z with X, Y, and D,

and subsequently activating said reactivity function moiety X by exposure to light of λ-ι; in a second binding step, contacting said mixture with a second compound (II) E-S-F-S-Q (I I)

wherein S has the meaning indicated above, E is said second connecting moiety able to selectively connect to the first binding moiety D in the first compound, F is cleavable by light of a wavelength λ ₂ , and Q is a sorting function able to pull out the reacted molecule from a complex mixture, e.g. by attachment to a surface.

- Subsequent to addition of the second compound, reaction conditions are set so that D selectively engages with E, facilitating reaction of first and second compound, thereby linking any captured protein to Q.

In a following first isolation step, molecules comprising the sorting function Q are separated from the mixture as a reaction fraction; thereafter, F is cleaved by exposure to light of λ ₂ ; and in a second isolation step, subsequent removal of molecules comprising Q from the reaction fraction renders isolated proteins.

Separating the selectivity and reactivity functions into one compound and the sorting function into another compound has the advantage of allowing for smaller and more apolar capture compounds to be designed, thereby facilitating their entry into cells and organs. The connecting moieties D and E can be small apolar moieties such as, by way of non-limiting example, a terminal alkyne group. This group will permeate intact membranes far easier than a capture compound bearing a biotin moiety or oligonucleotide as Q.

In one embodiment, X and F are activated or cleaved, respectively, at the same or similar wavelengths λ-ι and λ ₂ . This enables using the same light source for both steps. "Similar" in the present context signifies that the wavelengths do not have to be the same, but may differ by some few dozen nm. In one embodiment, the wavelengths λ-ι and ₂ are selected thus that a common laboratory UV light source shows sufficient intensity at both wavelengths to allow for activation of X and cleavage of F, respectively. One example for such light source is the „caprobox" (caprotec bioanalytics GmbH, Berlin). Another example is a common trans- illuminator for DNA agarose gel analysis, which generates light of ca. 360 nm and 254 nm. In one embodiment, the wavelengths λ-ι and λ ₂ differ equal or less than 100 nm. In one embodiment, the wavelengths λ-ι and λ ₂ differ equal or less than 50 nm. In one embodiment, the wavelengths λ-ι and λ ₂ differ equal or less than 25 nm.

The photo-activatable reactivity function X is a moiety that can be stimulated to generate a reactive species such as a nitrene, a carbene or a radical by electromagnetic radiation such as visible light or UV light. Such reactive species will quickly form a covalent bond with a range of suitable partners, for example by addition to a C=C-double bond, or by insertion into a O-H, S-H, N-H or C-H bond. Non-limiting examples for activation reactions for reactivity functions are the generation of a carbene by photolysis of a diazo compound, for example a diazirine, or the generation of a nitrene from an azide, or the generation of a (di-)radical from a carbon-hetero atom double bond as is the case for photolysis of benzophenone, any of which reactive intermediate species will quickly go on to react further with -by way of non-limiting example- an aromatic amino acid side chain, or an alcohol-, amino- or thiole function of a sterically proximate amino acid.

Non-limiting examples for reactivity functions are aryltrifluoromethyl diazirines (III), aryl azides (IV) oder benzophenones (V):

The covalent link of the reactivity function to compound (I) can be realized through other than the indicated positions, for example through position 2 or 3 in the ring. The aromatic rings may be substituted by, for example, cyano groups, nitro groups or hydroxyl groups.

In one embodiment, X is an aryltrifluoromethyl diazirine or alkyl diazirine, an aryl azide or a benzophenone.

The selectivity function Y in principle may be any molecular moiety with affinity to target (off- target-) proteins in the micromolar, nanomolar or sub-nanomolar range. Interaction of Y with a target protein will restrict linkage of the first compound (I) to certain proteins. The term "target" protein in this context is somewhat arbitrary. When used in the context of pharmaceutical drug development, the term "target" protein is used to designate the protein that the drug is meant to interact with, or assumed to interact with, thus forming the desired target. Any protein that is not known to be targeted by definition is a "non-target" or "off- target" protein, which however may turn out to be important for the indication in development, or another medical indication, thereby changing the designation to "target" as a function of changing purpose of the drug, or a shift in understanding of the molecular underpinnings of the disease being targeted.

