Labeling of biomolecules

Abstract

The method for reversibly labeling a biomolecule includes providing a linker molecule (11) comprising a first functional group (LG) containing a reaction center, a second functional group (FG) containing a reaction center, and a cleavable portion (e.g., a hydrolyzable portion) (A-B-C). The method also includes forming a covalent bond between the biomolecule (10) and the reaction center of the first functional group (LG), forming a covalent bond between the first label (L1) and the reaction center of the second functional group (FG), cleaving the cleavable portion (A-B-C) of the linker molecule (11), such as hydrolyzing the hydrolyzable portion of the linker molecule (11) to remove the first label (L1) and to form a third functional group (W) containing a reaction center, and forming a covalent bond between another molecule and the reaction center of the third functional group (W) to reform the cleavable portion, such as the hydrolyzable portion (A-B-C).

Description

[0001] The present invention relates generally to the labeling of biomolecules. More particularly, although not exclusively, the present invention relates to methods of reversibly labeling biomolecules.

[0002] Bioconjugation is a chemical strategy in which a covalent bond is formed between a biomolecule and a second molecule. The second molecule can act as a tag or marker, in which case the second molecule will impart new properties to the biomolecule. The second molecule can impart properties suitable for measuring the location and / or concentration of the biomolecule, for example, to track cellular events, determine enzyme function, measure drug delivery, etc. The second molecule can be, for example, a fluorescent molecule, a radioactive substance, and / or a different biomolecule suitable for detection assays. Alternatively, the second molecule can be selected so that it makes the biomolecule more soluble or more stable in a particular environment. In other cases, the second molecule can be used to target another biomolecule.

[0003] Some simple conjugation chemical reactions dominate the site-selective chemical modifications (or functionalizations) applied to biomolecules (e.g., Hermanson, G.T. Bioconjugate Techniques, 2013; Bioconjugated Oligonucleotides: Recent Developments and Therapeutic Applications. Bioconjug. Chem. 2019; Contemporary Approaches to Site-Selective Protein Modification. Nat. Rev. Chem. 2019). These chemical reactions lay the foundation for a variety of manipulations, classifications, and labeling experiments that subsequent analysis often relies on.

[0004] However, the focus of these approaches has always been on establishing a stable association between the target biomolecule and the required functional moiety. Therefore, functionalization can limit the study of biomolecules to a single analytical procedure. This approach is inconsistent with recent cell biology research, where an integrated approach to observe cell behavior is becoming increasingly important.

[0005] Recent efforts to address one or more of these problems include the following prior art. Bernades et al. have reported the development of a bifunctional linker that simultaneously allows site-specific protein modification and palladium-mediated bioorthogonal decaging. This is achieved through a thioether binding motif in a propargyl carbamate linker and readily available palladium complexes (Bernades et al. Chem. Sci., 2018, 9, 4185, Angew. Chem. Int. Ed. Vol 57, 36, 2018; Chem. Sci., 2016, 7, 4589-4593).

[0006] It has been reported that the use of cleavable diazo bonds in the capture and release of proteins is a tool for monitoring endogenous methyltransferase activity (Lou et al., J. Am. Chem. Soc. 2012, 134, 36, 14905-14912). After labeling and capture, the protein can be released by cleaving the diazo group with sodium dithionite.

[0007] A reversible mTAG labeling strategy has been reported, in which ultraviolet light is used to remove the labeling moiety (see Chem. Commun., 2018, 54, 449-451). The light-triggered removal is absolute, which means that repeated labeling of DNA molecules is necessary. In addition, the labeling moiety does not contain functionality for further functionalization.

[0008] Therefore, a first non-exclusive object of the present invention is to provide a bioorthogonal method for reversibly linking a biomolecule to a second molecule, such as a label. One of the other non-exclusive objects of the present invention is to provide a bioorthogonal method for labeling, delabeling and relabeling a biomolecule in an infinite cycle so that multiple analyses can be performed sequentially on a single sample. Other non-exclusive objects of the present invention are to provide a bioorthogonal method for recovering a second or other molecule.

[0009] Therefore, a first aspect of the present invention provides a method for reversibly labeling a biomolecule, the method comprising:

[0010] a. providing a linker molecule, the linker molecule comprising a first functional group containing a reaction center, a second functional group containing a reaction center and a cleavable portion such as a hydrolyzable portion;

[0011] b. forming a covalent bond between the biomolecule and the reaction center of the first functional group;

[0012] c. forming a covalent bond between the first label and the reaction center of the second functional group;

[0013] d. cleaving the cleavable portion of the linker molecule, for example, hydrolyzing the hydrolyzable portion of the linker molecule to remove the first label and to form a third functional group comprising a reactive center;

[0014] e. Forming a covalent bond between the other molecule and the reactive center of the third functional group to reform a cleavable moiety, such as a hydrolyzable moiety.

[0015] Advantageously, the cleavable portion or bond of the linker molecule in the method of the present invention, such as a hydrolyzable portion or bond, can be used to selectively write, modify, erase and rewrite functional sites onto biomolecules in an integrative manner. The method of the present invention can be used, for example, for single cell analysis where materials are limited. By using the method of the present invention to combine a variety of analytical methods, a new and more comprehensive understanding of cell behavior is possible. More advantageously, after the desired analysis has been performed, the linker molecule, the marker and the biomolecule can be recovered.

[0016] In some embodiments, the cleavable moiety is a hydrolyzable moiety. The hydrolyzable moiety can be a Schiff base, such as an imine moiety, an oxime moiety, and / or a hydrazone moiety.

[0017] In some embodiments, the hydrolyzable bond may comprise a disulfide (SS) bond.

[0018] In some embodiments, the other molecule may include a fourth functional group that includes a reactive center. The fourth functional group may be the same functional group as the second functional group.

[0019] In some embodiments, the method may further include step f. forming a covalent bond between the second label and the reaction center of the fourth functional group. In some embodiments, the method may further include step g. cleaving the cleavable moiety, such as hydrolyzing the hydrolyzable moiety, such as the Schiff base moiety, to remove the second label and reform the third functional group. In some embodiments, the method may further include step h. forming a covalent bond between other molecules and the third functional group to reform the cleavable moiety, such as the hydrolyzable moiety, such as the Schiff base moiety. In some embodiments, the other molecule of step e can be the same type as step h.

[0020] In some embodiments, step c is performed before step b. In some alternative embodiments, step b is performed before step c.

[0021] In some embodiments, step b may include a prior or simultaneous step of removing the leaving group to expose the reactive center of the first functional group. In some embodiments, step c may include a prior or simultaneous step of removing the leaving group to expose the reactive center of the second functional group.

[0022] In some embodiments, other molecules may comprise a marker. The method may include step i. forming a covalent bond between other molecules comprising a marker and a third functional group to reform a cleavable portion, e.g., a hydrolyzable portion, e.g., a Schiff base portion. It should be understood that the substance may replace the reaction product of step c in the method of the present invention. For example, a cleavable portion, e.g., a hydrolyzable portion, may be hydrolyzed to remove the marker and to form a functional group (e.g., a third functional group) comprising a reaction center. The method may also include forming a covalent bond between other molecules and a reaction center of a functional group (e.g., a third functional group) to reform a cleavable portion, e.g., a hydrolyzable portion.

[0023] In some embodiments, steps c, d, e, f and g may be performed sequentially and repeated in an infinite or closed loop. Advantageously, this enables sequential reversible labeling of biomolecules with a first label, a second label and an nth label.

[0024] The linker molecule may have the following general formula:

[0025]

[0026] Wherein FG represents the second functional group containing the reaction center;

[0027] Z represents a non-reactive group selected from one of an aliphatic bond or an aromatic bond;

[0028] ABC together represent a cleavable moiety, such as a hydrolyzable moiety (e.g., a Schiff base moiety);

[0029] Y represents a non-reactive group selected from an aliphatic bond or an aromatic bond;

[0030] LG represents the first functional group including the reaction center.

[0031] In some embodiments, the reaction center of the first, second or third or nth functional group may comprise a carbocation. In some alternative embodiments, the first, second, third or nth functional group may represent a reaction center, for example, the reaction center may comprise an azide moiety, for example, in a click chemistry reaction with an alkyne moiety, for example, on a first label. The reaction center may comprise an alkene or alkyne moiety, for example, which reacts with an azide moiety, for example, on a first label.

[0032] In some embodiments, the second functional group FG can be selected from one of the following: a halide (e.g., a F, Cl, Br, or I atom), an unsaturated bond (e.g., an olefin, an alkyne), an azide, an activated ester, an activated carbonate / ester, a carbamate / ester, an epoxide, an isothiocyanate / ester, or an isocyanate / ester moiety.

[0033] Preferably, the second functional group FG is an azide moiety.

[0034] In some embodiments, the first functional group LG is a functional group or chemical entity that allows for site-selective labeling of biomolecules by chemical or enzymatic methods.

[0035] In some embodiments, the first functional group LG can be selected from one of the following: halide, sulfonate (eg, mesylate, tosylate), iodine salts, acetates, acrylates (e.g., methacrylates), acrylamides (e.g., methacrylamide), maleimides (e.g., bromomaleimide, dibromomaleimide), bromopyridazinediones, propargyl esters, propargyl amides, α-bromoesters, α-bromoamides, 3-arylpropiolonitrile, -CH2-substituted trivalent sulfonium ions, halides (e.g., F, Cl, Br or I atoms), unsaturated bonds (e.g., olefins, alkynes), azides, activated salts and activated carbonates, carbamates, epoxides, isothiocyanates or isocyanate moieties.

