Improved o 6-alkylguanine-DNA alkyltransferase substrates

Abstract

The invention relates to alkylguanine transferase substrate molecules. In general formula (I) SB is a moiety that can be enzymatically cleaved from the rest of the molecule by an enzyme having an alkyltransferase activity selected from the group consisting of a 2-aminopyrimidinyl moiety, an aminopyridinyl moiety, and a 2-aminopurinyl moiety. Also described are molecules of general formula (II) and general formula (III) L is an optional linker, RN is H, methyl or ethyl or their deuterated or fluorinated derivatives, and R is selected from a detectable label and a selectively reactive moiety.

Description

[0001] Improved O6-Alkylguanine-DNA Alkyltransferase Substrates

[0002] Field

[0003] The present invention relates to improved substrates for the SNAP and CLIP family of alkyltransferase protein tags.

[0004] This application claims the right of priority of European Patent Application EP 24172286.7 filed 24 April 2024, which is incorporated by reference herein.

[0005] Background

[0006] O6-alkylguanine-DNA alkyltransferase (hAGT) is a DNA repair suicide protein that protects DNA from lesions and subsequent mutations caused by xenobiotic alkylating reagents. In this process, an O6-alkyl group present on a guanine residue is irreversibly transferred to a reactive Cys in position 145 of the hAGT. A wide range of nucleobase-like substrates and some non-nucleobase- substrates has been explored. hAGT reacts with benzyl guanine (BG) in vitro and in vivo, the mechanism appears to involve the protein gliding over the sugar backbone of the DNA minor groove until steric clash senses the alkylated guanine base, which subsequently is flipped to be positioned into a binding cleft of the protein and to react with Cys-145. An important residue for binding is Tyr-114, which establishes a hydrogen bond with N3of guanine. The reaction of endogenous hAGT protein with BG is about two orders of magnitude slower than that with BG- oligonucleotides, which is probably due to the protein’s binding affinity towards DNA.

[0007] Human O6-alkylguanine DNA alkyltransferase (hAGT) was engineered to react with conjugates of benzyl guanine (BG); the mutated AGT is referred to as SNAP herein. En route SNAP, labelling of hAGT mutants by BG-conjugates was improved with directed evolution and protein engineering approaches. Benzyl guanine tetramethyl rhodamine (BG-TMR) as SNAP substrate shows labelling with a second-order rate kinetic constant of 4.29 x 105M'1s'1(Keppler et al., A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 2003, 21 (1 ), 86-9; Correa et al., Substrates for improved live-cell fluorescence labelling of SNAP-tag. Current pharmaceutical design 2013, 19 (30), 5414-5420; Wilhelm et al., Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag. Biochemistry 2021 , 60 (33), 2560-2575).

[0008] CLIP-Tag (CLIP) was derived by rational reengineering of SNAP to react with O6-benzyl cytosine (BC) conjugates. CLIP bears 8 mutations in comparison with SNAP and it reacts with BC-TMR with a second-order rate kinetic constant of 1 .85 x 104M'1s'1.

[0009] Towards CLIP, rational protein- and substrate design has been applied for improvements in orthogonality and labelling speed. It has been postulated that slower reaction with conjugates of BC with SNAP and hAGT is due to the fact that BC does not bear nitrogen at the respective position to establish interaction with Tyr-114. By introducing 8 mutations, reactivity towards BG conjugates was reduced and towards BC conjugates was increased, which enabled simultaneous selective labelling of both SLPs. Highly important for potential labelling in clinically relevant samples or animals is CLIP / AGT orthogonality, where BC conjugates do not significantly react with endogenous hAGT (Gautier et al. An engineered protein tag for multiprotein labelling in living cells. Chem. Biol. 2008, 15 (2), 128-36).

[0010] Substrates of engineered AGT are disclosed, inter alia, in US8367361 B2 to Johnsson et al. (also published as WO02083937A2; the first SNAP tag patent); US8227602B2 to Gautier et al. (also published as W02008012296A1 ; CLIP and CLIP substrates); US8163479B2 to Jaccard et al.; US8178314B2 to Kindermann et al., all of which are incorporated by reference in their entirety herein.

[0011] Basic structures of known CLIP and SNAP substrates are: with R being a fluorescent dye or reactive moiety.

[0012] Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods for SNAP and CLIP tag-mediated labelling. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0013] Summary of the Invention

[0014] One aspect of the invention relates to an alkylguanine transferase substrate molecule of the general formula (I) ) wherein: - SB is a moiety that can be enzymatically cleaved from the rest of the molecule by an enzyme having an alkyltransferase activity selected from the group consisting of a 2- aminopyrimidinyl moiety, an aminopyridinyl moiety, and a 2-aminopurinyl moiety;

[0015] - L is an (optional) linker,

[0016] - RNis selected from the group consisting of H, methyl (CH3), ethyl, fluorinated methyl and fluorinated ethyl, and

[0017] - R is selected from a detectable label and a selectively reactive moiety.

[0018] Another aspect of the invention relates to an alkylguanine transferase substrate molecule of the general formula (II)

[0019] Yet another aspect of the invention relates to an alkylguanine transferase substrate molecule of the general formula (III) wherein L, RNand R have the same meanings as in formula (I). Further disclosed are specific compounds and useful intermediates.

[0020] Terms and definitions

[0021] General

[0022] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0023] The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of or “consisting of.”

[0024] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0025] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0026] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0027] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0029] The term alkylguanine transferase substrate molecule in the context of the present specification relates to a molecule which attaches to a polypeptide comprising any one of SEQ ID NO 001 , 002, 003, or 004.

[0030] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

[0031] Cell Biology, diagnostic method inventions: Markers, ligands

[0032] The term Detectable Label in the context of the present specification relates to a moiety R, with distinctive properties that facilitate the detection and quantification of an alkyltransferase fusion protein to which R becomes attached, in a biological sample. The label R allows to visually or instrumentally identify the presence or concentration of the target molecule. Common types of detectable labels include fluorescent dye molecules, radioactive isotopes, colloidal gold particles, and biotin, among others. These labels emit signals or exhibit distinct properties that can be measured or visualized to determine the presence and quantity of the target molecule in the sample.

[0033] The term fluorescent dye in the context of the present specification relates to a small molecule capable of fluorescence in the visible or near infrared spectrum. Examples for fluorescent labels or labels presenting a visible color include, without being restricted to, fluorescein isothiocyanate (FITC), rhodamine, allophycocyanine (APC), peridinin chlorophyll (PerCP), phycoerithrin (PE), alexa Fluors™ (Life Technologies, Carlsbad, CA, USA), dylight fluors™ (Thermo Fisher Scientific, Waltham, MA, USA) ATTO Dyes (ATTO-TEC GmbH, Siegen, Germany), BODIPY Dyes (4,4- difluoro-4-bora-3a,4a-diaza-s-indacene based dyes) and the like.

[0034] Organic Chemistry

[0035] The formulae of the present specification follow the convention of organic chemistry to not show hydrogen atoms on carbon scaffolds. Carbon is tetravalent and bonds not shown are assumed to be hydrogen unless shown otherwise. Hydrogen can be exchanged for deuterium without changing the bulk chemical properties of the molecule; however, in the case of dye or drug molecules, the exchange of hydrogen for deuterium may lead to changes in the spectral properties or receptor interactions of the molecule. Unless explicitly stated otherwise herein, the disclosure of a formula showing, explicitly or implicitly by the convention restated in the first sentence of this paragraph, encompasses molecules in which one or several of the hydrogen atoms are exchanged for deuterium.

[0036] A linker or linker moiety in the context of the present specification relates to a moiety connecting two constituent functional parts of the molecules disclosed herein (the aminobenzyl nitrogen and the functional group R). In certain embodiments, a linker moiety is characterized by a molecular mass of <1000 g / mol and consists of carbon, nitrogen, oxygen, and optionally sulphur and phosphorus atoms in the direct chain. The chain may be saturated or unsaturated, and may bear substituents or oxygen atoms. In particular embodiments, the linker is characterized by a molecular mass of <5000 g / mol. In more particular embodiments, a linker consists of 2, 3, 4, 5 or 6 C, N and / or O atoms in the linking chain (plus hydrogen atoms H or oxygen O as applicable). Linker moieties useful for the present invention include, but are not limited to, Cv-Ce alkyl, CO-Alk-NH, CO-Alk-CO- , CO-Alk-O-, CO-Alk-CO-, NH-Alk or O-Alk or NH-NH-CO-Alk, with Aik being a Ci, C2, C3, C4, Cs, or Ce unsubstituted alkyl or a C2, C3, C4, Cs, or Ce amino- and / or hydroxysubstituted alkyl.

[0037] The term alkyl in the context of the present specification relates to a saturated linear, branched or (partially or completely) cyclic hydrocarbon, wherein in certain embodiments one carbon-carbon bond may be unsaturated and one CH2 moiety may be exchanged for oxygen (ether bridge) or nitrogen (NH, or NR with R being methyl, ethyl, or propyl; amino bridge). The term unsubstituted Cnalkyl when used herein in the narrowest sense relates to the moiety -CnH2n- if used as a bridge between moieties of the molecule, or -CnH2n+i if used in the context of a terminal moiety. It may still contain fewer H atoms if used in the context of a cyclical structure.

[0038] The term C1-C4 alkyl in the context of the present specification relates to a saturated linear or branched hydrocarbon having 1 , 2, 3 or 4 carbon atoms. Non-limiting examples for a C1-C4 alkyl are methyl, ethyl, propyl, prop-2-enyl, n-butyl, 2-methylpropyl, tert-butyl, cyclo-butyl, cyclo-propyl, methyl-cyclo-propyl. In certain embodiments, a C1-C4 alkyl is a methyl, ethyl, propyl or butyl moiety.

[0039] A C1-C6 alkyl in the context of the present specification relates to a saturated linear or branched hydrocarbon having 1 , 2, 3, 4, 5 or 6 carbon atoms. Non-limiting examples for a C1-C6 alkyl include the examples given for C1-C4 alkyl above, and additionally n-pentyl, 2-methylbutyl, 3-methylbutyl, 1 ,1 -dimethylpropyl, 1 ,2-dimethylpropyl, 1 ,2-dimethylpropyl, cyclo-pentyl, cyclo-hexyl, methyl-cyclo- pentyl. In certain embodiments, a C5 alkyl is a pentyl or cyclopentyl moiety and a Ce alkyl is a hexyl or cyclohexyl moiety.

[0040] Where used in the context of chemical formulae, the following abbreviations may be used: Me is methyl CH3, Et is ethyl -CH2CH3, Prop is propyl -(CH2)2CH3 (n-propyl, n-pr) or -CH(CH3)2 (isopropyl, i-pr), but is butyl -C4H9, -(CH2)3CH3, -CHCH3CH2CH3, -CH2CH(CH3)2or -C(CH3)3.

[0041] In the context of the present specification, the term alkyl also encompasses unsaturated hydrocarbons such as alkene and alkyne.