The reactivity function X upon activation forms a covalent bond to any protein in its proximity. In absence of the selectivity function the capture compound would capture a more or less representative selection of any proteins present in the sample. The selectivity function restricts the linkage of the capture compound to such proteins that specifically interact with the selectivity function Y. One important field of application for the instant invention is drug development, in particular the analysis of drug-protein interactions and the improvement of specificity and selectivity for the (desired) drug-target interaction, and likewise the modification of (in most cases, undesired) drug-non-target interactions. For embodiments addressing such applications, Y is a pharmaceutical drug, drug development candidate or drug metabolite or prodrug. Other important exemplary applications are herbicides, pesticides, fungicides or other small organic molecules of relevance in any biomedical context.

In one embodiment, the selectivity function Y is a moiety of a molecular mass of less than 1000 u. In one embodiment, the selectivity function Y is a moiety of a molecular mass of less than 700 u. In one embodiment, the selectivity function Y is a moiety of a molecular mass of less than 500 u. In one embodiment, Y is a molecular moiety obeying the "Lipinski" rule, i.e. Y has a molecular mass between 160 u and 500u, comprises up to five hydrogen bond donators (e.g., oxygen and or nitrogen atoms with one H attached), up to ten hydrogen bond acceptors (e.g., oxygen or nitrogen atoms) and an octanol-water partition coefficient logP of below 5,6 (any of these characteristics applied to the isolated Y moiety, without regard to the rest of compound I).

In one embodiment, Y is a physiologically active peptide. In one embodiment, Y is a physiologically active peptide having a molecular mass of less than 3500 u. In one embodiment, Y is a peptide having 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

Binding of the selectivity function to the (non/off-) target protein(s) is effected by non-covalent interaction. The affinity of the "naked" selectivity function (measured with no first compound / capture compound attached) is characterized by a dissociation constant below 100 pmol/l. In some embodiments, the dissociation constant is below 10 pmol/l, <1 pmol/l, <100 nmol/l, <10 nmol/l or <1 nmol/l.

In some embodiments, the sorting function Q is a small molecule having strongly binding, highly selective interaction partners. One important example for a sorting function is biotin, which binds to streptavidin at a dissociation constant of about 10 ^"15 mol/l. In embodiments where Q is biotin, captured proteins can be isolated from the mixture by attachment to streptavidin-covered magnetic particles during the isolation step. Further examples for sorting functions having strongly binding, highly affine partners, particularly those readily applied to separation as commercially available reagent preparations, are hexahistidine tags (His6) or small molecules for which highly affine antibodies are available (e.g. fluorescein, TAMRA (rhodamine) or BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). In some embodiments, Q is an oligonucleotide composed of the canonic desoxyribonucleoside building blocks adenosine, thymidine, cytidine and guanosine or their analogues such as peptide nucleic acid (PNA) or locked nucleic acid, C2-C4-methylen-bridged ribose analogues of DNA (LNA). Such oligonucleotides bind to oligomers with revers-complimentary sequence highly selectively. In some embodiments, such oligonucleotides as sorting function Q are 4 to 20 monomers in length.

Alternatively, Q may be formed by a surface, for example the surface of a particle, or a surface of a microtiter well or a chip surface. In such embodiments, the second compound could be described by a formula E-S-F-S-(surface). In other words, instead of providing a second compound having a sorting function Q that is able to bind selectively to a surface, the compound is provided with the surface already attached.

In embodiments where Q is the surface of a particle, the particle may be removable from the mixture selectively, for example by virtue of its magnetic, electrostatic, inductive or optical properties. In one embodiment, the surface is the surface of a magnetic particle.

In some embodiments the surface is a stationary surface, for example the surface of a microtiter well or a microfluidics chip, or the surface of a material contained in a separation column. In these embodiments, the mixture obtained in the first binding step is washed onto the surface, the reaction conditions for reaction between D and E are provided and thus any captured protein is attached to the surface. Components of the mixture that have not attached to the surface after the first two steps are washed away; a distinct separation step is not necessary as the separation step is effected during movement of the sample over the surface.

In some embodiments, molecules comprising Q are removed during the second isolation step by applying a magnetic force to remove magnetic particles to which the protein-capture compound complex has bound. Magnetic separation achieves isolation both in embodiments where Q is a magnetic particle, i.e. the second compound (II) components E-S-F-S- are attached covalently to the particle, or where Q is biotin, and prior to the first isolation step, streptavidin coated magnetic particles have been added to the mixture to extract any protein- capture compound complex.

In some embodiments, Q is a fluorescent dye and molecules comprising Q are removed during the second isolation step by treatment with an antibody to that dye which is coupled to a magnetic particle, or to a surface.