[0036] In some embodiments, the first functional group LG can comprise a diazonium salt or a diazocarboxylate, for example, to react with a phenol on a biomolecule (eg, tyrosine).

[0037] In a preferred embodiment, the first functional group LG comprises a nitrile moiety, eg, 3-arylpropionitrile, for example to react site-selectively with a thiol (eg, cysteine) on a biomolecule.

[0038] In some embodiments, the first functional group LG comprises a leaving group, which leaves during the reaction to reveal a reaction center.

[0039] Preferably, the reaction center of the first functional group LG reacts with a thiol moiety on a biomolecule.

[0040] In one embodiment, the first functional group LG is a halogen, such as a bromine atom, and the reactive center is a carbocation.

[0041] In one embodiment, the first functional group LG is a chemical moiety suitable for enzyme-mediated labeling of biomolecules. Examples of the chemical moiety include, but are not limited to, the following methods: modified nucleotides for polymerase-directed labeling (see Holliger et al. Chem. Comm. 2009) or for terminal deoxynucleotidyl transferase-directed labeling (see Hollenstein et al ChemBiochem 2019); S-adenosylmethionine derivatives for MTA enzyme labeling (see Deen, J. et al Angew Chem Int Ed 2016); glycosyl donors, including glycosyl halides, polysaccharides, oxazolines and other glycosides for glycosylation; related enzymes include glycosidases, endoglycosynthases, glycosynthases, glycoceramidases or glycosyltransferases (see Wang et al. Chem. Rev. 2018); sortase substrates (e.g., LPTXG) for sortase A directional attachment (see Distefano et al Chem. Rev. 2018); Q-tags and K-tags (e.g., LLQG) for transglutaminase directional attachment (see Distefano et al Chem. Rev. 2018); tyrosine and derivatives for tyrosine ligase directional labeling (see Distefano et al Chem. Rev. 2018); Farnesyl diphosphate (FPP) derivatives for farnesyl transferase directional labeling (see Distefano et al Chem. Rev. 2018); al Chem. Rev. 2018); myristoyl-CoA derivatives, which are used for targeted labeling of N-myristoyltransferase (see Distefano et al Chem. Rev. 2018).

[0042] In other preferred embodiments, the first functional group LG is an S-adenosylmethionine derivative. In another preferred embodiment, the first functional group LG is an S-adenosyl-l-homocysteine ​​derivative.

[0043] It should be understood that the second functional group FG and / or the first functional group LG can react with the marker or biomolecule by a chemical reaction (e.g., click chemistry) or a chemical enzymatic reaction (e.g., in an enzyme-mediated reaction). Any suitable functional group can be selected as FG and / or LG for the reaction, which is bioorthogonal to the cleavable portion (e.g., hydrolyzable portion) ABC. For example, suitable reactions involving the first functional group LG or the second functional group FG and the biomolecule and the marker include click chemistry, cycloaddition, Staudinger reaction, epoxide ring-opening reaction, nucleophilic substitution and / or nucleophilic addition, respectively.

[0044] Preferably, FG and LG are different types of functional groups.

[0045] In a preferred embodiment, FG is an azide moiety and LG is an S-adenosyl-L-homocysteine ​​moiety. In another preferred embodiment, FG is an azide moiety and LG is a bromine moiety.

[0046] In some embodiments, the linker molecule may have the following general formula:

[0047]

[0048] wherein FG, Z, ABC, LG are as defined above; and U represents an unsaturated bond selected from one of the following: an alkene, an alkyne, an alkenyl or an aromatic group, a carbon atom containing a carbonyl group, a sulfur atom containing one or two S=O bonds;

[0049] M and N independently represent a non-reactive group selected from a hydrogen atom, a deuterium atom, an aliphatic group, and an aromatic group.

[0050] For example, both M and N may represent hydrogen atoms (or one or more deuterium atoms) to form a methylene (e.g., CH2) bond. Alternatively, one of M or N may represent an aliphatic or aromatic group to form a CHR bond. Alternatively, both M and N may represent an aliphatic or aromatic group (R 1 , R 2 ) to form CR 1 R 2 key.

[0051] In some embodiments, Z represents a non-reactive aliphatic or aromatic bond comprising 1 to 20 atoms (e.g., carbon atoms, oxygen atoms, and / or nitrogen atoms). The aliphatic and / or aromatic bond may comprise a hydrocarbon backbone and / or a polyether backbone. Additionally or alternatively, Z may be an aromatic bond comprising an aromatic moiety, such as a C6H4 aromatic ring comprising two substituents, such as an ortho-, meta-, or para-substituted aromatic ring.

[0052] In some embodiments, Z represents a polyether chain, for example, a polyethylene glycol chain comprising up to 10 ethylene glycol monomers, for example 9, 8, 7, 6, 5, 4, 3, 2 or 1 ethylene glycol monomers. Additionally or alternatively, Z may comprise an aromatic group, for example a C6H4(C=O)NH group.

[0053] For example, a linker molecule may have the following general formula:

[0054]

[0055] wherein FG, ABC, Y and LG are as defined above; q represents a number from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In some embodiments, q is 2 or 3.

[0056] For example, in some embodiments, the linker molecule has the following general formula:

[0057]

[0058] wherein FG, q, ABC, Y, U, M, N and FG are as defined above or below.

[0059] In some embodiments, the hydrolyzable moiety is a Schiff base moiety. In some embodiments, the Schiff base moiety ABC represents one of the following moieties:

[0060]

[0061] Where R x represents one of a hydrogen atom, a deuterium atom, an aliphatic bond, or an aromatic bond.

[0062] In some embodiments, Y represents a non-reactive aliphatic or aromatic chain, for example, comprising 1 to 15 atoms in the backbone, for example, 1 to 10 or 1 to 5 atoms in the backbone of the linker molecule. In some embodiments, Y represents a non-reactive aliphatic or aromatic chain comprising 1 to 15 CH2 moieties (e.g., 1 to 10 CH2 moieties, or 1 to 5 CH2 moieties).

[0063] In some embodiments, the third functional group is formed by hydrolysis of a hydrolyzable moiety (e.g., a Schiff base as described above), for example, using hydroxylamine. The third functional group may include an NH2 moiety.

[0064] In a preferred embodiment, the Schiff base moiety is an N-substituted hydrazone or an O-substituted oxime.

[0065] In some embodiments, the linker molecule has the following general formula:

[0066]

[0067] wherein p represents the number of CH2 groups, and p is 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5. In some embodiments, p represents 4, i.e., Y represents (CH2)4.

[0068] In some embodiments, U is an alkyne. In some embodiments, the linker molecule has the following general formula:

[0069]

[0070] wherein FG, Z, ABC, Y and LG are as defined above.

[0071] In some alternative embodiments, U is an olefin. In some embodiments, the linker molecule has the following general formula:

[0072]

[0073] wherein FG, Z, ABC, Y and LG are as defined above.

[0074] In some embodiments, the biomolecule is a peptide, ie, a polymer of amino acids. In some embodiments, the peptide contains amino acids that include a thiol moiety, such as a cysteine ​​moiety.

[0075] In some embodiments, the first functional group LG comprises a moiety that can act as a leaving group in a nucleophilic substitution reaction with a thiol moiety. In some embodiments, LG is one of the following: a halide (e.g., F, Cl, Br, or I), a sulfonate (e.g., mesylate, tosylate), an iodide. salts, acetates, acrylates (e.g., methacrylates), acrylamides (e.g., methacrylamide), maleimides (e.g., bromomaleimide, dibromomaleimide), bromopyridazinediones, propargyl esters, propargyl amides, α-bromoesters, α-bromoamides, 3-arylpropiolonitrile, -CH2-substituted trivalent sulfonium ions, unsaturated bonds (e.g., olefins, alkynes), azides, activated esters and activated carbonates, carbamates, epoxides, isothiocyanates or isocyanate moieties.

[0076] In some embodiments, the linker molecule used to label amino acids and / or peptides and / or proteins has the following general formula:

[0077]

[0078] Wherein FG represents the second functional group;

[0079] Z represents a non-reactive group selected from one of an aliphatic bond or an aromatic bond;

[0080] ABC represents a cleavable moiety, such as a hydrolyzable moiety, such as a Schiff base moiety;

[0081] Y represents a non-reactive group selected from one of an aliphatic bond or an aromatic bond; and

[0082] U represents an unsaturated bond selected from one of the following: an alkene, an alkyne, an aryl group, a carbon atom containing a carbonyl group, a sulfur atom containing one or two S═O bonds.

[0083] In some embodiments, U may represent an alkyne. In some embodiments, the linker molecule may have the following general formula:

[0084]

[0085] In some alternative embodiments, U may represent an alkene. In some embodiments, the linker molecule may have the following general formula:

[0086]

[0087] In some embodiments, the linker molecule may have the following general formula:

[0088]

[0089] wherein the cleavable moiety, e.g., the hydrolyzable moiety is a Schiff base moiety comprising a C=NXCQ moiety;

[0090] U represents an unsaturated bond selected from one of the following: an alkene, an alkyne, an aryl group, a carbon atom containing a carbonyl group, a sulfur atom containing one or two S=O bonds;

[0091] p represents a number from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5; for example, p may be 4;

[0092] Q represents one of the following: an oxygen atom or two hydrogen atoms or a deuterium atom independently bonded to a carbon center;

[0093] X represents one of an oxygen atom or a nitrogen atom;

[0094] Z represents a non-reactive group selected from one of an aliphatic bond or an aromatic bond;

[0095] FG represents a second functional group.