[0042] The term alkyne in the context of the present specification relates to a hydrocarbon comprising a triple bond. Unsubstituted alkyne is of formula -CHC- when being located intramolecularly, and of formula -CHCH (-C2H) when being a terminal moiety. An unsubstituted alkyne consists of C and H only. A substituted alkyne may comprise substituents as defined herein for substituted alkyl. A particular embodiment of alkyne of interest in so-called “click” reaction is cyclooctyne, due to its unique ability to undergo rapid and highly specific [3+2] cycloaddition reactions with azides without the need for a catalyst. Cyclooctyne derivatives have been developed to enhance reactivity and selectivity further. These derivatives, such as difluorinated cyclooctyne (DIFO), azadibenzocyclooctyne (DIBAC), and bicyclo[6.1 .0]nonyne (BCN), offer varying rates of reaction and solubility properties.

[0043] The term substituted alkyl in its broadest sense refers to an alkyl as defined above in the broadest sense, which is covalently linked to an atom that is not carbon or hydrogen, particularly to an atom selected from N, O, F, B, Si, P, S, Cl, Br and I, which itself may be -if applicable- linked to one or several other atoms of this group, or to hydrogen, or to an unsaturated or saturated hydrocarbon (alkyl or aryl in their broadest sense). In a narrower sense, substituted alkyl refers to an alkyl as defined above in the broadest sense that is substituted in one or several carbon atoms by groups selected from amine NH2, alkylamine NHR, imide NH, alkylimide NR, amino(carboxyalkyl) NHCOR or NRCOR, hydroxyl OH, oxyalkyl OR, oxy(carboxyalkyl) OCOR, carbonyl O and its ketal or acetal (0R)2, nitril CN, isonitril NC, cyanate CNO, isocyanate NCO, thiocyanate CNS, isothiocyanate NCS, fluoride F, choride Cl, bromide Br, iodide I, phosphonate PO3H2, PO3R2, phosphate OPO3H2 and OPO3R2, sulfhydryl SH, suflalkyl SR, sulfoxide SOR, sulfonyl SO2R, sulfanylamide SO2NHR, sulfate SO3H and sulfate ester SO3R, with R being defined further in the description. In certain embodiments, R is itself an unsubstituted or substituted Ci to C12 alkyl in its broadest sense, and in a narrower sense, R is methyl, ethyl or propyl unless otherwise specified.

[0044] The term aryl in the context of the present specification relates to a cyclic aromatic C5-C10 hydrocarbon. Examples of aryl include, without being restricted to, phenyl and naphthyl.

[0045] In the context of the present specification, the term aryl encompasses hydrocarbon-based aryl and heteroaryl.

[0046] The term heteroaryl in the context of the present specification relates to a cyclic aromatic C2-C9 hydrocarbon that comprises at least one heteroatom (e.g. N, O, S). Examples for heteroaryl include, without being restricted to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazin, quinoline, benzofuran and indole.

[0047] The term hydroxyl-substituted [group] refers to a moiety or group that is modified by one or several hydroxyl groups OH. Non-limiting examples of hydroxy-substituted alkyl include -CH2OH, -(CH2)2OH, -(CH2)3OH, -CH2CH(OH)CH3, -(CH2)4OH, -CH(CH2OH)CH2CH3, - CH2CH(CH2OH)CH3, -CH(OH)(CH2)2OH, -CH2CH(OH)CH2OH, -CH2CH(OH)(CH2)2OH and - CH2CH(CH2OH)2for terminal moieties and -CHOH-, -CH2CHOH-, -CH2CH(OH)CH2-, - (CH2)2CHOHCH2-, -CH(CH2OH)CH2CH2-, -CH2CH(CH2OH)CH2-, -CH(OH)(CH2CHOH-, - CH2CH(OH)CH2OH, -CH2CH(OH)(CH2)2OH and -CH2CHCH2OHCHOH- for a hydroxyl substituted alkyl moiety bridging two other moieties.

[0048] The term amino-substituted [group] refers to a moiety or group that is modified by one or several amino groups NH2. Non-limiting examples of amino-substituted alkyl include -CH2NH2, - CH2NHMe, -CH2NHEt, -CH2CH2NH2, -CH2CH2NHMe, -CH2CH2NHEt, -(CH2)3NH2, -(CH2)3NHMe, -(CH2)3NHEt, -CH2CH(NH2)CH3, -CH2CH(NHMe)CH3, -CH2CH(NHEt)CH3, -(CH2)3CH2NH2, -(CH2)3CH2NHMe, -(CH2)3CH2NHEt, -CH(CH2NH2)CH2CH3, -CH(CH2NHMe)CH2CH3, -CH(CH2NHEt)CH2CH3, -CH2CH(CH2NH2)CH3, -CH2CH(CH2NHMe)CH3, -CH2CH(CH2NHEt)CH3, -CH(NH2)(CH2)2NH2, -CH(NHMe)(CH2)2NHMe, -CH(NHEt)(CH2)2NHEt, -CH2CH(NH2)CH2NH2, -CH2CH(NHMe)CH2NHMe, -CH2CH(NHEt)CH2NHEt, -CH2CH(NH2)(CH2)2NH2, -CH2CH(NHMe)(CH2)2NHMe, -CH2CH(NHEt)(CH2)2NHEt, -CH2CH(CH2NH2)2, -CH2CH(CH2NHMe)2and -CH2CH(CH2NHEt)2for terminal moieties and -CH2CHNH2-, -CH2CHNHMe-, -CH2CHNHEt- for an amino substituted alkyl moiety bridging two other moieties.

[0049] The term amine-substituted [group] refers to a moiety or group that is modified by one or several amine groups -NHR or -NR2, or derivatives thereof, with each R being defined further in the description. The term ester refers to a group of -CO-O-R or -O-CO-R, with R being defined further in the description.

[0050] The term ether refers to a group having one oxygen in between two saturated carbon atoms.

[0051] An amide refers to a group of -CONHR, with R being defined further in the description.

[0052] The term carbamate refers to a compound of the general formula R2NC(0)0R and structure >N-C(=O)-O-, which can be understood to be formally derived from carbamic acid (NH2COOH). The term may include salts with the carbamate anion R2NCOO- (e.g. ammonium carbamate) with each R being defined independently as laid out further in the description.

[0053] Detailed Description of the Invention

[0054] One aspect of the invention relates to an alkylguanine transferase substrate molecule of the general formula (I) ) wherein:

[0055] - SB is a moiety that can be cleaved from the rest of the molecule by an alkyltransferase activity selected from the group consisting of a 2-aminopyrimidinyl moiety, an aminopyridinyl moiety, and a 2-aminopurinyl moiety;

[0056] - RNis selected from the group consisting of H, methyl (CH3), ethyl, fluorinated methyl and fluorinated ethyl, and

[0057] - L is an (optional) linker, and

[0058] - R is selected from a detectable label and a selectively reactive moiety.

[0059] The term fluorinated methyl or ethyl where used in the context of the present specification, refers to a methyl or ethyl that is modified by one or several fluoride groups F. Examples of fluorinated alkyl methyl and ethyl include -CH2F, -CHF2, -CF3, -(CH2)2F, -(CHF)2H, -(CHF)2F, -C2F5.

[0060] In particular embodiments of any group of molecules represented by a formula containing the substituent RNherein, RNis selected from methyl, ethyl and hydrogen.

[0061] RNmay be deuterated partially or in full.

[0062] In particular embodiments, the molecule is described by the general formula l-H: wherein SB, L and R can take the same meanings as disclosed for formula I.

[0063] The inventors also generated variations of general formula I where, instead of the fluorine attached to the phenyl ring at position 3, the phenyl ring was substituted with Br or Cl in this position. These modifications, however, were found to increase cross reactivity with hAGT. Bisfluoride substitution in both 3, 3’ positions by F is tolerated, but slows down reaction with SNAP in cell free systems and in cellular assays. Small heterocycles such as thiazine and oxazine instead of the fluorophenyl moiety are tolerated, but were not found to give higher activity or selectivity than mono F benzyl (see Fig. 4 and 5).

[0064] The inventors also generated variants in which position 2 and 2’ of the phenyl ring were modified. None improved the performance (see compounds S28 to S31 , Fig. 4 and 5).

[0065] In particular embodiments, the molecule according to this aspect of the invention is described by an aminopyridinyl moiety of the general structure (I A) wherein RN, L and R can take any of the meanings assigned above for formula (I).

[0066] In more particular embodiments, the molecule according to this aspect of the invention is described by an aminopyridinyl moiety of the general structure (I AH) wherein L and R can take any of the meanings assigned above for formula (I).

[0067] The inventors generated compounds representing the variation of pyrimidinyl SNAP tag substrate moieties disclosed in W02006114409A1 . They found that in general, all variations of the general formula were improved upon combination with the fluorinated benzyl according to general formula I / 1 B:

[0068] Thus, in particular embodiments, the alkylguanine transferase substrate molecule according to this aspect of the invention is described by the general structure (I B) wherein RN, L and R can take any of the meanings assigned above for formula (I), and wherein X is selected from CF3, Cl, CN, N3, CH3, OCH3 and H.

[0069] In more particular embodiments, the alkylguanine transferase substrate molecule according to this aspect of the invention is described by the general structure (I BH) wherein L and R can take any of the meanings assigned above for formula (I), and wherein X is selected from CF3, Cl, CN, N3, CH3, OCH3 and H.

[0070] In more particular embodiments thereof, X is CF3. In other more particular embodiments thereof, X is Cl.

[0071] In other more particular embodiments thereof, X is OCH3.

[0072] In other more particular embodiments thereof, X is H.

[0073] The inventors expect a similar improvement of performance when combining the 3-fluorinated benzyl according to general formula I with guanine as cleaved substrate moiety. Guanine derivatives however have not been pursued in the context of the experimental work underlying the present specification; they lack stability in comparison with the molecules shown in the synthetic examples, and they usually show lower permeability in cellular assays.

[0074] In particular embodiments, the molecule according to this aspect of the invention is described by the general structure (I C) wherein L, RNand R can take any of the meanings assigned above for formula (I).

[0075] In particular embodiments, RN is H, i.e. the compound is described by general formula (I CH):

[0076] Again, L is an optional linker, exemplary embodiments of which are further described below, and R is selected from a detectable label and a selectively reactive moiety, exemplary embodiments of which are also further described below.

[0077] The use of pyridine, instead of pyrimidine, as a CLIP tag substrate was surprisingly found to improve upon known substrates in the absence of the fluorinated benzyl moiety of formula (I). The main exponent of this series is the 4-amino pyridine analogue. It has high pKa (~7), and its reactivity and permeability are significantly higher due to these changes. The inventors have investigated Cl- and F- pyridine analogues (see attachments), but they are not reactive, likely due to a change in nitrogen pKa (from 7 to 5).