Subsequently, irradiation of the surface with light of the second wavelength λ ₂ under continued flow renders a fraction containing the isolated protein, ready for analysis. One advantage of such embodiments is that the method can be automated easily, without requiring much manual handling or complex robotics. According to a third alternative, Q can be a fluorescent dye molecule. Examples are the dyes known as FITC, TAMRA, Cy2, Cy3, Cy5 or Dy547. These dyes are particularly suited to applications where the isolated proteins are analysed by two-dimensional gel

electrophoresis. Fluorescently tagged proteins may be isolated by extraction using dye- specific antibodies.

The method of the invention is applicable to mixtures of any origin that contain protein molecules. One particular field of application is the isolation of proteins from cells having an intact cellular architecture. The separation of the functionalities X, Y and Q of the "classic" capture compound onto two different molecules enables smaller molecules to be used. These diffuse better and are more easily permeate across membranes into cells and intracellular compartments. There, they can interact with proteins in their physiologically relevant context. Similarly, the method of the invention can be advantageously used on tissue samples, cell lysates or homogenates.

A further advantage is based on the smaller size of the first compound (I), which avoids steric interactions of the sorting function that might decrease the specific binding of the selectivity function Y to the protein. Furthermore, absence of the sorting function Q during the activation of X avoids capture of proteins as a result of interaction of a protein's ability to bind to the sorting function -rather than to Y-, which is of particular importance when using biotin as a sorting function. A number of intracellular proteins do bind biotin and as a result, appear as "false positives" in capture assays using biotin directly linked to the capture compound.

The reaction partners D and E that mediate coupling of the first and second compound of the invention can be any variants of the reactions commonly referred to as "click" chemistry. The reaction most often used to date in biological chemistry is the 1 ,3-dipolar cycloaddition reaction between an azide and an alkyne developed by Huisgen (Rostovtsev et al., Angew. Chem. 2002; van Berkel et al., ChemBioChem 2008, 9, 1805-1815) (scheme 1 , a).

Alternatively, the following reactions are contemplated:

- Staudinger ligation (Saxon & Bertozzi, Science 2000, 287, 2007-2010; Kohn &

Breinbauer, Angew. Chem. 2004, 116, 3168-3178; Scheme 1 b upper panel) between an azide and an arylcarboxylic acid methyl ester-5-ortho-phosphine,

- Staudinger phosphite reaction (Serwa et al., Angew. Chem. 2009, 121 Scheme 1 b

lower panel) between an azide and a phosphite ester,

Diels-Alder reaction with the ene component as function D (Song et al., Angew. Chem. 2008, 120, 2874-2877; J. Am. Chem. Soc. 2008, 130, 9654-9655; Devaraj et al., Bioconjugate Chem. 2008, 19, 2297-2299) (Scheme 1 c), or as function E (not catalysed: Marchan et al., Nucl. Acids Res. 2006, 34, e24 1-9; Ti as catalyst: Litz, Molecules 2007, 12, 1674-1678, 13; Serganov et al., Nat. Struct. Mol. Biol. 2005, 12, 218-224) (Scheme 1 d);

Ene reaction or photo-thio-en reaction (Killops et al., J. Am. Chem. Soc. 2008, 130, 5062-5064; Schema 1 e).

a)

Conditions: 1) CuSO sodium ascorbate

Conditions: (1) no catalyst/additive

catalysis

e) other coupling techniques

photo- thio- en- click Conditbns: (1) h x (350 nm)

Conditions: (1) no catalyst/additive

Scheme 1

According to one embodiment, D and E are each selected from an azide and a member of the group comprising an alkyne, a 2-phosphinobenzoate and a phosphite ester. In other words, either D or E is an azide, and the other partner is an alkyne, a 2-phosphinobenzoate and a phosphite ester. Alternatively, D and E can be selected from either of the groups: a double bond and a 1 ,3 diene system, a thiol and an alkene, or a double bond with at least one allylic hydrogen and an azo compound. According to some embodiments, the following combinations are employed:

A=B double bond (En) A=B-D=E (diene) Diels-Alder

4 with A, B, D, E independently being C or N

C=C-C=C (Dien) C=C double bond (En) Diels-Alder

5

C=C double bond (En) C=C-C=C (diene) Diels-Alder

6

C=C R-SH thiol-ene

7

R-SH C=C thiol-ene

8

Table 1

In one embodiment, D and E are selected from the group of reaction partners comprising:

- an azide and a member of the group comprising an alkyne, an aryl carboxlic acid methyl ester ortho-phosphine and a phosphite ester,

- a double bond and a 1 ,3-diene system in s-cis conformation,

- a double bond having an allylic hydrogen atom and a diazo compound.