[0096] In some embodiments, the linker molecule has the following general formula:

[0097]

[0098] The Schiff base part is a -C=NNC=O bond;

[0099] p represents a number from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5; for example, p may be 4;

[0100] q represents a number from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2; for example, q may represent 2 or 3;

[0101] FG represents a second functional group.

[0102] In some embodiments, the linker molecule has the following general formula:

[0103]

[0104] wherein the cleavable moiety, e.g., the hydrolyzable moiety is a Schiff base moiety comprising a -C=NO- bond;

[0105] p represents a number from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5; for example, p may be 4;

[0106] q represents a number from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2; for example, q may be 2 or 3;

[0107] FG represents a second functional group.

[0108] In some embodiments, the linker molecule has the following general formula:

[0109]

[0110] Where p is 4 and q is 2 or 3.

[0111] In some embodiments, the linker molecule has the following general formula:

[0112]

[0113] Where p is 4, and q is 2 or 3.

[0114] In some embodiments, the biomolecule is a polynucleotide, such as a DNA strand or an RNA strand.

[0115] In some embodiments, the first functional group LG comprises a CH2-substituted trivalent sulfonium ion containing an R group, for example, wherein the R group is represented by FG-ZABCYU-CH2-, which can be used to form a covalent bond between the CH2 group of the linker molecule and an atom of the biomolecule capable of alkylation. For example, the linker molecule can be a derivative of S-adenosyl-l-homocysteine, wherein LG is S-adenosyl-l-homocysteine. In this embodiment, the reaction center of the first functional group LG is the carbon atom of the R group bonded to the sulfonium center.

[0116] In some embodiments, the linker molecule is a cofactor for an enzyme-mediated reaction. In some embodiments, the linker molecule is an analog of the S-adenosyl-L-methionine cofactor.

[0117] In some embodiments, step b. forming a covalent bond between the biomolecule and the first functional group can further include providing a catalyst, such as an enzyme, such as a DNA methyltransferase capable of transferring an alkyl group from the S-adenosyl-l-methionine cofactor analog.

[0118] In some embodiments, the linker molecule has the following general formula:

[0119]

[0120] Wherein FG represents the second functional group;

[0121] Z represents a non-reactive group selected from one of an aliphatic bond or an aromatic bond;

[0122] ABC represents a cleavable moiety, such as a hydrolyzable moiety, such as a Schiff base moiety;

[0123] Y represents a non-reactive group selected from one of an aliphatic bond or an aromatic bond;

[0124] U represents an unsaturated bond selected from one of the following: an alkene, an alkyne, an aryl group, a carbon atom containing a carbonyl group, a sulfur atom containing one or two S=O bonds;

[0125] k represents an integer of 1 or 2; and

[0126] W - It is a counter ion.

[0127] Counterion W - It can be carbonate anion (CO3 2- ), bicarbonate (HCO3 - ), tetrafluoroborate anion (BF4 - ), hexafluorophosphate anion (PF6 - ), acetate (OAc - ), trifluoroacetate anion, formate anion, halide (such as F - , Cl - Br - ,I - The following embodiments include sulfonium ions and also include counter ions, although they are not shown.

[0128] Advantageously, the linker molecule can be used to write, erase and rewrite polynucleotides in the method according to the present invention. In the prior art, the established and emerging methods for studying deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein usually rely on its conjugation with functional chemical groups (such as biotin or fluorophore). These functionalities have realized the operation from conventional purification using immunoprecipitation to advanced analytical research (such as fluorescence imaging). However, the array of biological conjugation chemistry methods currently used to connect chemical functionality with biomolecules is inert to a large extent. Therefore, conjugates focus subsequent work on a single application or analysis.

[0129] Methyltransferases (MTases) are emerging as important tools for site-selective modification of DNA, RNA, and proteins. In nature, methyltransferases catalyze the highly specific transfer of methyl groups from the S-adenosyl-l-methionine cofactor to DNA or RNA. The introduction of methyl groups into these classes of biomolecules helps regulate gene expression levels within cells. This can modify chromatin structure through histone methylation, or directly methylate DNA and RNA.

[0130] In mTAG labeling, an S-adenosyl-l-methionine cofactor analog is employed in which the methyl group is exchanged for a different moiety, such as a linker moiety or a label. A methyltransferase can then be used to alkylate a target biomolecule with the different moiety using the modified cofactor. By manipulating the chemical structure of the naturally occurring S-adenosyl-l-methionine cofactor, this labeling process can be used as a method for covalently introducing functional groups into biomolecules. The linker moiety may contain additional functionalities that can be used to further functionalize the biomolecule, such as with a marker, tag, or other biomolecule. One of the most common applications explored using this approach is the introduction of clickable groups into DNA to introduce fluorophores for mapping.

[0131] EP1712557 describes methods for labeling biopolymers using mTAG. It was shown that the reactivity of S-adenosyl-l-methionine cofactor analogs towards extended saturated alkyl transfer to DNA biopolymers can be improved by adding unsaturated carbon-carbon bonds such as olefins, alkynes or aromatic substituents at the beta position to the sulfonium center.

[0132] There have been several reports on methods for reversibly labeling polynucleotide biomolecules. For example, it is also known to use mTAG labeling followed by coupling with a cross-linking reagent containing cleavable disulfide bonds using the redox reagent dithiothreitol (DTT). This allows the capture and release of DNA molecules for sequencing. However, disulfide chemistry has many disadvantages that limit its use in biological systems. For example, thiols are present in most biological samples and buffers. This results in a lower efficiency of labeled biomolecules due to competing reactions. In addition, disulfide chemistry cannot be applied to proteins because the oxidation state of cysteine ​​usually determines the 3D structure of the protein, so changes in the oxidation state lead to protein denaturation.

[0133] The methods of the invention using such linker molecules are bioorthogonal.

[0134] Advantageously, linker molecules according to embodiments of the invention comprising a trivalent sulfonium ion comprise an unsaturated moiety U in the β-position of the sulfonium center. This enhances the reactivity of the linker molecule towards alkylation of the polynucleotide biomolecule using a methyltransferase.

[0135] In some embodiments, U may represent an alkyne. In some embodiments, the linker molecule may have the following general formula:

[0136]

[0137] wherein FG, Z, ABC, Y, and k are as defined above.

[0138] In some alternative embodiments, U may represent an alkene. In some embodiments, the linker molecule may have the following general formula:

[0139]

[0140] wherein FG, Z, ABC, Y, and k are as defined above.

[0141] In some embodiments, the linker molecule may have the following general formula:

[0142]

[0143] wherein the cleavable moiety, e.g., the hydrolyzable moiety is a Schiff base moiety comprising a C=NXCQ moiety;

[0144] p represents a number from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5; for example, p may be 4;

[0145] Q represents one of the following: an oxygen atom or two hydrogen atoms or a deuterium atom independently bonded to a carbon center;

[0146] U represents an unsaturated bond selected from one of the following: an alkene, an alkyne, an aryl group, a carbon atom containing a carbonyl group, a sulfur atom containing one or two S=O bonds;

[0147] X represents one of an oxygen atom or a nitrogen atom;

[0148] Z represents a non-reactive group selected from one of an aliphatic bond or an aromatic bond;

[0149] k represents an integer of 1 or 2; and

[0150] FG represents a second functional group.

[0151] In some embodiments, the linker molecule has the following general formula:

[0152]

[0153] wherein the cleavable moiety, e.g., the hydrolyzable moiety is a Schiff base moiety comprising a -C=NNC=O bond;

[0154] p represents a number from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5; for example, p may be 4;

[0155] q represents a number from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2; for example, q may represent 2 or 3;

[0156] k represents an integer of 1 or 2; and

[0157] FG represents a second functional group.

[0158] In some embodiments, the linker molecule has the following general formula:

[0159]

[0160] wherein the cleavable moiety, e.g., the hydrolyzable moiety is a Schiff base moiety comprising a -C=NO- bond;

[0161] p represents a number from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5; for example, p may be 4;

[0162] wherein q represents a number from 1 to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; or a number from 1 to 10; or a number from 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2; for example, q may be 2 or 3;

[0163] k represents an integer of 1 or 2; and

[0164] FG represents a second functional group.

[0165] In some embodiments, the linker molecule has the following general formula:

[0166]

[0167] wherein p is 4 and q is 2 or 3; k is as defined above.

[0168] In some embodiments, the linker molecule has the following general formula:

[0169]

[0170] wherein p is 4 and q is 2 or 3; k is as defined above.

[0171] In some embodiments, hydrolyzing the cleavable portion of the linker molecule, e.g., the hydrolyzable portion, e.g., the Schiff base portion, to remove a label, e.g., a first label, or an nth label; and to form a third functional group can comprise treating with hydroxylamine, e.g., in an ammonium acetate buffer solution, e.g., at pH 4.