[0078] Thus, another aspect of the invention relates to an alkylguanine transferase substrate molecule of the general formula (II)

[0079] L is an optional linker,

[0080] R is selected from a detectable label and a selectively reactive moiety. In particular embodiments, the alkylguanine transferase substrate molecule of the general formula (II) is described by II H: ) wherein:

[0081] RNis selected from the group consisting of H, methyl (CH3), ethyl, fluorinated methyl and fluorinated ethyl,

[0082] L is an optional linker, and

[0083] R is selected from a detectable label and a selectively reactive moiety.

[0084] Additionally, the inventors found that a methoxy moiety in position X of the pyrimidinyl group imparts favourable cell penetration properties also in the absence of the fluorinated benzyl moiety of formula (I). Thus, yet another aspect of the invention relates to an alkylguanine transferase substrate molecule of the general formula (III) wherein

[0085] RNis selected from the group consisting of H, methyl (CH3), ethyl, fluorinated methyl and fluorinated ethyl, L is an optional linker, and

[0086] R is selected from a detectable label and a selectively reactive moiety.

[0087] In particular embodiments, RNof the compounds of formula III is hydrogen, i.e. wherein L and R can take any of the meanings assigned above for formula (I). The Linker Moiety L

[0088] The term linker or linker moiety L as used herein is an optional flexible moiety connecting the substrate-benzyl (fluorobenzyl as relates to the first aspect of the invention) and the detectable label or click reactive moiety, without impeding their respective functionality. Linker units are chosen in the context of the envisioned application, i.e. in the transfer of the substrate to a fusion protein comprising the SNAP or CLIP tag peptide moiety. They may also increase the solubility of the substrate in the appropriate solvent. The linkers used are chemically stable under the conditions of the actual application. The linker does not interfere with the reaction with tag nor with the functionaliy of R, but may be constructed such as to be cleaved at some point in time after the reaction of the compound of formula (I) with the alkyltransferase moiety (the tag).

[0089] In certain embodiments, L is characterized by a molecular mass of <1000 g / mol and consists of carbon, nitrogen, oxygen, and optionally sulphur and phosphorus atoms in the direct chain. The chain may be saturated or unsaturated, and may bear substituents, particularly substituents selected from oxo (=0), hydroxy and amino. In particular embodiments, a linker is characterized by a molecular mass of <500 g / mol. In certain embodiments, L is a linear chain of 10-15 atoms in length, particularly atoms selected from C, O, N and S. In certain embodiments, L comprises alkyl, trans-alkylene, and / or ether moieties and optionally one or several methyl substituents. In certain embodiments, L is 10-11 atoms in length. In certain embodiments, L is an unbranched chain that does not comprise methyl substituents. In certain particular embodiments, L essentially consists of alkyl, trans-alkylene, and / or ether moieties and optionally one or several methyl substituents. In more particular embodiments, a linker consists of 2, 3, 4, 5 or 6 C, N and / or O atoms in the linking chain (plus hydrogen atoms H or oxygen O as applicable). Linker moieties useful for the present invention include, but are not limited to, Cv-Ce alkyl, CO-Alk-NH, CO-Alk-CO-, CO-Alk-O-, CO-Alk- CO-, NH-Alk or O-Alk or NH-NH-CO-Alk, with Aik being a Ci, C2, C3, C4, Cs, or Ce unsubstituted alkyl or a C2, C3, C4, Cs, or Ce amino- and / or hydroxysubstituted alkyl.

[0090] In particular embodiments of any of the aspects of the invention disclosed herein, a linker moiety L is present in the alkylguanine transferase substrate molecule according to the invention, and L is a chain of 2 to 50 atoms selected from C, O, N and S.

[0091] In more particular embodiments, L is a Ci to C10 substituted alkyl, particularly a Ci to C10 substituted alkyl having one more methylene groups exchanged by a group independently selected from -CO-, -O-, -CONH-, -HNC(O)O-, -SS-, -NH-, -NN-.

[0092] In certain embodiments, L comprises alkylene (saturated), trans-alkenyl (mono-unsaturated), and / or ether moieties and optionally one or several methyl substituents. In certain embodiments, L is 10-11 atoms in length. In certain embodiments, L is an unbranched linker that does not comprise methyl substituents. In certain particular embodiments, L essentially consists of alkyl, trans-alkylene, and / or ether moieties and optionally one or several methyl substituents. In particular embodiments, L is a straight or branched chain alkylene group with 1 to 300 carbon atoms. In other particular embodiments, L is a straight or branched chain alkylene group with 1 to 300 atoms of mass >12, wherein one or more carbon atoms are replaced by oxygen, in particular wherein every third carbon atom is replaced by oxygen, which is a poly(ethylene glycol) group having 1 to 100 ethyleneoxy units.

[0093] L as described in the foregoing may comprise amide (-CONH-) or ester (-COO-) functions linking one part of L to another, or to either of the N-benzyl or the functional moiety R.

[0094] L may contain one or more unsaturated bonds.

[0095] L may further comprise a cycloalkyl or aryl, including heteroaryl, moiety. L may in particular comprise a triazole moiety such as is generated by reaction of an ethynyl and an azide moiety.

[0096] L may also comprise a disulfide group as part of the linking chain, which renders the linker cleavable under selective conditions.

[0097] In certain embodiments, L is a straight chain alkylene group with 1 to 25 carbon atoms wherein carbon atoms are optionally replaced by an amide function -NRNCO- or CONRN, with RNselected from H and C1-C4 alkyl. L may also comprise a carbamate group -HNC(O)O-.

[0098] Cyclic substructures in a linker L reduce the molecular flexibility as measured by the number of rotatable bonds within L, which in certain applications may lead to a better membrane permeation rate, important for all in vivo labelling applications.

[0099] Substituents that may be present in the chain include, but are not limited to, lower alkyl, particularly methyl, lower alkoxy, particularly methoxy, lower acyloxy, particularly acetoxy, or halogenyl, particularly chloro.

[0100] L may further comprise, or be fully composed of, amino acids, particularly proteinogenic amino acids. In certain embodiments, L comprises or exclusively consists of 1 to 10 amino acids. In particular embodiments, L comprises or exclusively consists of 1 to 10 amino acids selected from G, A, S and T.

[0101] In particular embodiments, L is a photocleavable linker. Photocleavable linkers comprise a reactive function selected from o-nitro-phenyl, phenacyl, alkoxybenzoin, benzylthioether and pivaloyl glycol.

[0102] In other particular embodiments of any of the aspects of the invention disclosed herein, a linker moiety L is not present.

[0103] The functional moiety R

[0104] R is a moiety that facilitates the detection and / or manipulation of the tag protein to which it is attached. R, also referred to herein in generic terms as a label, is chosen so that the labelled fusion protein carrying label L is easily detected or separated from its environment. R can be chosen by those skilled in the art dependent on the application for which the fusion protein is intended. Examples of labels include the members of the following group:

[0105] (1 ) a spectroscopic probe such as a fluorophore, a chromophore, a magnetic probe or a contrast reagent;

[0106] (2) a radioactively labelled moiety or a chelate binding a radioactive isotope, or a heavy metal isotope detectable by mass spectroscopy;

[0107] (3) a moiety which is one part of a specific binding pair, and which is capable of specifically binding to the other partner of that pair. Such specific binding pairs are well known in the art and include, for example, biotin, which can bind to avidin or streptavidin, or a “click” reactive moiety.

[0108] (4) a moiety which is capable of crosslinking to other biomolecules as known to those skilled in the art [Nadeau and Carlson in Protein-Protein interactions: a molecular cloning manual ; Ed. E Golemis, Cold Spring Harbor Laboratory Press ; pp. 75-92 (2002)];

[0109] (5) a moiety that is suspected to interact with other biomolecules, for example a pharmacophore or drug candidate, particularly a small molecule drug candidate. In particular embodiments, such small molecule drug candidate fulfils the criteria summarized as Lipinski’s Rules of five (the drug candidate fulfils at least three of the following rules: number of H-bond donors is < 5; number of H-bond acceptors is < 10; molecular mass is <500Da; octanol / water partition coefficient < 5).;

[0110] (6) a molecule which is capable of generating hydroxyl radicals upon exposure to H2O2 and ascorbate such as a tethered metal-chelate [Hori and Baichoo in Protein-Protein interactions: a molecular cloning manual ; Ed. E Golemis, Cold Spring Harbor Laboratory Press; pp. 288- 311 (2002)];

[0111] (7) a molecule which is capable of generating reactive radicals upon irradiation with light such as malachite green [Jay and Sakurai, Biochim. Biophys. Acta M39-48 (1999)];

[0112] (8) a molecule covalently attached to a solid support, where the support may be a glass slide, a microtiter plate or any polymer in general known to those proficient in the art;

[0113] (9) a nucleic acid or a derivative thereof capable of undergoing base-pairing with its complementary strand;

[0114] (10) a lipid or other hydrophobic molecule with membrane inserting properties.

[0115] When the label R is a molecule that can cross-link to other proteins, e.g. a molecule containing functional groups such as maleimides, active esters or azides and others known to those proficient in the art, contacting such labelled alkyltransferase substrates with alkyltransferase fusion proteins that interact with other proteins {in vivo or in vitro) leads to the covalent cross-linking of the alkyltransferase fusion protein with its interacting protein via the label. This allows the identification of the protein interacting with the alkyltransferase fusion protein. Labels L for photo cross-linking are e.g. benzophenones. In a special aspect of cross-linking the label R is a molecule which is itself an alkyltransferase substrate leading to dimerization of the alkyltransferase fusion protein. The chemical structure of such dimers may be either symmetrical (homodimers) or unsymmetrical (heterodimers).

[0116] Other labels R considered are for example fullerenes, boranes for neutron capture treatment, nucleotides or oligonucleotides, e.g. for self-addressing chips, peptide nucleic acids, and metal chelates, e.g. platinum chelates that bind specifically to DNA. A particular biomolecule with desirable enzymatic, chemical or physical properties is methotrexate. Methotrexate is a tight- binding inhibitor of the enzyme dihydrofolate reductase (DHFR). Compounds of formula (I) wherein R is methotrexate belong to the well-known class of so-called "chemical inducers of dimerization" (CIDs). Using fusion proteins of alkyltransferase with the DNA-binding domain LexA, and adding DHFR with the transcriptional activation domain B42 to the in vivo labeling of the alkyltransferase fusion protein with a compound of formula (I) wherein R is methotrexate induces the coupling ("dimerization") of the alkyltransferase -LexA fusion protein and DHFR-B42 fusion protein, leading to spatial proximity of LexA and B42 and subsequent stimulation of transcription.

[0117] R can be a detectable label

[0118] Detectable labels include any label that can be visualized by optic or electronic means, directly or indirectly. Direct detection is possible, inter alia, by optical measurement, mass spectrometry, autoradiography. Indirect detection may be achieved by using a label to which a ligand bearing a directly detectable label may be attached, such as biotin (which may bind fluorescently labelled streptavidin), or a nucleic acid, which may selectively hybridize to a fluorescently labelled probe.