In some embodiments, the alkyne function D or E is an annular alkyne under strain, for example a cyclooctyne. "Strain-promoted" [3+2] cycloaddition reactions are well known in the art; they confer the advantage of proceeding in the absence of copper ions, which are toxic for most living systems.

In some embodiments, the photocleavable group F is cleavable at wavelengths of between 300 nm and 400 nm. This wavelength can be used for activating reactivity groups in commercially available reagents and devices. In some embodiments, F is an o-

nitrobenz lether (cleavage at 320-350 nm) or a substituted acetophenone.

Scheme 2

(B, Het are oxygen, nitrogen or sulphur; S signifies a spacer as specified above; R is H or alkyl, R' is H or OH)

In formula (I), Z is the central atom or function bridging the moieties X, Y and D (and thereby, Q in the protein-capture compound complex after reaction with compound (II)). The functional moieties X, Y, D/E, F and Q are linked to each other by bridging moieties (spacers) S. In its most simple manifestation, Z is a carbon atom, for example the central carbon atom of an alpha amino acid. Proteinogenic amino acids having a side chain such as lysine or serine are suitable core molecules for first compounds (I) since they provide three functions that can be easily derivatized and coupled to moieties independently from each other. If, for instance, lysine is used, then the central alpha carbon atom can be viewed as the core Z, and the carboxy function, the amino function and the butylamine function can be viewed as the respective spacers S.

Similarly, Z can be a tertiary amine bearing the three functions X, Y and D.

In some embodiments, Z is a trisubstituted phosphorus atom, for example an phosphine oxide of the formula

O

I I

xs- ^p — ^SD

SY wherein S, X, Y and D have the meaning defined above.

In some embodiments, Z is a silicium atom.

In some embodiments, the core of compound (I) is a ring. Non-limiting examples are a phenyl ring or a ribose. Here, Z formally is one of the ring members, and at least two of the spacers bridging the functional moieties X, Y and D are bridged or joined to each other. Compounds of annular core structure are similarly suited for the method of the invention.

S can be any chemical moiety that is suited to link the functional moieties X, Y and D (and F, E and Q). In some embodiments, each S is a linear chain of carbon and hetero atoms (oxygen, nitrogen, sulphur, silicium, phosphorus). In some embodiments, each S is a chain of 2, 3, 4, 5, 6 7, 8, 9, 10, 1 1 or 12 atoms. The chain may be substituted by carbon or hetero atoms and contains the hydrogen atoms corresponding to the oxidation state of each non- hydrogen atom. One example for S is the methoxy moiety forming the side chain of serine, a CO-NH or NH-CO moiety (linkage by amide on the nitrogen or carboxy carbon), a CO-0 or O-CO moiety. When designing compounds I and II, ease of chemical synthesis, sterical freedom of functions X, Y, D, E and Q, particularly the interaction of Y with the protein to be isolated, and the requirements regarding diffusion into cells or other biological structures will be taken into account.

Examples for S are

with n = 0 to n = 20. Preferred embodiments have n = 0, n = 1 , n = 2, n = 3, n = 4 or n = 5.

X and Y should be linked to Z by S moieties that are sufficiently long to enable both X and Y functions to interact with protein surfaces independently from each other; on the other hand S should be minimized to allow for diffusion into cells or organelles, where such diffusion is expected. In some embodiments, X and Y are linked to Z by S that together comprise 8 to 15 bridging atoms.

In some embodiments, S comprises a alkyl silicium compound that can be selectively cleaved by fluorides.

In some embodiments, proteins isolated by the method of the invention are analysed by mass spectrometry. In one embodiment, isolated proteins are separated prior to mass spectrometry by a further separation method such as chromatography. Isolated proteins can be digested or fragmented by proteinase treatment prior to analysis and identification.

According to a second aspect of the invention, a compound for use in a method as described above is provided. Said compound -the second compound of the method described above- can be characterized by a general formula E-S-F-S-Q. Therein, any S independent of other S is a spacer molecule as defined above. E is the second connecting moiety engaging with said first connecting moiety, and for which alternatives have been discussed above. E is able to selectively engage with connecting moiety D at reaction conditions at which D and E do not react with polypeptides.