[0172] In some alternative embodiments, hydrolyzing the Schiff base moiety may include heating the linker molecule and / or treating with an acid.

[0173] In the method of the present invention, the reaction center of the first functional group reacts with the biomolecule to form a covalent bond. The biomolecule comprises a functional group that can react with the first functional group of the linker molecule to form a covalent bond. This may comprise a chemical reaction or an enzyme-mediated reaction. Advantageously, the first functional group of the linker molecule may be selected to react with a specific functional group on a specific biomolecule. In some embodiments, the covalent bond formed between the biomolecule and the reaction center of the first functional group may be, for example, a carbon-carbon bond, a carbon-nitrogen bond, a carbon-sulfur bond, or a carbon-oxygen bond.

[0174] In some embodiments, the second and / or fourth functional group FG can be selected from one of the following: halides (e.g., F, Cl, Br, or I atoms), unsaturated bonds (e.g., olefins, alkynes), azides, activated esters, activated carbonates / esters, carbamates, epoxides, isothiocyanates / esters, or isocyanates / ester moieties. In the method of the present invention, the reaction center of the second functional group reacts with the first label to form a covalent bond. The first label comprises a functional group that can react with the first functional group of the linker molecule to form a covalent bond. Advantageously, the second functional group of the linker molecule can be selected to react with a specific functional group on a specific label. In some embodiments, wherein the second functional group comprises, for example, an azide moiety, the functional group of the first, second, or nth linker can be an alkynyl group, which can react with the azide moiety by click chemistry. In some embodiments, the covalent bond formed between the linker molecule and the label can be a carbon-nitrogen bond, for example, formed by reacting an azide and an alkyne.

[0175] In some embodiments, one or more labels, such as the first label, the second label, and / or the nth label, may comprise one or more of a fluorescent molecule, a radioactive substance, and / or a biological molecule such as biotin.

[0176] The third functional group is formed by hydrolysis of a hydrolyzable portion of the linker molecule, such as a Schiff base portion. In some embodiments, the third functional group comprises an NH2 group of an O-substituted hydroxylamine portion or an N-substituted hydrazone portion.

[0177] The third functional group can be used in a method of reforming a hydrolyzable moiety, such as a Schiff base moiety, by reacting with other molecules to form a covalent bond. In some embodiments, the other molecule comprises an aldehyde moiety that can react with the third functional group, such as an NH2 moiety, to reform a Schiff base. The covalent bond can comprise a carbon-nitrogen double bond, i.e., C=N.

[0178] In some embodiments, the other molecule is represented by the following formula:

[0179]

[0180] Where R 2 represents one of a hydrogen atom, a deuterium atom, an aliphatic or aromatic moiety, or a heteroatom (e.g., oxygen, nitrogen, sulfur);

[0181] R 3 represents one of an aliphatic moiety or an aromatic moiety;

[0182] FG represents a second functional group, for example, selected from one of an azide, an alkyne, an isothiocyanate or an isocyanate moiety or a label.

[0183] In some embodiments, the other molecule is represented by the following formula:

[0184]

[0185] Other aspects of the invention provide a method for reversibly labeling a polynucleotide molecule, such as DNA, comprising:

[0186] a. providing a linker molecule (compound A) having the following general formula:

[0187]

[0188] Wherein R represents a transferable group;

[0189] FG represents the second functional group;

[0190] Z represents a non-reactive group selected from one of an aliphatic bond or an aromatic bond;

[0191] ABC represents a cleavable moiety, such as a hydrolyzable moiety, such as a Schiff base moiety;

[0192] Y represents a non-reactive group selected from one of an aliphatic bond or an aromatic bond;

[0193] U represents an unsaturated bond selected from one of the following: an alkene, an alkyne, an aryl group, a carbon atom containing a carbonyl group, a sulfur atom containing one or two S=O bonds;

[0194] k represents an integer of 1 or 2;

[0195] b. forming a covalent bond between the polynucleotide molecule and the R group of compound A using a DNA methyltransferase capable of using compound A as a cofactor and under conditions that allow the R group of compound A to be transferred to the polynucleotide molecule;

[0196] c. forming a covalent bond between the first label and the second functional group FG of compound A;

[0197] d. hydrolyzing a cleavable portion of the linker molecule, such as a hydrolyzable portion, such as a Schiff base portion, to remove the first label and form an O-substituted hydroxylamine or N-substituted hydrazone;

[0198] e. Forming a covalent bond between the aldehyde portion of the other molecule and the O-substituted hydroxylamine or N-substituted hydrazone to reform the Schiff base portion.

[0199] Another aspect of the present invention provides a method for reversibly labeling a polynucleotide molecule such as DNA, the method comprising the following steps in a specified order:

[0200] a. incubating the polynucleotide molecule with compound A in the presence of a methyltransferase capable of using compound A as a cofactor and under conditions that allow the R group of compound A to be transferred to the polynucleotide molecule to form an alkylated nucleotide molecule "R-polynucleotide", wherein compound A is represented by the following general formula;

[0201] Compound A:

[0202]

[0203] Wherein FG represents the second functional group;

[0204] Z represents a non-reactive group selected from one of an aliphatic bond or an aromatic bond;

[0205] ABC represents a cleavable moiety, such as a hydrolyzable moiety, such as a Schiff base moiety;

[0206] Y represents a non-reactive group selected from one of an aliphatic bond or an aromatic bond;

[0207] U represents an unsaturated bond selected from one of the following: an alkene, an alkyne, an aryl group, a carbon atom containing a carbonyl group, a sulfur atom containing one or two S=O bonds;

[0208] k represents an integer of 1 or 2;

[0209] b. incubating the alkylated polynucleotide molecule "R-polynucleotide" with a first label, the first label comprising a functional group capable of reacting with the functional group FG of the R group to form a covalent bond between the "R-polynucleotide" and the first label to form a first conjugate;

[0210] c. cleaving a cleavable moiety, such as a hydrolyzable moiety, such as a Schiff base moiety, of the first conjugate to remove the first label;

[0211] d. reactivating the cleavable moiety, e.g., the hydrolyzable moiety, e.g., the Schiff base moiety to reform the "R-polynucleotide";

[0212] e. Incubating the "R-polynucleotide" with a second label comprising a functional group capable of reacting with the functional group FG of the R group to form a covalent bond between the "R-polynucleotide" and the second label to form a second conjugate.

[0213] In some embodiments, the method may further include cleaving the Schiff base moiety of the second conjugate to remove the second label.

[0214] Advantageously, the method according to the invention is bioorthogonal, in other words, it can be performed in biological systems without interfering with natural biochemical processes. Even more advantageously, once reversed, the modification of a polynucleotide biomolecule such as DNA is relatively small and hydrophilic, meaning that it does not affect the way the DNA interacts in solution or with enzymes.

[0215] The linker molecule comprises a cleavable portion, such as a hydrolyzable portion, such as a Schiff base portion, which is cleavable or hydrolyzable so that the label can be reversibly conjugated to the biomolecule. Advantageously, the conditions required for cleaving or hydrolyzing the cleavable portion or the hydrolyzable portion (e.g., Schiff base portion) are mild and do not destroy the structure of the biomolecule. Interestingly, Schiff base chemistry is not common in biomolecules, unlike prior art methods such as the use of disulfide bonds. More advantageously, hydrolyzable portions such as oximes and hydrazones are stable at physiological pH.

[0216] The covalent bonds formed using linker molecules are reversible and rewritable. Advantageously, label molecules can be used for repeated modification of biomolecules, such as labeling, capturing, releasing, refunctionalization, for example, fluorescent labeling and / or imaging.

[0217] Analogs of the S-adenosyl-l-methionine cofactor can be designed to include linker molecules of any suitable structure.

[0218] Advantageously, the second functional group of the linker molecule can be used to further functionalize a biomolecule, e.g., a polynucleotide, e.g., using click chemistry. This can be used for DNA capture, DNA complexing, drug attachment, and / or fluorescent labeling.

[0219] Within the scope of the present application, it is explicitly intended that the aspects, embodiments, examples and alternatives set forth in the preceding paragraphs, in the claims and / or in the following description and in the accompanying drawings, in particular their respective features, may be taken independently or in any combination. In other words, all embodiments and / or the features of any embodiment may be combined in any manner and / or composition unless these features are incompatible. To avoid doubt, the terms "may", "and / or", "such as", "for example" and any similar terms as used herein should be interpreted as non-restrictive, and therefore no features described in this manner need be presented. In fact, without departing from the scope of the present invention, any combination of optional features is explicitly envisioned, whether or not these are explicitly claimed. The applicant reserves the right to change any initially filed claims or to submit any new claims accordingly, including the right to amend any initially filed claims to be subordinate to any other claims and / or to include any features of any other claims, although protection was not initially claimed in this manner.