[0119] In certain particular embodiments, the functional moiety R is a spectroscopic probe (dye molecule). In certain embodiments, the functional moiety R is a probe that can be excited by electromagnetic radiation and where excitation can subsequently be used for its detection in biological samples.

[0120] In certain embodiments, the functional moiety R is an organic dye moiety characterized by a molecular mass of between 300 g / mol and 1300 g / mol. In certain embodiments, the functional moiety R is a fluorescent organic dye moiety. In certain particular embodiments, R is a fluorophore, particularly a fluorescent dye molecule selected from the family of xanthene or polymethine dyes.

[0121] Xanthene dyes include rhodamine, silicon rhodamine, carborhodamine, or sulforhodamin dyes. Particular examples of xanthene type dyes with improved permeability or particular spectroscopic properties that are considered for practice of the present invention include those disclosed in W02013029650A1 , W02020115286A2, WO2019122269A1 .

[0122] A fluorogenic polymethine dye molecule as disclosed in PCT / EP2024 / 052060 is also considered as a particular useful embodiment of R for the present invention. Particular fluorophores considered as examples of R include: Alexa Fluor™ dyes, including Alexa Fluor 350, 488, 532, 546, 555, 635 and 647 (Invitrogen Corp., Carlsbad, CA 92008, USA, see also Panchuk-Voloshina, N. et al., J. Histochem. & Cytochem. 47:1 179-1 188, 1999); coumarins such as 7-dimethylamino-coumarin-4-acetic acid (succinimidyl ester supplied as product D374 by Invitrogen Molecular Probes), 7-amino-4-methyl-coumarin-3-acetic acid and 7-diethylamino- coumarin-3-carboxylic acid; Cyanine-3 (Cy 3), Cyanine 5 (Cy 5) and Cyanine 5.5 (Cy 5.5) (Amersham - GE Healthcare, Solingen, Germany); ATTO 488, ATTO 532, ATTO 600 and ATTO 655 (Atto-Tec, D57076 Siegen, Germany); DY-505, DY-547, DY-632 and DY-647 (Dyomics, Jena, Germany); and 5(6)-carboxyfluorescein and difluoro-5(6)-carboxyfluorescein (Oregon Green).

[0123] In particular embodiments of any of the aspects of the invention disclosed herein, R is a fluorescent dye moiety.

[0124] In certain embodiments, R is a dye, particularly a fluorescent dye, comprising a triarylmethane or xanthene type fluorophore. In certain particular embodiments, R is selected from a rhodamine, a silicon rhodamine, a fluorescein, a Janelia Fluor dye, an olefinic silicon rhodamine derivative with an exocyclic double bond, a cell permeable (MaP) xanthene fluorophore dye, a carbopyronine, a carbocyanine (particularly a Cy3, or a Cy5 dye), a pyrene, a Bodipy fluorophore, a coumarine, a rhodol, and an Alexa™ dye.

[0125] In certain embodiments, R is a rhodamine selected from carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR) and the isothiocyanate derivative TRITC, sulforhodamine 101 , Texas Red, and Rhodamine Red.

[0126] In certain embodiments, R is a silicon rhodamine, a rhodamine wherein the central oxygen atom is replaced by SiR’2 (with R’ selected from Ci to C4 alkyl).

[0127] In certain embodiments, R is a fluorescein selected from 3',6'-dihydroxyspiro[isobenzofuran- 1 (3H),9'-[9H]xanthen]-3-one, fluorescein isothiocyanate (FITC) and, 6-FAM phosphoramidite.

[0128] The Janelia Fluor family of molecules comprises rhodamine-type dyes having an azetidine moiety formed around the nitrogen atoms the outer rings. In certain embodiments, R is a Janelia Fluor dye selected from JF646, JF635, JF585, JF549, JF525, and JF503.

[0129] In certain embodiments, R is an olefinic silicon rhodamine derivative with an exocyclic double bond having a structure described in WO 2019122269 A1 , incorporated herein by reference.

[0130] In certain embodiments, R is a cell permeable (MaP) xanthene fluorophore dye selected from MaP510, MaP555, MaP618, and MaP700 as disclosed in bioRxiv preprint doi: https: / / doi.org / 10.1101 / 690867 or W02020115286, incorporated herein by reference.

[0131] In certain embodiments, R is a carbocyanine selected from tetramethylindo(di)-carbocyanine, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, NIR-820, ICG, Cypate, and CyTE-822.

[0132] In certain embodiments, R is a Bodipy fluorophore is selected from BODIPY™ FL, BODIPY R6G, BODIPY TMR, BODIPY 581 / 591 , BODIPY TR, BODIPY 630 / 650, and BODIPY 650 / 665. In certain embodiments, the Alexa dye is selected from Alexa Fluor™ 350, Alexa Fluor™ 350, Alexa Fluor™ 405, Alexa Fluor™ 430, Alexa Fluor™ 488, Alexa Fluor™ 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, and Alexa Fluor 790.

[0133] In certain embodiments, the moiety R is a fluorescent dye described by the general formula

[0134] "Class- cal" modan.nes X JF-Dyes.

[0135] R can be a reactive moiety

[0136] The ability to covalently couple the molecule of the invention to a range of compounds or functionalities is an important aspect of the invention. Thus, the labelled alkyltransferase fusion protein may be attached to a moiety that facilitates selective attachment to a pre-selected partner by means of selective chemical reaction, either by conventional activated groups such as N- hydroxysuccinimide (NHS), or more recent “click chemistry”, such as may be copper-catalyzed ethine-azide reactions. R can be used to separate molecules on a solid phase, by coupling to the analyte or solid phase.

[0137] In certain embodiments, the functional moiety R is a synthetic ligand capable of binding to a biomolecule with an affinity of at least (i.e. a numerical value below) 100 pmol / L. In particular embodiments, the functional moiety R is a synthetic ligand capable of binding to a biomolecule with an affinity characterized by a Kd of <1 .0 pM. Such ligand allows coupling of a biomolecule. One application is the commercial provision of pre-synthesized SNAP / CLIP-Tag substrates (ligands), to which biomolecules or reactive partners can be coupled by selective, highly reactive reaction partners (i.e. “click chemistry” partners).

[0138] In particular embodiments of any of the aspects of the invention disclosed herein, the linkable moiety of R may be selected from a. an azide moiety, b. an alkyne moiety, particularly a cyclooctyne moiety; c. biotin; d. an unprotected or protected amine moiety; e. a sulfonamide moiety, f. a carboxylic acid or an activated form of a carboxylic acid, particularly an N- hydroxysuccinimide moiety, g. an ester moiety, h. an aldehyde, i. a thiol, and j. an isothiocyanate.

[0139] In certain embodiments, the linkable moiety of R is an unprotected or protected amine moiety, facilitating the attachment of the compound in a ready-to-use format to a suitable chemical function on a substrate, for example by attaching it to an activated carboxylic acid on the substrate. In turn, certain embodiments provide the linkable moiety of R as a carboxylic acid or an activated form of a carboxylic acid, rendering easy attachment to an amine or hydroxyl moiety on a substrate. In certain embodiments, the linkable moiety of R is an NHS moiety.

[0140] In certain embodiments, the linkable moiety of R is a sulfonamide moiety. Certain sulfonamides are amenable to Huisgen 1 ,3-dipolar cycloaddition.

[0141] In certain embodiments, the linkable moiety of R is an N3 moiety. In certain embodiments, the linkable moiety of R is an alkyne moiety. Azide groups react with carbon-carbon triple bonds by way of 1 ,3-dipolar cycloaddition. The catalyzed coupling of an alkyne to an azide facilitates the attachment of the compound to a broad range of substrates by simple “click chemistry” reaction. One particularly useful example of an alkyne is a cyclooctyne moiety.

[0142] In certain embodiments, the linkable moiety of R is an ester moiety or an activated ester, readily reacting with nucleophiles such as amines. In certain embodiments, the linkable moiety of R is an aldehyde, which can, inter alia, form Schiff base adducts with amines. In certain embodiments, the linkable moiety of R is a thiol. In certain embodiments, the linkable moiety of R is an isothiocyanate. Isothiocyanates can undergo click-type reactions with thiols.

[0143] In certain embodiments, the linkable moiety of R is a norbornene, which is versatile in its reactivity and can engage in reactions with a range of partners in the context of click chemistry. Some of the key reactions and partners include Tetrazines in Diels-Alder Reactions, leading to the formation of highly stable adducts. This reactivity is particularly exploited in bioconjugation.

[0144] The skilled practitioner is able to select combinations of the reactive group and features of the molecule that will assure that the moieties do not lead to self-reaction, for example by avoiding the use of an amino group in the linker if the reactive group is selected N-hydroxyl succinimide.

[0145] Other applications for non-covalent, highly specific and reversible interaction of the compounds disclosed herein include purification.

[0146] R can be a drug or drug candidate molecule

[0147] In certain embodiments, the functional moiety R is a pharmaceutical drug or a pharmaceutical drug candidate fulfilling the so-called Lipinski rule-of-five. The Lipinski rule states that the pharmaceutical drug or a pharmaceutical drug candidate has no more than 5 hydrogen bond donors, no more than 10 hydrogen bond acceptors, a molecular mass of less than 500 daltons, and an octanol-water partition coefficient (log P) that does not exceed 5.

[0148] In certain embodiments, the functional moiety R is an oligopeptide or a polypeptide.

[0149] In certain embodiments, the functional moiety R is a nanoparticle. A nanoparticle is a particle of matter that is between 1 and 100 nm in diameter. Advantageous applications of nanoparticle technology include those using gold nanoparticles and quantum dots, nanoscale semiconductor materials. Many methods for covalent linking of organic linkers to nanoparticles are known in the art, which include but are not limited to sulfur (SH) mediated bonds to metal surfaces.

[0150] In certain embodiments, the functional moiety R is a nucleic acid oligomer. The tagged nucleic acid could thus be directed at, and reversibly attached to, a protein or another structure bearing the alkyltransferase tag.

[0151] In certain embodiments, the functional moiety R is a carbohydrate.

[0152] In certain embodiments, the functional moiety R is a lipid.

[0153] In certain embodiments, the functional moiety ZR is a sensor. In certain embodiments, the functional moiety R is a fluorescent sensor. In certain embodiments, the fluorescent sensor is an environment-sensitive or analyte-binding small chemical to detect the presence of a particular substance of interest by the use of fluorescence. In certain embodiments, the fluorescent sensor works via a fluorescent readout change to indicate changes of an analyte concentration or other external factor of interest. In certain embodiments, the functional moiety R is a natural product. In certain embodiments, the functional moiety R is a metabolite, a vitamin, a provitamin, or an enzymatic co-factor having a molecular mass of <1000 g / mol. In certain embodiments, the functional moiety R is a vitamin, a provitamin, or an enzymatic co-factor having a molecular mass of <1000 g / mol.

[0154] The invention further encompasses the specific compounds disclosed herein, namely the compounds of formula: 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 16, 17, 18.