F is the function cleavable by light of wavelength λ ₂ , and Q is a sorting function. All functions or moieties are contemplated as described in detail for the method of the invention above.

In one embodiment, Q is biotin. In one embodiment, Q is a fluorescent dye. In one embodiment, Q is an oligonucleotide or a LNA or PNA oligomer. In one embodiment, Q is a magnetic particle.

According to a third aspect of the invention, a reagent combination for use in a method of the invention is provided. This reagent combination comprises

- a first compound described by a general formula (I)

(I) wherein

- X is a reactivity function moiety activated at a first wavelength λ-ι,

- Y is a selectivity function comprising a moiety of a molecular mass of less than

3500 u,

- Z is a carbon, nitrogen, phosphorus or silicon atom; D is a first connecting moiety, which is able to selectively engage with a second connecting moiety E at reaction conditions at which D and E do not react with polypeptides;

- each S independently is a covalent linking moiety comprising carbon, nitrogen, oxygen, sulphur and/or silicon atoms bridging Z with X, Y, and D,

- a second compound according to the second aspect of the invention.

The reagent combination can be provided as a kit of parts, as a pre-packaged assortment of reagents or as an informal combination of the individual reagents.

In some embodiments, reagent combinations are chosen so that X and F are activatable and cleavable, respectively, at a similar wavelength. In some embodiments, X is selected from the group comprising aryltrifluoromethyl diazirine, aryl azide and benzophenone. In some embodiments, Y is a moiety of a molecular mass of less than 1000 u.

Wherever alternatives for single features such as, for example, a reactivity function X, selectivity function Y, sorting function Q or spacer S are laid out herein as "embodiments", it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

Non-limiting examples of compounds useful for practicing the method of the invention, or for use as compounds in combinations of the invention, are:

Scheme 1

As one example of a first compound I, 3 was synthesized according to scheme 1 from 1 and 2 and isolated by MPLC.

MS: C ₄ 5H ₆ 2F ₃ N ₁₂ Oi ₃ P calculated [M-H ⁺ ]: 1067.43, found: 1067.43

Scheme 2

As one example of a first compound I, 5 was synthesized according to scheme 2 from 4 and obtained as a colourless solid after purification by MPLC.

NMR (MeOH-cU): 7.47/7.16 (ΑΑ'/ΒΒ', 4H), 4.29 (dd, 1H), 3.39 (t, 2H), 3.15 (dt, 2H), 2.75 (t, 2H), 2.51- 2.49 (m, 4H), 2.35 (t, 2H), 2.29 (m, 1H), 2.13 (t, 2H), 2.09 (t, 2H), 1.97 (dd, 1H), 1.73-1.66 (m, 2H), 1.66-1.59 (m, 2H), 1.59-1.52 (m, 2H), 1.50 (dd, 1 H), 1.52-1.46 (m, 2H), 1.42-1.36 (m, 4H), 1.30-1.22 (m, 2H), 1.04 (s, 3H).

Scheme 3

As one example of a second compound II, 8 was synthesized according to scheme 3 by condensation of 6 and 7 and obtained as a yellowish-green solid after purification by MPLC.

NMR (MeOH-d ₄ ): 7.79 (s, 1H), 7.35 (s, 1H), 4.97 (br. s, 2H), 4.47 (br. dd, 1H), 4.28 (dd, 1H), 4.25-4.23

(m, 2H), 4.12 (s, 2H), 3.99 (s, 3H), 3.91-3.88 (m, 2H), 3.74-3.72 (m, 2H), 3.66-3.62 (m, 8H), 3.60 (t, 2H), 3.55 (t, 2H), 3.46 (t, 2H), 3.35 (t, 2H), 3.34-3.32 (m, 2H), 3.17 (ddd, 1H), 2.91 (dd, 1H), 2.69 (d,

1H), 2.19 (t, 2H), 1.69 (m, 1H), 1.66-1.59 (m, 2H), 1.57 (m, 1H), 1.45-1.36 (m, 2H).

Scheme 4

Triazol 11 was obtained according to scheme 4 after reaction of 30 min as one example for a product of the method of the invention.

MS: C77H-1 -12F3N20O25PS calculated:! 836.8, found: 1836.6

The product is readily cleaved by irradiation at 350 nm for 10 min, agitating the mixture every 2 min to allow for uniform exposure of the sample. Beads were washed and the washing solution submitted to LC-MS.