[0220] Some embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0221] Figure 1 is a schematic representation of reversible and rewritable modification of biomolecules according to one embodiment of the present invention;

[0222] Figure 2A is a schematic representation of a linker molecule according to one embodiment of the present invention;

[0223] Figure 2B is a schematic representation of reversible and rewritable modification of biomolecules according to other embodiments of the present invention;

[0224] Figure 3 is a reaction scheme for forming a linker molecule precursor containing hydrazone Schiff base 21A;

[0225] Figure 4 is a reaction scheme for forming a linker molecule precursor containing oxime Schiff base 21B;

[0226] Figure 5 is a reaction scheme for forming S-adenosyl- l -methionine cofactor analogs;

[0227] Fig. 6A is a graphic representation of the limit determination;

[0228] Figure 6B Analysis of enzymatic DNA labeling by gel electrophoresis is shown;

[0229] Figure 7 shows the analytical HPLC chromatogram after rewriting the original functionality of DNA;

[0230] Figure 8 is a schematic representation of dual functionalization of DNA; and

[0231] Fig. 9 is a reaction scheme for forming other linker molecules.

[0232] Reference Figure 1 , shows a schematic diagram 1 of a reversible and rewritable modification of a biomolecule 10 according to one embodiment of the present invention.

[0233] Shown are a biomolecule 10 and a linker molecule 11. The linker molecule 11 comprises a first functional group LG containing a reaction center, a second functional group FG containing a reaction center, a hydrolyzable portion (e.g., a Schiff base portion) ABC, non-reactive groups Y and Z, and an unsaturated bond U. Also shown are a first label L1 and a second label L2.

[0234] The method of reversibly labeling a biomolecule 10 comprises the following steps, which are labeled in the figure:

[0235] a) providing a linker molecule 11, the linker molecule 11 comprising a first functional group LG containing a reaction center, a second functional group FG containing a reaction center, and a hydrolyzable portion, such as a Schiff base portion, ABC;

[0236] b) forming a covalent bond between the biomolecule 10 and the reaction center of the first functional group LG;

[0237] c) forming a covalent bond between the first label L1 and the reaction center of the second functional group FG;

[0238] d) hydrolyzing the hydrolyzable portion ABC of the linker molecule 11 to remove the first label L1 and to form a third functional group W comprising a reaction center;

[0239] e) Formation of a covalent bond between the other molecule (not shown) and the reactive center of the third functional group W to reform the hydrolyzable moiety ABC.

[0240] Advantageously, the covalent bond formed between the reactive center of the first functional group LG of the linker molecule 11 and the biomolecule 10 adds chemical functionality to the biomolecule 10 in the form of the further functional groups FG and the hydrolyzable moiety ABC.

[0241] The method of the present invention may further comprise the optional step i. forming a covalent bond between another molecule comprising a label (eg, the second label L2) and the reaction center of the third functional group W to reform the hydrolyzable moiety ABC.

[0242] In some embodiments, step c can be performed before step b.

[0243] First reference Figure 2A , two linker molecular structures are shown; 21A and 21B.

[0244] Also refer to Figure 2B , shows a schematic diagram 2 of a reversible and rewritable modification of a biomolecule 20 according to other embodiments of the present invention.

[0245] A biomolecule 20 and a linker molecule 21 are shown. The linker molecule 21 may be Figure 2A Any of the molecules shown in . The linker molecules 21A, 21B each comprise a first functional group LG, a second functional group FG and a hydrolyzable portion ABC.

[0246] A first marker L1 ' and a second marker L2' are also shown.

[0247] In this embodiment, the biomolecule 20 is a DNA molecule and the linker molecule 21 is an analog of the S-adenosyl-l-methionine cofactor.

[0248] The first functional group LG of the linker molecules 21A and 21B is S-adenosyl-l-homocysteine, the second functional group FG of the linker molecules is an azide moiety, and the hydrolyzable moiety is one of a hydrazone (linker 21A) or an oxime (linker 21B).

[0249] In this embodiment, the hydrolysis of the hydrolyzable moiety (e.g., Schiff base) ABC further comprises treatment with hydroxylamine, e.g., in an ammonium acetate buffer solution, e.g., at pH 4. The hydrolyzable moiety ABC is hydrolyzed to provide a third functional group W comprising a reactive center; an N-substituted hydrazone (wherein the Schiff base is hydroxylamine) or an O-substituted hydroxylamine (wherein the Schiff base is an oxime).

[0250] Other molecules 22 used in step e to form a covalent bond with the NH2 group of the N-substituted hydrazone (21A) or the O-substituted hydroxylamine (21B) to reform the Schiff base moiety of the hydrazone (21A) or the oxime (21B) are also shown. Figure 2A middle.

[0251] In this embodiment, the functional groups of the first label L1' and the second label L2' are both alkyne moieties, which form CN covalent bonds with the reaction center (azide moiety) of the second functional group FG through click chemistry reaction.

[0252] In this embodiment, step b of method 2 further comprises providing a catalyst, such as an enzyme, such as a DNA methyltransferase capable of transferring an alkyl group from the S-adenosyl-l-methionine cofactor analog.

[0253] The reversible and rewritable modification of the DNA molecule 20 includes the following steps, which are labeled on the diagram:

[0254] a) Providing a linker molecule 21A or 21B

[0255] The linker molecule 21A or 21B comprises an S-adenosyl-l-homocysteine ​​moiety, which comprises a CH2-substituted trivalent sulfonium ion moiety LG, an azide moiety FG and a hydrazone (21A) or an oxime (21B) or a Schiff base moiety ABC;

[0256] b) Site-selective methyltransferase-directed writing of DNA molecule 20

[0257] forming a covalent bond between the DNA molecule 20 (at one or more of cytosine C5, cytosine N4, or adenosine N6) and the carbon of the CH2 group of the linker molecule 21A or 21B;

[0258] c) Modification of DNA molecules by azide-alkyne cycloaddition 20

[0259] forming a CN covalent bond between the alkyne portion of the first label L1' and the azide of the linker molecule 21A or 21B;

[0260] d) Functionality introduced by dynamic exchange erasure

[0261] The hydrazone (21A) or oxime (21B) Schiff base portion of the linker molecule 21A or 21B is hydrolyzed to form an N-substituted hydrazone (21A) or an O-substituted hydroxylamine (21B).

[0262] e) Rewriting of intermediate DNA molecules through Schiff base formation

[0263] A covalent bond is formed between another molecule (not shown) and the NH2 group of the N-substituted hydrazone (21A) or O-substituted hydroxylamine (21B) to reform the Schiff base moiety of the hydrazone (21A) or oxime (21B).

[0264] Optionally, step i may involve further functionalization of the DNA intermediate by standard conjugation techniques.

[0265] Other molecules, such as the other molecules of step e, or the other molecules of step i, comprise an aldehyde moiety for reacting with the NH2 functionality of the third functional group to reform the Schiff base.

[0266] To further illustrate the present invention, reference is also made to the following non-limiting examples:

[0267] Synthesis of Precursor 1 for Synthesis of Linker 21A

[0268] Reference Figure 3 , shows a reaction scheme for forming the precursor 1 of the linker molecule containing the hydrazone Schiff base 21A. The precursor 1 was synthesized using the following scheme.

[0269] 1.1 Synthesis of 8-hydroxyoct-6-ynoic acid 7.

[0270] Under argon, in anhydrous THF (42ml), a solution of 6-heptynoic acid (2g, 15.87mmol) was prepared, HMPA (34.9mmol, 6.13ml) was added thereto and the solution was cooled to -78°C. nBuLi (1.6M in hexane, 34.9mmol, 21.8ml) was added dropwise thereto while keeping the temperature below -60°C. The solution was then warmed to -40°C and stirred for 1 hour. After 1 hour, paraformaldehyde (1.47g, 47.6mmol) was added by a powder funnel under an argon stream. The reaction mixture was then warmed to 45°C and kept for 4 hours. After the reaction, the mixture was quenched to pH 4 to 5 with 1M HCl and extracted with EtOAc. The solvent was then dried and EtOAc was removed by rotary evaporation to obtain a crude product. Purification was completed using flash column chromatography (silica gel, Hex: EtOAc, 6: 4):

[0271] Yield = 68%, Rf = 0.27 (Hex:EtOAc, 6:4);

[0272] 1 H NMR (300MHz, DMSO-d6) δ12.03 (s, 1H), 5.03 (s, 1H), 4.02 (d, J=2.6Hz, 2H), 2. 29-2.14(m, 4H), 1.63-1.50(m, 2H), 1.50-1.39(m, 2H); MS: m / z[MH]=155.46.

[0273] 1.2 Synthesis of tert-butyl 2-(8-hydroxyoct-6-ynyl)hydrazine-1-carboxylate 8

[0274] 8-Hydroxyoctyl-6-ynoic acid 7 (1.35 g, 8.65 mmol) and t-butyl carbazate (1.4 g, 10.38 mmol) were dissolved in 2:1 THF:H2O (13.5:6.75 ml). EDC.HCl (1.87 g, 9.52 mmol) was slowly added thereto over 15 minutes. The mixture was stirred for 3 hours and then extracted with EtOAc. The organic layer was washed with 0.1 M HCl, water and brine, then the organic layer was collected, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the product as a white solid:

[0275] Yield = 63%; 1H NMR (400MHz, DMSO-d6) δ9.47 (s, 1H), 8.66 (s, 1H), 5.04 (t, J=5.9Hz, 1H), 4.02 (dt, J=5.9, 2.2Hz, 2 H), 2.19 (tt, J=7.1, 2.2Hz, 2H), 2.06 (t, J=7.2Hz, 2H), 1.58 (p, J=7.3Hz, 2H), 1.50-1.32 (m, 12H); 13 C NMR (101MHz, DMSO) δ172.01, 84.36, 80.94, 79.42, 49.59, 33.06, 28.53, 28.08, 24.70, 18.24; MS: m / z [M+Na] = 294.15.