[0155] The invention further encompasses the intermediate compound useful in the synthesis of an alkylguanine transferase substrate molecule according to the invention, selected from the group consisting of the compound of formula 33, 34, 35, 38, 187.

[0156] Further encompassed by the invention are the following embodiments:

[0157] 1 . A molecule of the general formula (I) ) wherein:

[0158] - SB is selected from the group consisting of a 2-aminopyrimidinyl moiety, an aminopyridinyl moiety, and a 2-aminopurinyl moiety;

[0159] - RNis selected from the group consisting of H, methyl (CH3), ethyl, fluorinated methyl and fluorinated ethyl, and

[0160] - L is an optional linker,

[0161] - R is selected from a detectable label and a selectively reactive moiety.

[0162] 2. The molecule according to embodiment 1 , wherein the molecule is described by the general structure (I A) having the same meaning as defined in embodiment 1 .

[0163] 3. The molecule according to embodiment 1 , wherein the molecule is described by the general structure (I A) with L, RNand R having the same meaning as defined in embodiment 1 .

[0164] The molecule according to embodiment 3, wherein the molecule is described by the general structure (I AH) wherein L and R have the meaning assigned in embodiment 1 .

[0165] The molecule according to embodiment 1 , wherein the molecule is described by the general structure (I B) wherein L, RNand R have the meaning assigned in embodiment 1 , and wherein X is selected from CF3, Cl, CN, N3, CH3, OCH3 and H. The molecule according to embodiment 5, wherein the molecule is described by the general structure (I BH) wherein L and R have the meaning assigned in embodiment 1 , and wherein X is selected from CF3, Cl, CN, N3, CH3, OCH3 and H. The molecule according to embodiment 5 or 6, wherein X is CF3. The molecule according to embodiment 5 or 6, wherein X is Cl. The molecule according to embodiment 5 or 6, wherein X is OCH3. The molecule according to embodiment 5 or 6, wherein X is H. The molecule according to embodiment 1 , wherein the molecule is described by the general structure (I C) wherein L, RNand R have the meaning assigned in embodiment 1 . The molecule according to embodiment 11 , wherein the molecule is described by the general structure (I CH) wherein L and R have the meaning assigned in embodiment 1 . A molecule of the general formula (II) wherein L, RNand R have the meaning assigned in embodiment 1 . The molecule according to embodiment 13, wherein the molecule is described by the general structure (II H) wherein: wherein L and R have the meaning assigned in embodiment 1 . A molecule of the general formula (III) wherein L, RNand R have the meaning assigned in embodiment 1 . The molecule according to embodiment 15, wherein the molecule is described by the general formula III H: wherein L and R have the meaning assigned in embodiment 1 . The molecule according to any one of the preceding embodiments, wherein a linker moiety L is present, and L is a chain of 2 to 50 atoms selected from C, O, N and S. The molecule according to embodiment 17, wherein L is a Ci to C10 unsubstituted or substituted alkyl. The molecule according to embodiment 18, wherein L is a Ci to C10 substituted alkyl having one more methylene groups exchanged by a group independently selected from - CO-, -O-, -CONH-, -HNC(O)O-, -SS-, -NH-, -NN-. The molecule according to any one of the preceding embodiments 1 - 16, wherein a linker moiety L is not present. The molecule according to any one of the preceding embodiments, wherein R is a fluorescent dye moiety. The molecule according to embodiment 21 , wherein the fluorescent dye moiety is a rhodamine, particularly a rhodamine selected from carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR) and the isothiocyanate derivative TRITC. The molecule according to any one of the preceding embodiments 1 to 20, wherein R is a click reactive moiety, particularly a moiety selected from the group consisting of: a. an azide moiety, b. an alkyne moiety, particularly a cyclooctyne moiety; c. biotin; d. an unprotected or protected amine moiety; e. a sulfonamide moiety, f. a carboxylic acid or an activated form of a carboxylic acid, particularly an N- hydroxysuccinimide moiety, g. an ester moiety, h. an aldehyde, i. a thiol, and j. an isothiocyanate. The invention further encompasses the following compounds:

[0166] 1. A compound of formula:

[0167] mpound, selected from the group consisting of: n. the compound of formula 33: o. the compound of formula 34: p. the compound of formula 35: q. the compound of formula 38: r. the compound:

[0168] Wherever alternatives for single separable features 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. Thus, any of the alternative embodiments for a detectable label may be combined with any of the alternative embodiments of linker and these combinations may be combined with any alkyl transferase substrate moiety mentioned herein. The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

[0169] Description of the Figures

[0170] Fig. 1 shows labelling kinetics of SNAP2.0 with TF-, BG- and CF3P-TMR measured by stopped-flow fluorescence anisotropy. The panels present anisotropy traces (points) and predications of fits based on the kinetic model (lines) of SNAP2.0 labelling reaction. Labelling was performed at different concentrations of SNAP protein and a constant substrate concentration of 1 pM. The experiment was performed as previously described. (Wilhelm 2021 , ibid.) The lowest concentrations correspond to the lowest curves in the graph, and higher concentrations correspond to higher curves in order as seen in the level part of the graph.

[0171] Fig. 2 shows labelling performance of new SNAP-TMR substrates - quantification of imaging results: Violin plot of TMR / GFP labelling ratios.

[0172] Fig. 3 shows labelling performance of new CLIP-TMR substrates - quantification of imaging results: Violin plot of TMR / GFP labelling ratios.

[0173] Fig 4 shows the core structures of molecules encompassed by the inventions provided herein. The numbering coded by “S” refers to the numbers used to designate results in Fig. 5.

[0174] Fig. 5 shows the result of labelling experiments for the structures shown in Fig. 4, relative to the efficiency of BC, which is set to 1 .

[0175] Fig. 6 Live cell imaging of SNAP - substrates: U2OS cells were used (wild type and U2OS with stable expression of mEGFP-SNAP or mEGFP-CLIP). All images were taken under the same imaging conditions in the respective channel (laser intensity and detection settings). Cells were treated with 100 nM of substrates, 2 h incubation, unreacted substrates were quenched with mixture of SNAP and CLIP proteins (each 1 pM) and washed 2x for 10 min with imaging medium.

[0176] Fig. 7 Live cell imaging of CLIP - substrates: U2OS cells were used (wild type and U2OS with stable expression of mEGFP-SNAP or mEGFP-CLIP). All images were taken under the same imaging conditions in the respective channel (laser intensity and detection settings). Cells were treated with 100 nM of substrates, 2 h incubation, unreacted substrates were quenched with mixture of SNAP and CLIP proteins (each 1 pM) and washed 2x for 10 min with imaging medium. Examples

[0177] Example 1: Performance of new substrates in comparison with conventional substrates

[0178] New SNAP substrate TF-TMR (6) shows second order labelling kinetic constant kappof 9.00 (±0.02) x 106M'1s'1in SNAP2.0 mutant. In comparison, determined kinetic constants show superiority of the new substrate over CF3P-TMR tested in parallel in the same assay (kapp= 1 .36 (± 0.03) x 106M'1s'1) and over BG-TMR (kapp= 5.74 (± 0.01 ) x 105Ms’1).

[0179] New substrate CP-BF-TMR (5) outperforms CP-TMR in labelling in cellulo (See Table 1 , Fig. 2). Difference in fluorescence intensity ratio of TMR / GFP after 2 h of incubation of 100 nM is 2.5x and it indicates that new substrates has a higher permeability and / or labelling speed than CP-TMR. TF- TMR (6) shows TMR / GFP intensity ratio 2.47x higher than CP-TMR when tested in paralell (Table 1 , Fig. 2).

[0180] Comparison of performance in live cell imaging of SNAP - substrates CP-TMR, CP-BF-TMR, TF- TMR: U2OS cells were used (wild type and U2OS with stable expression of mEGFP-SNAP). Images in the GFP and TMR channel of a fluorescence microscope were taken under the same imaging conditions (laser intensity and detection settings). Cells were treated with 100 nM of substrates, 2 h incubation, unreacted substrates were quenched with mixture of SNAP and CLIP proteins (each 1 pM) and washed 2x for 10 min with imaging medium. When comparing different substrates, it can be observed clearly that the substrate molecule according to the invention performs significantly better, giving a stronger signal, than CP-TMR. GFP channel just shows localisation of SNAP protein, since it is expressed as a fusion of mEGFP and SNAP.

[0181] Table 1. Labelling performance of new SNAP-TMR substrates - quantification of imaging results (TMR / GFP ratio represents signal intensity corrected by expression level): descriptive statistics of analysed cells.

[0182] CLIP substrate 4-amino-pyridine analogue 7 is a substrate of CLIP2.0 with high labelling speed kapp= 1.01 (± 0.03) x 106M'1s'1. New fluorinated substrate 8 showed labelling speed with kapp= 3.43 (± 0.04) x 106M'1s'1. With the same protein mutant and assay, BC-TMR showed labelling speed with kapp= 2.81 (± 0.10) x 104M'1s'1. Both new substrates Py-TMR and PF-TMR outperformed BC-TMR in cellulo, showing difference in fluorescence intensity ratio after 2 h of incubation of 100 nM of 3.88X for Py-TMR and of 4.48X (See Table 2, Fig. 3). Comparison of performance in live cell imaging of CLIP - substrates BC-TMR, Py-TMR and PF- TMR: U2OS cells were used (wild type and U2OS with stable expression of mEGFP-CLIP). Again, images in the GFP and TMR channel of a fluorescence microscope were taken under the same imaging conditions (laser intensity and detection settings). Cells were treated with 100 nM of substrates, 2 h incubation, unreacted substrates were quenched with mixture of SNAP and CLIP proteins (each 1 pM) and washed 2x for 10 min with imaging medium. Again, when comparing different substrates, the substrate molecule according to the invention performs significantly better, giving a stronger signal, than BC-TMR:

[0183] Table 2. Labelling performance of new CLIP-TMR substrates - quantification of imaging results (TMR / GFP ratio represents signal intensity corrected by expression level): descriptive statistics of analysed cells.

[0184] New non-fluorescent substrates (15-18) were tested in competition in vitro assay to assess their labelling performance and compared with conventional non-fluorescence substrate substrates bearing the same tags (acetyl, Ac; norbornene, Nor; phenylazide, PhN3 and bicyclo 6.1.0 non-4- yn-9-ylmethyl, BCN) as previously described (Wilhelm et al., ibid.). New non-fluorescent substrates (TF conjugates) showed high labelling speed and they outperformed BG and CP conjugates (PhNa (18) and BCN (17) analogues) or showed comparable second order kinetic constants (Ac (15) and Nor (16) analogues) in both mutants SNAP and SNAP2.0 (See Table 3).

[0185] Table 3. Labelling performance of new non-fluorescent substrates in comparison with conventional substrates in SNAP and SNAP2.0 mutants: (a) tabularly presented values; graphical overview of values.

[0186] Kapp is given in unit: (M'1s'1).

[0187] Example 2 Demonstration of substrate performance in live cell labelling by confocal microscopy

[0188] Live cell imaging of SNAP - substrates: As shown in Fig. 6, the compounds according to the invention perform superior over prior art compounds. Additionally, Fig. 6 and 7 illustrate that for dye moieties different from tetramethylrhodamine TMRm such as siliconrhodamine SiR or carborhodamine CPY, the fluorobenzyl substrates work as well, and show orthogonality to CLIP.

[0189] Example 3: Experimental part

[0190] In vitro assays

[0191] Comparative kinetics assay

[0192] The comparative analysis of the kinetic properties of the substrates for hAGT mutants was conducted in triplicate within a 384-well plate with black non-binding flat bottom (Corning Inc.) using a microplate reader (Spark 20M, Tecan), by recording changes in fluorescence polarization over time (FP), maintained at a temperature of 37°C with a humidity control system. To ensure constant measurement conditions for all proteins / substrate combinations, the protein substrate (final concentration 20 nM) was added to the well with protein (final concentration 50 nM) via an injector, resulting in a final volume of 50 pL. The time elapsed between injection and the initial measurement was manually recorded and used later for correction. The G-factor was calculated based on a buffer and free fluorophore substrate control and the gain was set to 50%.

[0193] The calculated Kapp(apparent rate constant) and standard deviation (SD) were normalized by division by the corresponding Kapp value obtained for the hAGT mutant reference in each reaction (BC-TMR for CLIP-Tag and CP-TMR for SNAP-Tag and hAGT). The data points were then fitted to a one-phase association linear equation using R-Studio.

[0194] FP = FPo + (FP max—FPo)(1 - ekt)

[0195] In this equation, the variables include FPo (baseline measurement without protein), FPmax (maximum signal in FP), t (time), and k (rate constant in s'1). The k-value, when divided by the protein concentration, yields the Kappin M'1s'1.

[0196] In cello assays

[0197] Generation of stable cell lines

[0198] This protocol outlines the steps for generating and validating stable cell lines for gene expression studies.

[0199] Cell Line Generation

[0200] 1 . U2OS (Flp-ln ™ ) stable cell lines expressing mEGFP-SNAP and mEGFP-CLIP were generated. 2. The gene of interest (GOI) was cloned into the pcDNA5-FRT expression vector.

[0201] 3. Host cells were cultured in T25 flasks at 70% confluency one day before transfection.

[0202] 4. Transfection was performed by mixing pcDNA-FRT-GOI and pOG44 in 400 pL OptiMEM containing transfection reagents P3000 (8 pL, Invitrogen) and Lipofectamine 3000 (8 pL, Invitrogen) as recommended.

[0203] 5. The DNA-P3000-Lipofectamine 300 complex was added to the cells and incubated overnight at 37°C with 5% CO2.

[0204] 6. After 24 hours, the growth medium was changed.

[0205] Selection

[0206] 1 . Hygromycin (Gibco Life Technologies) (100 pg / mL) was added to the growth medium to initiate selection.

[0207] 2. Positive cells expressing the protein of interest (POI) were sorted using the FACS (BD Biosciences) Melody system with specific filter settings for mEGFP (FITC).

[0208] 3. Prior to sorting, cells were washed with PBS, trypsinized, and resuspended in FACS buffer.

[0209] 4. The cell suspensions were filtered before FACS sorting.

[0210] 5. After another round of hygromycin selection, cells were cultured in DMEM with 10% FBS in T75 flasks.

[0211] Validation

[0212] 1 . To verify the proper integration of the GOI, 100 pL of cells were taken and grown to confluence in a 96-well plate.

[0213] 2. Cells were subjected to lysis (56 °C for 1 h followed by 95 °C for 10 min, using DNA lysis buffer (30 pL).

[0214] 3. After DNA extraction, PCR analysis was performed to amplify specific primers for the detection of the FRT sites (primers SV40 Forward and Hygromycin Reverse).

[0215] 4. Agarose gel electrophoresis was used to visualize the PCR results.

[0216] Confocal microscopy - live cell imaging

[0217] U2OS cells, which had previously been engineered to stably express either mEGFP-SNAP or mEGFP-CLIP (as detailed above), were cultured to a state of 70% confluency. Prior to their use in the final experiment, these cells underwent 2-3 passages. For the experimental setup, 10,000 cells were carefully seeded into a 96-well plate with cell culture-grade quality. Subsequently, they were left to incubate overnight to allow for cell attachment. After 16 hours, the growth medium was aspirated, and 200 pL of pre-dissolved substrate conjugates, at a final concentration of 100 nM, were added to each well. The well plates were then incubated for 2 hours at 37 °C.

[0218] Following incubation, the cells were subjected to a series of preparatory steps. These included: a) removal of medium with substrates and addition of quenching solution - mixture of SNAP and CLIP proteins (each 1 pM solution in imaging medium) and incubation for 15 min at 37 °C; b) double wash with imaging medium (for 15 min at 37 °C), each time 200 pL. Imaging medium consists of full DMEM medium without phenol red. Cells were imaged using confocal Stellaris 5 Leica microscope. Stable cell were imaged with GFP channels and channel of respective fluorophore used for labelling (TMR, CPY or SiR) and wild type U2OS cells using respective channel of a fluorophore and bright field module.

[0219] The images were then further analyzed using Imaged, segmentation was done by automated pipeline in CellProfiler. (Stirling et al. 2021 )

[0220] Violin plots, as well as statistical analysis, were conducted using GraphPad Prism 10.

[0221] Syntheses procedures

[0222] General information

[0223] Reagents

[0224] All the reagents were obtained from various suppliers, such as Acros Chemicals, Alfa Aesar, Apollo Scientific, ABCR, Carbolution Chemicals, Carbosynth, Grussing, Manchester Organics, Merck, Novabiochem, Sigma-Aldrich, TCI Europe, VWR. Analytical grade solvents were used for flash chromatography without any purification. Anhydrous solvents were purchased for the reactions. Dissolved gases were removed from the solvents by either applying 3 cycles of freezing, pumping and thawing or by flushing N2 for at least 15 minutes. Deionized water was used for all the experiments.

[0225] Reaction conditions

[0226] All reactions were performed in heat dried glassware under atmosphere of N2 if not stated otherwise.

[0227] Reaction monitoring

[0228] Reactions were monitored by thin-layer chromatography (TLC) performed on TLC-aluminum sheets (Silica gel 60 F254, Merck). Compounds were visualized using ultraviolet light irradiation at 254 nm and 366 nm or by using the following staining agents (dip, dry & heat development).

[0229] Staining solution: KMnO4 (1 g), K2CO3 (6.6 g), 5% NaOH (1 .7 mL) in H2O (90 mL).

[0230] The reactions were monitored by using liquid chromatography-mass spectrometry (LCMS-2020, Shimadzu). A C18 1 .9 pm, 2.1 x 50 mm column (Supelco) was used for product visualization on an analytical reversed phase LC-MS equipped with an SPD-20AV UV-VIS photodiode array detector. A linear gradient of 10-95 % MeCN / FhO with constant 0.1 % v / v formic acid additive was applied in a 6 min run with 1 mL / min flow rate. Isolation of products.

[0231] Purification and isolation of compounds

[0232] An UltiMate 3000 (Thermo Fisher Scientific) was used for preparative RP-HPLC (Prep-HPLC) with a C18 5 pm, 21.2 x 250 mm column (Supelco, flow rate 8 mL / min), solvent A: 0.1 % v / v TFA in H2O, solvent B: MeCN. A freeze drying lyophilizer (Christ) equipped with a high-vacuum pump (Vacuubrand) was used for lyophilizing the compounds purified by Prep-HPLC. The compounds were dried, purified and spectroscopically characterized, and the yields were reported. Silica gel (230-400 mesh, Silicycle) was used for flash column chromatography (FCC) on an automated purification system (Biotage Isolera One).

[0233] Structural characterization of products

[0234] A Bruker DPX 400 was used for recording1H,13C and19F nuclear magnetic resonance (NMR) spectra at room temperature. Appropriate residual solvent peaks (CDCh, MeOH-D4, MeCN-D3, DMSO-D6) were used for referencing the spectra, which were reported in ppm (5-values). The spectra were recorded at 298 K, unless otherwise stated. CDCI3 was stored over molecular sieves at 4 °C. A maXis II™ ETD with electron spray ionization (ESI) (Bruker) was used for measuring high-resolution mass spectra (HRMS).

[0235] Synthesized molecules - new compounds or new synthesis routes

[0236] Building blocks - benzyl alcohols tert-butyl (2-fluoro-4-(hydroxymethyl)benzyl)carbamate (21 ):

[0237] Nitrile 19 (0.25 g, 1 .4 mmol, 1 equiv.) was dissolved added to LiAIFU (0.264 g, 6.9 mmol, 5 equiv.), suspended in THF (~50 mL) at 0 °C. After stirring for 20 min at 0 °C, solution was let to heat to 25 °C and then it was refluxed for 16 h. Reaction mixture was cooled to 0 °C, and remaining LiAIFU was quenched by careful addition, under nitrogen atmosphere, of EtOAc (~10 mL), water, after which all volatiles were removed under reduced pressure to obtain crude amino-alcohol (20), which was used in the next step without further purification.

[0238] Crude amine 20 was dissolved in 1 ,4-dioxane / water (1 :1 , ~50 mL) and cooled down to 0 °C, to which EtsN (2.82 g, 2.79 mmol, 2 equiv.) and BOC2O (0.456 g, 2.09 mmol, 1 .5 equiv.) were added. Reaction mixture was let to reach 25 °C while stirring. Volatiles were removed under reduced pressure and aqueous residue was extracted with EtOAc (2x ~50 mL), anhydrated with solid Na2SO4 and purified by FCC to obtain brownish solid product 21 (304 mg, 85.3% yield).

[0239] TLC: Rf- 0.33 (EtOAc / n- Hexane = 3:7).

[0240] 1H NMR (400 MHz, CDCIs) <5 = 7.17 (s, 2H), 6.98 (s, 2H), 5.24 (s, 1 H), 4.55 (s, 2H), 4.24 (d, >6.1 , 2H), 3.76 (s, 1 H), 1.41 (s, 9H) ppm.13C{1H} NMR (101 MHz, CDCI3) <5 = 162.0, 159.6, 156.2, 156.1 , 143.1 , 143.0, 140.1 , 138.0, 129.5, 129.5, 127.4, 127.1 , 124.6, 124.5, 122.2, 122.2, 122.1 , 113.6, 113.4, 79.7, 64.5, 63.8, 63.7, 44.3, 38.3, 38.3, 28.3, 28.2 ppm.

[0241] 19F NMR (376 MHz, CDCh) 5 = -119.3 ppm.

[0242] HRMS (El+) m / z: [M + Na]+, Calculated for Ci3Hi8NO3FNa 278.1163, found 278.1161 .