[0276] 1.3 Synthesis of tert-butyl 2-(8-bromooctan-6-ynyl)hydrazine-1-carboxylate 1

[0277] A solution of tert-butyl 2-(8-hydroxyoctyl-6-alkyne acyl)hydrazine-1-carboxylate 8 (300mg, 1.11mmol) was prepared in anhydrous DCM (3.33ml) and cooled on ice. Triphenylphosphine (437mg, 1.67mmol) was added and dissolved, and once dissolved, tetrabromomethane (552mg, 1.67mmol) was slowly added. The reaction was then warmed to room temperature and stirred for 1 hour. The solvent was removed under reduced pressure after the reaction, and the crude mixture was purified by flash column chromatography (silica gel Hex: EtOAc, 7: 3):

[0278] Yield = 55%; Rf = 0.15 (Hex:EtOAc 7:3); 1 H NMR (300MHz, DMSO-d6) δ9.48 (s, 1 H), 8.67 (s, 1H), 4.21 (t, J=2.3Hz, 2H), 2.27 (tt, J=6.9, 3.4Hz, 2H), 2.06 (t, J=7.4Hz, 2H), 1.65-1.31 (m, 13H); 13 C NMR (101MHz, DMSO) δ171.4, 155.2, 87.7, 78.9, 76.3, 54.9, 39.5, 32.5, 28.0, 27.3, 24.1, 17.9, 17.2; MS: m / z [M+Na] = 355 / 357.08.

[0279] Synthesis of Precursor 4 for Synthesis of Linker 21B

[0280] Reference Figure 4, shows a reaction scheme for the precursor 4 used to form the linker molecule containing the oxime Schiff base 21B. Precursor 4 was synthesized using the following scheme.

[0281] 2.1 Synthesis of 7-bromo-hept-1-yne 9

[0282] Prepare a solution of 6-heptyne-1-alcohol (5g, 44.6mmol) in anhydrous DCM (60ml) and cool on ice. Add triphenylphosphine (17.6g, 67mmol) thereto, slowly add tetrabromomethane (22.2g, 67mmol) after complete dissolution. The reaction mixture is warmed to room temperature and stirred for 1 hour. After completion, remove the solvent under reduced pressure. Hexane is added to the crude product to form a white suspension. Filter the hexane fraction, collect, and then remove the solvent. The remaining oily residue is purified by flash column chromatography with hexane:

[0283] Yield = 91%, Rf = 0.45 (hexane); %); vmax (neat) / cm-1 540 (C-Br); 1 H NMR (300MHz, DMSO-d6) δ3.53 (t, J=6.7Hz, 2H), 2.75 (t, J=2.7Hz, 1H), 2.23-2.10 (m, 2H), 1.89-1.74 (m, 2H), 1.50-1.43 (m, 4H).

[0284] 2.2 Synthesis of 8-bromooctan-2-yn-1-ol 10

[0285] A solution of 7-bromohept-1-yne 9 (20.56mmol, 3600mg) was prepared in anhydrous THF (12.3ml) and cooled to -78°C under argon. A solution of nBuLi in hexane (1.6M, 13ml) was added dropwise thereto while keeping the temperature below -60°C. The reaction mixture was then warmed to 0°C in an ice bath, at which time paraformaldehyde (1718mg, 55.5mmol) was added under an argon stream and stirred for 30 minutes. The mixture was then warmed to room temperature and stirred, with the temperature being maintained at less than 30°C until the exothermic reaction ceased. The mixture was then heated to 45°C for 2 hours. Once completed, the reaction was extracted with ether and saturated NH4Cl. The organic layer was collected, and the solvent was removed under reduced pressure to obtain a crude product as an oil. Once dried, purification was completed by flash column chromatography (silica gel, hexane: ethyl acetate, 9:1). The product as a colorless oil was then collected:

[0286] Yield = 55%, Rf = 0.15 (Hex: EtOAc 9: 1), 1H NMR (300MHz, DMSO-d6) δ5.04 (t, J=5.7Hz, 1H), 4.03 (dt, J=5.5, 2.1Hz, 2H), 3.54 (t, J=6.7Hz, 2H), 2.20 (m, 2H), 1.88-1.75 (m, 2H), 1.52-1.40 (m, 4H).

[0287] 2.3 Synthesis of tert-butyl ((8-hydroxyoct-6-yn-1-yl)oxy)carbamate 11

[0288] To a solution of N-Boc hydroxylamine (890 mg, 6.55 mmol) in DMF (4.3 ml) was added 8-bromooct-2-yn-1-ol (10) (1200 mg, 5.85 mmol) and 1,8-diazacyclo[5.4.0]undec-7-ene (1000 mg, 6.55 mmol). The solution was stirred at 50 °C for 20 hours. Once complete, the reaction was extracted with DCM and 15% citric acid solution. The organic phase was dried and collected, and the solvent was removed under reduced pressure. A colorless oil was collected as a crude product. It was further purified by flash column chromatography (silica gel, hexane: ethyl acetate, 8:2). The product was collected as a colorless oil:

[0289] Yield = 73%, Rf = 0.27; 1 H NMR (300MHz, DMSO-d6) 69.91 (s, 1H), 5.03 (t, J=5.9Hz, 1H), 4.02 (dt, J=5.9, 2.2Hz, 2H) , 3.66 (t, J=6.2Hz, 2H), 2.17 (tt, J=6.7, 1.7Hz, 2H), 1.40 (m, 15H); MS: m / z[M+H]=258.2.

[0290] 2.4 Synthesis of tert-butyl ((8-bromooct-6-yn-1-yl)oxy)carbamate 4

[0291] A solution of tert-butyl ((8-hydroxyoct-6-alkynes-1-yl)oxy)carbamate 11 (1 g, 3.89 mmol) was prepared in anhydrous DCM (5.2 ml) and cooled on ice. Triphenylphosphine (1.53 g, 67 mmol) was added thereto. After complete dissolution, tetrabromomethane (1.94 g, 67 mmol) was slowly added. The reaction mixture was warmed to room temperature and stirred for 1 hour. After completion, the solvent was removed under reduced pressure. Purification was completed using flash column chromatography (silica gel, hexane: ethyl acetate, 8:2):

[0292] Yield = 67%, Rf 0.52 (Hex:EtOAc, 8:2); vmax (neat) / cm-1 1712 (C=O), 607 (C-Br); 1 H NMR (300MHz, DMSO-d6) δ9.90 (s, 1H), 4.21 (t, J=2.4Hz, 2H), 3.66 (t, J=6.2Hz, 2H), 2.25 (tt, J=6.9, 2.4Hz, 2H), 1.40 (m, 15H); 13 C NMR (101MHz, DMSO) δ156.04, 87.85, 79.37, 76.22, 75.05, 39.52, 28.05, 27.64, 27.04, 24.76, 18.06, 17.25; MS: m / z [M+Na]=342.35 / 344.35, [M- t BuOH]=246.38 / 248.38.

[0293] Examples of linker molecules for enzymatic labeling of polynucleotides: methyltransferase-directed labeling of polynucleotides

[0294] Synthesis of linker molecules 21A and 21B

[0295] Reference Figure 5 , shows a reaction scheme 5A for forming a linker molecule containing a hydrazone Schiff base 21A from precursor 1. Also shown is a reaction scheme 5B for forming a linker molecule containing an oxime Schiff base 21B from precursor 4.

[0296] 3) Synthesis of S-adenosyl-1-methionine cofactor analogs 21A and 21B

[0297] 3.1 General coupling process

[0298] Precursors 1, 4 were prepared and reacted with S-adenosyl-L-homocysteine ​​under acidic conditions to give reversible and rewritable Boc-protected AdoMet derivatives.

[0299] A solution of S-adenosyl-1-homocysteine ​​(15 mg, 0.04 mmol) was prepared in a 1:1 mixture of formic acid and acetic acid (300 μl). Precursor 1 or 4 (tert-butyl 2-(8-bromooctan-6-ynoyl)hydrazone-1-carboxylate or tert-butyl ((8-bromo-6-yn-1-yl)oxy)carbamate) (1.2 mmol, 30 equiv) was added dropwise on ice. The reaction mixture was warmed to 35° C. and stirred overnight. After stirring overnight, the reaction mixture was extracted with ether, and the aqueous layer was collected and dried by lyophilization: MS: m / z [M+H]=638 (2), [M+H]=624 (5).

[0300] 3.2 Cofactor deprotection

[0301] AdoMet analogs are deprotected under acidic conditions to reveal the hydrazone or alkoxyamine moiety.

[0302] The crude product was dissolved in TFA (400 μl) and stirred at room temperature for 2 hours. After the reaction the acid was removed under a stream of argon.

[0303] 3.3 Cofactor purification

[0304] Any excess precursor was removed by purification.

[0305] Both diastereomers of the deprotected cofactor could be separated by HPLC, their separation was not possible at a later stage.