[0243] Building blocks - nucleobases tert-butyl (4,6-dichloropyrimidin-2-yl)carbamate (23):

[0244] 22 23

[0245] 2-amino-4,6-dichloro pyrimidine 22 (4.2 g, 25.6 mmol, 1 eq.) was dissolved in DCM (50 mL) and oxalyl chloride (9 ml) was added followed by the addition of 3 drops of DMF, at 25 °C. The reaction mixture was refluxed for 16 h. After removing volatiles, the residue was dissolved in DCM and an excess (10 mL) of t-BuOH was added. The reaction was refluxed for an additional 16 h. Volatiles were removed, and the residue dissolved in DCM and purified by FCC to give colorless tick oily product 23 (4.5 g, 66.5% yield).

[0246] TLC: Rf- 0.75 (EtOAc / n- Hexane = 2:8).

[0247] 1H NMR (400 MHz, DMSO) 5 = 10.74 (s, 1 H), 7.53 (s, 1 H), 1 .46 (s, 9H) ppm.

[0248] 13C{1H} NMR (101 MHz, DMSO) 5 = 161 .2, 157.6, 150.0, 114.7, 80.5, 27.8 ppm.

[0249] HRMS (El+) m / z: [M + Na]+, Calculated for C9HiiCI2N3NaO2+286.0121 , found 286.0122. tert-butyl (4-chloro-6-(methylthio)pyrimidin-2-yl)carbamate (24):

[0250] Carbamate 23 (4.5 g, 17 mmol) was dissolved in DMF (-100 mL) and solid sodium methanethiolate (1 .2 g, 17 mmol, 1 equiv.), was added in one portion at 25 °C. Reaction mixture was stirred for 16 h after which DMF was removed under reduced pressure. Solid residue was dissolved in DCM (-200 mL) and washed with water (3 x -50 mL). Organic fraction was anhydrated with solid Na2SO4 and purified by FCC. TLC: Rf= 0.38 (EtOAc / n- Hexane = 1 :1 ).

[0251] 1H NMR 400 MHz, CDCh) 5 = 7.36 (s, 1 H), 6.86 (s, 1 H), 2.59 (s, 3H), 1 .55 (s, 9H) ppm.

[0252] 13C{1H} NMR (101 MHz, CDCI3) <5 = 173.7, 159.6, 156.5, 149.7, 111.9, 81.9, 28.2, 12.7 ppm.

[0253] HRMS (El+) m / z: [M + H]+, Calculated for CioHi5N302CIS+276.0568, found 276.0569. tert-butyl (4-chloro-6-(methylsulfonyl)pyrimidin-2-yl)carbamate (25):

[0254] Methylthiopyrimidine 24 (3.7 g, 13.6 mmol) was dissolved in DCM (-100 mL) and solid 3- chloroperoxybenzoic acid (16.8 g, 68.3 mmol, 5 equiv., 70% purity), was added in few small portions at 0 °C. Reaction mixture was stirred for 16 h letting it to reach 25 °C. Reaction mixture was cooled to 0 °C and quenched by addition of aqueous solution of Na2SO3. Organic fraction was separated and aqueous fraction was extracted with additional DCM (3 x ~150 mL). Combined organic fractions were anhydrated with solid Na2SO4 and purified by FCC.

[0255] 1H NMR (400 MHz, DMSO) <5 = 10.98 (s, 1 H), 7.72 (s, 1 H), 3.34 (s, 3H), 1.47 (s, 9H) ppm.

[0256] 13C{1H} NMR (101 MHz, DMSO) <5 = 167.2, 163.1 , 158.1 , 150.1 , 110.7, 80.7, 27.8 ppm.

[0257] HRMS (El+) m / z: [M + Na]+, calculated for CioHi4CIN3Na04S+330.0286, found 330.0288.

[0258] 4-(methylsulfonyl)-6-(trifluoromethyl)pyrimidin-2-amine (28):

[0259] 4-chloro-6-(trifluoromethyl)pyrimidin-2-amine 26 (2.0 g, 10.12 mmol, 1 eq.) was dissolved in DMF (-100 mL) and solid sodium methanethiolate (0.85 g, 12.149 mmol, 1 .2 equiv.), was added in one portion at 25 °C. Reaction mixture was stirred for 16 h after which DMF was removed under reduced pressure. Solid residue was dissolved in DCM (-200 mL) and washed with water (3 x -50 mL). Organic fraction was anhydrated with solid Na2SO4 and used in the next step without further purification after confirming conversion by LC / MS to methylthiopyrimidine 27.

[0260] Crude methylthiopyrimidine 27 was suspended in DCM / H2O (1 :1 , 50 mL) at 25 °C followed by addition of Na2WO4 (0.031 g, 0.096 mmol, 0.05 equiv.), H2O2(0.9 mL, 7.7 mmol, 4 equiv., 30% in H2O) and one drop of acetic acid. Reaction mixture was heated at 50 °C (oil bath) for 4 h. After cooling of reaction mixture to 0 °C, remaining hydrogen peroxide was quenched by addition of aqueous solution of Na2SOs. Organic fraction was separated and aqueous fraction was extracted with additional DCM (3 x -100 mL). Combined organic fractions were anhydrated with solid Na2SO4 and purified by using 25 g prepacked column (gradient 20-80% EtOAc / n- Hexane, over 30 column volumes) to give as white amorphous solid methylsulfon 28 (805 mg) in yield of 32% over 2 steps.

[0261] TLC: Rf- 0.65 (EtOAc / n- Hexane = 1 :1 ).

[0262] 1H NMR1H NMR (400 MHz, DMSO) 5 = 8.10 (s, 2H), 7.34 (s, 1 H), 3.32 (s, 3H) ppm.

[0263] 13C{1H} NMR (101 MHz, DMSO) 5 = 169.4, 164.1 , 158.9, 158.5, 121.8, 119.1 , 100.0, 39.6 ppm. 19F{1H} (376 MHz, CDCI3) 5 = -69.4 ppm.

[0264] HRMS (El+) m / z: [M + H]+, Calculated for CeHeFsIX^S 242.0206 found 242.0205.

[0265] Conjugate precursors tert-butyl (4-(((4-aminopyridin-2-yl)oxy)methyl)benzyl)carbamate (33): a) Bis-alcohol (29, 0.145 g, 1.07 mmol, 1.1 equiv.) was dissolved in round bottom flask in mixture of 1 ,4-dioxane and THF (1 :1 , 20 mL), cooled to 10 °C and solid NaH (0.045 g, 60%, 1 .2 equiv.) was added. After stirring for 30 min at the same temperature under N2 atmosphere, fluoropyridine 30 (0.107 g, 0.95 mmol, 1 equiv.) was added and reaction mixture was heated to reflux (-100 °C, oil bath) and stirred for 16 h. After cooling down to 25 °C, reaction was quenched with solid NH4CI (-100 mg) and volatiles were removed under reduced pressure. Crude residue was dissolved in

[0266] DCM (-100 mL) and washed with water (2 x -30 mL) and organic fraction was anhydrated with solid anhydrous Na2SO4. After removing the volatiles under reduced pressure, alcohol 31 was used in the next step without further purification. b) Alcohol 31 was dissolved in mixture of toluene and THF (4:1 , 20 mL) at room temperature (-25 °C) in the round bottom flask. To the reaction mixture DPPA (0.525 g, 1 .9 mmol, 2 equiv.) was added, followed by DBU (0.29 g, 1.9 mmol, 2 equiv.) and it was stirred under N2 atmosphere for 16 h. Reaction mixture was diluted with EtOAc (-50 mL) and washed with saturated solution of NaHCOs (3 x -20 mL), organic fraction was anhydrated with solid anhydrous Na2SO4, volatiles removed under reduced pressure and azide (32) was used in the next step without further purification. c) Crude azide (32) was dissolved in THF (20 mL) and tributylphosphine (0.256 g, 1.3 mmol, 1 .33 equiv.) was added in one portion at room temperature, while keeping round bottom flask open to reduce chance of over pressure in the flask caused by released gas. The reaction mixture was stirred for 2 h and water (3 mL) was added in one portion. After stirring for 16 h, NaOH (1 M, 5 mL) was added at 0 °C followed by BOC2O (0.43 g, 2.0 mmol, 2.0 equiv.) and stirring was continued for another 12 h, letting the reaction mixture to reach room temperature. Reaction mixture was extracted with EtOAc (3 x 30 mL), organic fractions were combined and anhydrated with solid anhydrous Na2SC>4. After removing the volatiles under reduced pressure, automated FCC using 20 g prepacked column (gradient 20% EtOAc in n-Hexane to 100% EtOAc, over 20 volumes of column) provided 95 mg of carbamate 33 as white amorphous solid (29.5% yield, over 3 steps).

[0267] TLC: Rf- 0.74 (EtOAc / n- Hexane = 7:3).

[0268] 1H NMR (400 MHz, CDCh) <5 = 7.68 (d, >5.7, 1 H), 7.26 (d, >8.1 , 2H), 7.14 (d, >7.9, 2H), 6.07 (dd, >5.8, 2.0, 1 H), 5.86 (d, >2.0, 1 H), 5.17 (s, 2H), 4.17 (d, >6.2, 4H), 1 .35 (s, 9H) ppm.

[0269] 13C NMR (101 MHz, CDCh) 5 = 165.0, 156.0, 155.5, 147.0, 138.4, 136.7, 128.1 , 127.4, 105.8, 93.9, 79.4, 67.1 , 44.4, 28.4.

[0270] HRMS (El+) m / z: [M + H]+, Calculated for Ci8H24N3O3+330.1812, found 330.1811. tert-butyl (4-(((2-((tert-butoxycarbonyl)amino)-6-chloropyrimidin-4-yl)oxy)methyl)-2- fluorobenzyl)carbamate (34):

[0271] Benzyl alcohol 21 (24 mg, 0.097 mmol, 1 equiv.) was dissolved in THF (5 mL) and cooled down to 0 °C. Sodium hydride (4.3 mg, 0.126 mmol, 1.3 equiv.) was added and suspension was stirred at the constant temperature for 30 min. Methyl sulfone 25 (30 mg, 0.097 mmol, 1 equiv.) was added in one portion and reaction mixture was let to reach 25 °C over 16 h. Remaining unreacted hydride and alkoxide was quenched by addition of solid NH4AC (~10 mg), volatiles were removed under reduced pressure and mixture was purified by automated FCC, 20 g prepacked column was used, gradient 10% EtOAc in n-Hexane to 50% EtOAc, over 20 volumes of column. Solid white product was obtained (33 mg, 70% yield).

[0272] TLC: Rf- 0.82 (EtOAc / n- Hexane = 3:7).

[0273] 1H NMR (400 MHz, CDCh) 5 = 7.38 - 7.21 (m, 3H), 7.13 (s, 2H), 6.37 (s, 1 H), 5.33 (s, 2H), 4.28 (d, >6.1 , 2H), 1 .47 (s, 9H), 1 .37 (s, 9H) ppm.