[0306] The crude reaction mixture was then dissolved in water (2 ml). Purification of the AdoMet analog was performed by preparative reverse phase HPLC (ACE 5 C-18 25×2.12 cm) with a gradient elution of 20 mM ammonium acetate pH 5.5 water (A) / MeOH (B) at a flow rate of 10 ml / min. Gradient system: 3 to 30% B in 30 min, 30 to 97% B in 30 min, hold at 97% B for 5 min, program aborted. Retention times: hydrazide iso.1=17.51 ​​min, iso.2=18.73 min, hydroxylamine iso.1=25.47 min, iso.2=28.24 min: MS: m / z [M+H]=538 (2), [M+H]=524 (5).

[0307] The deprotected AdoMet derivatives slowly degraded through multiple pathways, especially after freeze-drying, giving rise to additional peaks at higher retention times.

[0308] 3.4 Aldehyde coupling

[0309] To mitigate degradation, the AdoMet derivatives were reacted with commercially available benzaldehyde immediately after purification by HPLC to minimize side reactions due to the nucleophilic nature of the hydrazone and alkoxyamine moieties.

[0310] To the collected HPLC fractions was added Ald-PEG3-N3 (1.2 eq) and shaken at room temperature for 30 min. The fractions were then dried by lyophilization. Once dry, the solids were dissolved in 100 μl 0.1% acetic acid and stored at -20°C. The HPLC was performed by using ε260 = 15.400 dm -3 mol -1 cm -1 The concentration was determined by UV absorption analysis: MS: m / z [M+H]=867 (3), [M+H]=856 (6).

[0311] The resulting linker molecules 21A and 21B contain reactive terminal azides that can be readily conjugated to a range of functional groups, while condensation of aldehydes with hydrazones or alkoxyamines allows for the incorporation of dynamic functionality that can be reversibly functionalized.

[0312] A slight excess of aldehyde (1.2 equiv.) was used to ensure complete functionalization of the deprotected intermediate.

[0313] No degradation of the freeze-dried AdoMet derivatives 21A and 21B was observed.

[0314] Methyltransferase-directed labeling of polynucleotides using 21A and 21B

[0315] Restriction assays were used to demonstrate the activity of the methyltransferase in the presence of linker molecules 21A and 21B.

[0316] M. TaqI (N6-adenine DNA methyltransferase) was incubated with linker molecules 21A, 21B and plasmid pUC19 with four enzyme recognition sites (TCGA) at 50°C for 1 hour, see Fig. 6A .

[0317] Successful transfer of the functional group by M.TaqI resulted in protection of the plasmid from restriction digestion with R.TaqI, an endonuclease with the same target site as M.TaqI.

[0318] Reference Figure 7 B, shows gel electrophoresis of pUC19 after enzymatic treatment with M.TaqI and / or R.TaqI in the presence or absence of methionine (375 μl) or linker 21B.

[0319] In the absence of M.TaqI-mediated alkylation ( Figure 6B , lanes 4, 8 and 12), pUC19 was cut into fragments, the largest three of which could be identified by gel electrophoresis. M. TaqI-mediated binding to methionine ( Figure 6B , lane 10) or linker 21B ( Figure 6B Alkylation of the isoforms (lanes 1 to 3 and 5 to 7) resulted in partial to complete freedom from R.TaqI restriction, as observed by gel electrophoresis as predominantly open circular and supercoiled plasmid DNA. Neither isoform interfered with the ability of R.TaqI to digest plasmid DNA ( Figure 6B , lanes 4 and 8).

[0320] In the absence of methionine ( Figure 6B , Lanes 11 and 12), without M.TaqI ( Figure 6B , lanes 4, 8 and 12) and without R.TaqI ( Figure 6B , lane 9) and shows that labeling was successful.

[0321] To test the labeling efficiency of each isomer, a cofactor dilution series was run to highlight any differences in affinity to the enzyme, with the second fraction having higher activity. The preferred isomer, diastereomer II, was used for subsequent experiments.

[0322] Similar effects were observed with linker 21 A. Both linker molecules 21A and 21B have the potential to be used for dynamic labeling of biomolecules.

[0323] M.Mpel is a cytosine-C5 methyltransferase that targets CpG dinucleotides. pUC19 was incubated with mutant M.Mpel (Q136A, N347A) and linker 21B, followed by the addition of the restriction enzyme R.HaeII that targets a subset of CpG dinucleotides. Efficient alkylation of plasmid DNA was also observed in the presence of M.Mpel.

[0324] By targeting a copy of the M.TaqI sequence (TCGA) for 14 base pair oligonucleotides for alkyl transfer, evidence of the ability of methyltransferase to alkylate DNA in the case of joints 21A and 21B was obtained. The labeling of oligonucleotides was directly monitored using HPLC. Analyzed at a melting temperature higher than that of DNA, so that two chains can be clearly identified in the chromatogram. When compared with the retention time of unmodified DNA, when labeled with joints 21A and 21B, an obvious movement of the retention time was observed. The movement of two peaks was observed, which showed that M.TaqI was able to label two chains due to the palindromic nature of the sequence recognized by the methyltransferase. This movement is directly proportional to the size and properties of the transfer joint, and the AdoMet methylation causes a slight movement of the retention time, and the movement of the oxime derivative 21B is the largest. The presence of a small amount of erased oligoDNA was observed, and it may be due to the hydrolysis under HPLC conditions. MS was used to analyze each peak, which confirmed the properties of the side chain functionality introduced after successful labeling and incubation.

[0325] Reversible enzyme labeling protocol

[0326] according to Figure 2A and 2B For the embodiment shown, the following scheme may be used.

[0327] 1) Enzyme labeling (step b of the method of the present invention)

[0328] For each sample, a solution of oligobiological molecules (120 μl, 10 μM), buffer (40 μl, 10×NEB cutsmartbuffer), M.TaqI (45 μl), water (189 μl) and linker molecules (6 μl, 20 mM) was prepared. The samples were incubated at 50°C for 1.5 hours. Proteinase K (2.5 μl) was added after incubation, and the samples were incubated at 50°C for another 1 hour. The samples were then purified using Qiagen (RTM) Qiaquick nucleotide purification kit and eluted into 50 μl water, and their concentrations were measured by shimadzubiospec-nano. The samples that were not reversed were taken out and stored in a refrigerator until HPLC analysis.

[0329] 2) Schiff base reversal (step c of the method of the present invention)

[0330] To the hydrazone-labeled DNA, H2NOH.HCl aqueous solution (10 μl, 10 equivalents) was added. The pH of the solution was then adjusted using 100 mM ammonium acetate buffer (pH 4.0, 7 μl). The samples were then incubated at 50°C for 1.5 hours and then stored in a refrigerator until analysis.

[0331] 3) HPLC marker analysis

[0332] The results were analyzed by reverse phase HPLC (Phenomenex, Gemini, 5 μm, C18, ), eluted with 0.1 M triethylamine acetate buffer, pH 7.0 (A) / MeCN (B) gradient at a flow rate of 1 ml / min at 60°C to perform purification of labeled oligonucleotides. Gradient system A: 5 to 18% B in 25 minutes, to 100% for 5 minutes, kept at 100% for 10 minutes, reduced to 5% for 5 minutes. System B: 5 to 31% B in 50 minutes, to 100% for 10 minutes, kept at 100% for 5 minutes, reduced to 5% for 10 minutes. For unlabeled and methylated oligonucleotides, gradient A was used, and for all remaining samples, system B was used. Fractions were collected and analyzed by mass spectrometry.

[0333] Under these conditions, melting of the hydrazone-labeled oligonucleotide DNA and the two DNA strands could be observed independently. After the loss of the potential hydrophobic aldehyde, more than 85% of the functional linker was cleaved and the new HPLC peak shifted to a lower retention time.

[0334] Under these conditions, the oxime-labeled oligoDNA remained intact, consistent with the higher stability of this class of Schiff bases.

[0335] 4) Rewriting the original functionality through Schiff base formation

[0336] Hydrazide functionalized oligo DNA ( Figure 7 , line A) incubated in the presence of excess aldehyde 5 ( Figure 7 A significant shift in the retention time of the main peak associated with oligo DNA was observed ( Figure 7 , line B). Chromatogram obtained after incubation of oligo DNA with M.TaqI and adapter 21A ( Figure 7 , line C) shows good overlap of the peaks associated with the azide functionalized DNA at approximately 6.2 and 6.4 minutes, and similar proportions of this peak to the free hydrazide (at 3.8 and 4.0 minutes). The presence of a small amount of hydrazide functionalized oligoDNA is likely due to hydrolysis of the Schiff base under the HPLC conditions. Three additional peaks were observed after rewriting with the aldehyde, which overlapped with the peaks observed when the aldehyde was incubated with H2NOH·HCl ( Figure 7 , line D). The peaks were analyzed by LC-MS, which confirmed that the rewriting was successful and the nature of the chemical functionality on the oligoDNA.

[0337] 5) Further modification after Schiff base cleavage.

[0338] The DNA fragment generated by PCR contained 17 CpG sites, which were site-selectively labeled with M.MpeI. After labeling, it was incubated with H2NOH·HCl and reacted with the commercially available NHS-activated fluorophore Atto647N (further functionalized). The reaction was monitored by gel electrophoresis and showed specific conjugation of Atto647N to DNA modified with linker 21B. Although no red fluorescence was observed in the absence of Atto647N, this dye is positively charged and is able to non-specifically associate with DNA in the control sample. Comparison of red and green ( Green) channel intensity to evaluate the degree of labeling with ATO647N per unit DNA, which showed that the labeling degree of DNA that had undergone write-erasure was 3.8 times higher than that in the case of H2NOH·HCl treatment without an erase step, and the methyltransferase-directed labeling was more than 22 times higher than that in the case without a write step.

[0339] 6) Double modification

[0340] Sequential modification with complementary fluorescent dyes (write-modify-erase-further modification) was achieved by labeling short DNA fragments with two different fluorescent dyes. To this end, the DNA fragments were first incubated with M.MpeI and the dynamic linker 21A to generate azide-functionalized DNA ( Figure 8 , Image A, Step 1). This modification results in a slight shift in the migration time of the DNA on the gel ( Figure 8 , Image B, Step 2), but as expected, no fluorescence was observed ( Figure 8, Image C, Step 1). When the azide-functionalized DNA was modified with TAMRA-DBCO6, a further shift in migration time was observed ( Figure 8 , Image B, Step 2), but more importantly, the excitation from the DNA-associated TAMRA fluorophore can be clearly observed ( Figure 8 , Image C, step 2). TAMRA modification was erased by incubation with excess H2NOH·HCl. No fluorescence was observed from the resulting DNA fragments ( Figure 8 , Image C, Step 3), and observed to shift back to the original migration time ( Figure 8 , Image B, Step 3), which shows that the hydrazide linker has little effect on the physical properties of the DNA. Incubation of this hydrazide-functionalized DNA with NHS-activated Atto647N 7 resulted in a new shift in migration time ( Figure 8 , Image B, Step 4) and the corresponding fluorescence appearance, now visible under red illumination ( Figure 8 , Image C, step 4).

[0341] Modification was monitored using gel electrophoresis: DNA concentration: 7 ng / μL, release buffer: 10 mM ammonium acetate, pH 6.8, 1 M NaCl, 0.01% SDS. Staining. Bio-Rad PharosFX ( Excitation, trans-UV; excitation filter, 590 / 110nm; TAMRA: excitation, epi-green illumination; excitation filter: 602 / 50nm; Atto 647N 7: excitation, epi-red illumination; excitation filter: 700 / 50nm). The TAMRA channel is yellow and Atto 647N 7 is red for visualization. (B) channel and (C) composite images of TAMRA and Atto647n channels.

[0342] Example of chemical labeling of biomolecules: Cysteine ​​labeling of peptides

[0343] Reference Fig. 9 , showing the use of Figure 3 The synthetic route of precursor 1 to other linker molecules 31 described in the figure, wherein the first functional group LG is a bromine atom.

[0344] Synthesis of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-4-((2-(8-bromooct-6-ynoyl)hydrazoneylidene)methyl)benzamide

[0345] The activated linker (tert-butyl 2-(8-bromooctan-6-ynoyl)hydrazone-1-carboxylate) 1 (20 mg, 0.06 mmol) was dissolved in 500 μL TFA and stirred for 2 hours. TFA was evaporated to give 14 mg of a yellow oil. The crude product was then dissolved in 200 μL PBS, and a solution of Ald-Ph-PEG-azide (22) (21 mg, 1 equivalent) in PBS was added (pH 7). Immediately, a white precipitate appeared. The white solid was filtered and dissolved in DCM for purification by preparative HPLC (0 to 60% ACN 40 minutes). MS: m / z[M+Na]=586.8 / 588.8 to give the linker molecule 31.

[0346] 1) Chemically labeling amino acids (step b of the method of the present invention)

[0347] In the following scheme, linker molecule 31 is used to label cysteine ​​molecule 90.

[0348] The linker molecule 31 (1.2 mg) was dissolved in 7 M ammonia in methanol. N-acetylcysteine ​​(compound 90) (3 mg, 2.25 eq.) was added and the mixture was stirred at room temperature for 2 hours. After the reaction, compound 32 was formed, which was concentrated under pressure.

[0349] 2) Schiff base reversal (step c of the method of the present invention)

[0350] The Schiff base of compound 32 was hydrolyzed in the following scheme. Compound 32 was dissolved in release buffer (release buffer: 231 mg NH2OH in 200 μl ammonium buffer, pH 6) and heated at 50°C for 1 hour. The product (compound 33, 22) was purified by injection HPLC (analytical 0 to 60% ACN in 40 minutes and 100% ACN for 10 minutes).

[0351] The method of labeling amino acids according to this example of the present invention shows that the method of the present invention can be used to label and release and / or re-label amino acids containing thiol moieties other than cysteine. In addition, this example illustrates that according to an embodiment of the present invention, peptides containing amino acids containing thiol moieties (e.g., cysteine) can be labeled and released, as well as re-labeled.

[0352] Advantageously, the method according to the invention is bioorthogonal, in other words, it can be performed in biological systems without interfering with natural biochemical processes. Even more advantageously, once reversed, the modification made to a biomolecule, such as a polynucleotide biomolecule, such as DNA, is relatively small and hydrophilic, meaning that it does not affect the way the biomolecule interacts in solution or with enzymes.

[0353] The linker molecule comprises a hydrolyzable portion, such as a Schiff base portion, which is hydrolyzable so that the label can be reversibly conjugated to the biomolecule. Advantageously, the conditions required for hydrolyzing the Schiff base portion are mild and do not destroy the structure of the biomolecule. In addition, unlike prior art methods such as the use of disulfide bonds, Schiff base chemistry is not common in biomolecules. More advantageously, Schiff bases such as oximes and hydrazones are stable at physiological pH.

[0354] The covalent bonds formed using linker molecules are reversible and rewritable. Advantageously, the labeling molecules can be used for repeated modification of biomolecules, such as labeling, capturing, releasing, refunctionalization, such as for fluorescent labeling and / or imaging.

[0355] Analogs of the S-adenosyl-l-methionine cofactor can be designed to include linker molecules of any suitable structure.

[0356] Advantageously, the second functional group of the linker molecule can be used to further functionalize a biomolecule, such as a polynucleotide, for example using click chemistry. This can be used for DNA capture, DNA complexing, drug attachment and / or fluorescent labeling.

[0357] Those skilled in the art will appreciate that several variations of the above embodiments are contemplated without departing from the scope of the present invention. For example, the biomolecule need not be a polynucleic acid. In some embodiments, the first functional group of the linker molecule can be selected to be able to react with moieties on different biomolecules to form a covalent bond. For example, the biomolecule can include an azide moiety and the first functional group can include an alkyne moiety, or vice versa, which can form a covalent bond by click chemistry.

[0358] Those skilled in the art will also appreciate that any number of combinations of the foregoing features and / or features shown in the accompanying drawings provide significant advantages over the prior art and are therefore within the scope of the invention described herein.

Claims

1. A method for reversibly labeling a DNA molecule, the method comprising: a. Providing a linker molecule compound A having the following general formula: Wherein R represents a transferable group; FG represents a functional group selected from one of the following: alkyne, azide and activated ester; Z consists of a C6H4(C=O)NH group and a polyethylene glycol chain of up to 5 ethylene glycol monomers; -ABC- represents a Schiff base moiety selected from Y represents a non-reactive aliphatic bond consisting of 1 to 5 CH2 moieties; U is an alkene or an alkyne; and k is 2; b. forming a covalent bond between the DNA molecule and the R group of compound A using a DNA methyltransferase capable of using compound A as a cofactor and under conditions that allow the R group of compound A to be transferred to the DNA molecule; c. forming a covalent bond between the first marker and the functional group FG of compound A; d. hydrolyzing the Schiff base portion of compound A to remove the first label and to form an O-substituted hydroxylamine or N-substituted hydrazone; as well as e. forming a covalent bond between the aldehyde portion of the other molecule and the O-substituted hydroxylamine or the N-substituted hydrazone to reform the Schiff base portion, The first marker is a biomarker or a fluorescent marker.

2. The method of claim 1, wherein the other molecule comprises an alkyne, an azide, or an activated ester.

3. The method of claim 2, wherein the method further comprises step f. forming a covalent bond between the second label and the alkyne, azide or activated ester of the other molecule.

4. The method of claim 3, wherein the method further comprises cleaving the Schiff base moiety to remove the second label and to reform the O-substituted hydroxylamine or N-substituted hydrazone.

5. The method according to claim 4, wherein the method further comprises step h. forming a covalent bond between the aldehyde portion of the other molecule and the O-substituted hydroxylamine or N-substituted hydrazone to reform the Schiff base portion.

6. The method according to claim 1, wherein compound A has the following general formula:

7. The method according to claim 1, wherein compound A has the following general formula:

8. The method according to claim 1, wherein compound A has the following general formula: p represents a number from 1 to 5; q represents a number from 1 to 5; k is 2; and FG represents a functional group selected from alkyne, azide and activated ester.

9. The method of claim 1, wherein the functional group FG is an azide moiety.

10. The method according to claim 1, wherein compound A has the formula:

11. The method of claim 1, wherein forming a covalent bond between the first label and the functional group FG comprises reacting an azide moiety with an alkyne moiety to form a carbon-nitrogen covalent bond.

12. The method of claim 1, wherein cleaving the Schiff base moiety to remove the label and to form an O-substituted hydroxylamine or an N-substituted hydrazone comprises treating with hydroxylamine.

13. The method of claim 12, wherein the treatment is carried out in an ammonium acetate buffer solution at pH 4.

14. The method of claim 1, wherein the biomarker is biotin.

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