[0274] 13C NMR (101 MHz, CDCh) 5 = 170.5, 161.2, 156.7, 155.8, 149.7, 137.2, 137.1 , 129.9, 129.0, 124.2, 115.7, 115.5, 101.8, 101.8, 81.9, 81.8, 79.7, 67.9, 67.9, 38.5, 28.4, 28.2 ppm.

[0275] 19F NMR (376 MHz, CDCh) 5 = -118.8 ppm. HRMS (El+) m / z: [M + Na]+, Calculated for C22H28CIFN4NaO5+505.1624, found 505.1613. tert-butyl (4-(((2-amino-6-(trifluoromethyl)pyrimidin-4-yl)oxy)methyl)-2-fluorobenzyl)carbamate (35):

[0276] Carbamate 35 was synthesized following procedure described above for 34 from alcohol 21 and methylsulfone 28. White solid product (175 mg, 68% yield) was obtained after purification by automated FCC and additional purification by prep. HPLC.

[0277] TLC: Rf- 0.48 (EtOAc / n- Hexane = 3:7).

[0278] 1H NMR (400 MHz, CDCh) 5 = 7.36 (s, 1 H), 7.21 - 7.00 (m, 2H), 6.44 (s, 1 H), 5.35 (s, 2H), 4.37 (d, >5.9, 2H), 1.46 (s, 9H) ppm.

[0279] 13C{1H} NMR (101 MHz, CDCh) 5 = 170.7, 163.3, 163.2, 162.0, 159.6, 157.2, 156.8, 155.9, 137.4, 137.3, 130.0, 130.0, 128.5, 126.1 , 126.0, 123.6, 123.6, 121 .9, 119.2, 114.9, 114.7, 95.3, 95.3, 95.2, 95.2, 79.7, 68.1 , 67.3, 67.3, 38.4, 28.4, 28.2 ppm.

[0280] 19F NMR (376 MHz, CDCh) 5 = -76.55 (TFA), -124.42 - -124.48 (m) ppm.

[0281] HRMS (El+) m / z: [M + H]+, Calculated for C18H21N4O3F4 I7.1544, found 417.1542. tert-butyl (2-fluoro-4-(((4-iodopyridin-2-yl)oxy)methyl)benzyl)carbamate (37):

[0282] Iodide 37 was synthesized following procedure described above for 34 from alcohol 21 and fluoro- iodo-pyridine 36. Colorless to yellowish oily product was obtained, which solidified upon storage in fridge (320 mg, 78% yield) was obtained after purification by automated FCC.

[0283] TLC: Rf- 0.80 (EtOAc / n- Hexane = 3:7).

[0284] 1H NMR (400 MHz, CDCh) 5 = 7.75 (dd, >5.3, 0.7, 1 H), 7.39 - 7.13 (m, 4H), 7.13 - 6.98 (m, 2H), 5.25 (d, >2.2, 2H), 4.28 (d, >6.0, 2H), 1 .37 (s, 9H) ppm.

[0285] 13C{1H} NMR (101 MHz, CDCh) 5 = 163.6, 163.3, 162.1 , 159.7, 155.8, 147.0, 138.7, 138.6, 136.0, 130.0, 129.9, 128.3, 127.6, 126.3, 126.1 , 125.6, 125.4, 123.4, 120.7, 120.6, 114.8, 114.6, 106.5, 106.4, 79.7, 67.6, 66.8, 66.7, 38.5, 28.4, 28.4 ppm.19F NMR (376 MHz, CDCh) 6 = -119.03 (d, >18.9), -119.03 ppm.

[0286] HRMS (El+) m / z: [M + H]+, Calculated for Ci8H2iFIN2O3+459.0575, found 459.0571. tert-butyl (4-(((4-((tert-butoxycarbonyl)amino)pyridin-2-yl)oxy)methyl)-2-fluorobenzyl)carbamate (38):

[0287] Iodide 37 (0.3 g, 0.654 mmol, 1 equiv.) was dissolved in anhydrous 1 ,4-dioxane (~25 mL) followed by addition of Tris(dibenzylideneacetone)dipalladium(0) (89 mg, 0.098 mmol, 0.15 equiv.), tert- Butyl carbamate (115 mg, 0.98 mmol, 1.5 equiv.), Xantphos (170 mg, 0.294 mmol, 0.45 equiv.) and CsCOs (853 g, 2.6 mmol, 4 equiv.). Reaction mixture was refluxed under inert nitrogen atmosphere for 3 h, let to cool down to room temperature and diluted with EtOAc (-100 mL) and washed with saturated solution of NaHCOs, ammonium chloride and water (each -30 mL). Organic fraction was anhyd rated with solid Na2SO4 and purified by FCC to provide 256 mg of bis-carbamate 38 in 87% yield, as a brownish solid.

[0288] TLC: Rf- 0.65 (EtOAc / n- Hexane = 7:3).

[0289] 1H NMR (400 MHz, CDCh) 5 = 7.97 (d, >5.8, 1 H), 7.29 (d, >7.3, 2H), 7.13 (td, >8.7, 1.6, 3H), 7.01 - 6.81 (m, 2H), 5.33 (d, >2.0, 2H), 4.35 (d, >6.0, 2H), 1 .51 (s, 9H), 1 .45 (s, 9H) ppm.

[0290] 13C{1H} NMR (101 MHz, CDCh) 5 = 164.5, 155.9, 152.0, 148.1 , 148.0, 147.2, 147.2, 139.3, 139.3, 129.8, 129.7, 128.0, 123.1 , 123.1 , 114.5, 114.3, 107.6, 107.5, 98.4, 98.4, 81.5, 81.4, 79.6, 67.3, 66.4, 66.4, 38.5, 28.4, 28.4, 28.2, 28.2 ppm.

[0291] 19F NMR (376 MHz, CDCh) 5 = -119.20 (t, >9.2) ppm.

[0292] HRMS (El+) m / z: [M + H]+, Calculated for C23H3iN3O5F+448.2242, found 448.2243.

[0293] Conjugate 5: Active ester of TMR-6-C00H: TMR-6-C00H (13 mg, 0.032 mmol, 1.2 equiv.) was dissolved in DMF (~3 ml_) and DIPEA (13 mg, 0.1 mmol, 4 equiv.) was added followed by addition of PyAOP (17 mg, 0.32 mmol, 1.2 equiv.). Reaction mixture was stirred for 10 min.

[0294] Carbamate 34 (10 mg, 0.021 mmol, 1 equiv.) was dissolved in DCM (~5 mL) and cooled to 0 °C. TFA (2 mL) was added and reaction mixture stirred for 1 h at the constant temperature. After removing volatiles (at 25 °C), active ester of TMR was added at 25 °C to the crude ammonium residue in DMF (~ 3 mL) and reaction was stirred for 4 h. Unreacted components were quenched by addition of 0.5 mL of 10% AcOH in water, volatiles removed under reduced pressure and purified by preparative HPLC to provide 7 mg (48% yield) of fluorophore conjugate 5 as a red solid.

[0295] HRMS (El+) m / z: [M + H]+, Calculated for Cs / F^CIFNeOs 695.2180, found 695.2182.

[0296] Conjugate 6:

[0297] Conjugate (6) was synthesized like described above for 5. Solid red product was isolated by preparative HPLC (9 mg, 47% yield).

[0298] HRMS (El+) m / z: [M]+, Calculated CssF^NeOs 729.2443, found 729.2441.

[0299] Conjugate 7:

[0300] Conjugate (7) was synthesized like described above for 5. Solid red product was isolated by preparative HPLC (10 mg, 57% yield).

[0301] HRMS (El+) m / z: [M + H]2+, Calculated for CssHszNsOs2321 .6392, found 321 .6388. Conjugate 8:

[0302] Conjugate (8) was synthesized like described above for 5. Solid red product was isolated by preparative HPLC (7 mg, 92% yield). HRMS (El+) m / z: [M + H]+, Calculated for C38H35FN5C 660.2617, found 660.2623.

[0303] Conjugate 9:

[0304] Conjugate (9) was synthesized like described above for 5 (CPY-6-COOH, 5.4 mg, 0.012 mmol was used). Dark blue to violet product was isolated by preparative HPLC (7 mg, 77% yield). HRMS (El+) m / z: [M]+, Calculated for C4iH39F4N6O4+755.2963, found 755.2963.

[0305] Conjugate 10:

[0306] Conjugate (10) was synthesized like described above for 5 (CPY-6-COOH, 7 mg, 0.015 mmol was used). Dark blue to violet product was isolated by preparative HPLC (7 mg, 69% yield). HRMS (El+) m / z: [M]+, Calculated for C4iH42N5O4+668.3231 , found 668.3226. Conjugate 11 :

[0307] Conjugate (11 ) was synthesized like described above for 5 (CPY-6-C00H, 5 mg, 0.011 mmol was used). Dark blue to violet product was isolated by preparative HPLC (6 mg, 78% yield). HRMS (El+) m / z: [M]+, Calculated for C4oH42FN504+686.3137, found 686.3139.

[0308] Conjugate 12:

[0309] Conjugate (12) was synthesized like described above for 5 (SiR-6-COOH, 11 mg, 0.024 mmol was used). Dark blue product was isolated by preparative HPLC (10 mg, 54% yield). HRMS (El+) m / z: [M]+, Calculated for C40H39F4N6O4SF 771 .2733, found 771 .2734.

[0310] Conjugate 13:

[0311] Conjugate (13) was synthesized like described above for 5 (SiR-6-COOH, 5.2 mg, 0.011 mmol was used). Dark blue product was isolated by preparative HPLC (13 mg, 86% yield). HRMS (El+) m / z: [M]+, Calculated for C4oH42N504Si+684.3001 , found 684.3004. Conjugate 14:

[0312] Conjugate (14) was synthesized like described above for 5 (SiR-6-COOH, 11 mg, 0.024 mmol was used). Dark blue product was isolated by preparative HPLC (7 mg, 89% yield). HRMS (El+) m / z: [M]+, Calculated for C4oH4iN5F04Si+702.2906, found 702.2909.

[0313] Conjugate 15:

[0314] Carbamate 35 (15 mg, 0.036 mmol, 1 equiv.) was dissolved in DCM (~5 mL) and cooled to 0 °C. TFA (3 mL) was added and reaction mixture was stirred at the same temperature for 2 h. Volatiles were removed in flow of nitrogen and crude residue was used for coupling with acylating reagent (1 ,3-Dihydro-1 ,3-diacetyl-2H-benzimidazol-2-one) (9 mg, 0.043 mmol, 1 equiv.) like previously described.10(Kim, C. S.; Chung, I. H.; Cha, K. S.; Seo, J. H.; Kim, J. H.; Chung, B. Y., 1 , 3-Dihydro- 1 , 3-diacetyl-2H-benzimidazol-2-one: a new versatile and selective acetylating agent. Heterocycles 2000, 53 (3), 529.) in DCM as solvent (~5 mL) and using triethylamine as a base (7.3 mg, 0.072 mmol, 2 equiv.). Reaction mixture was stirred for 8 h. White amorphous product was isolated by preparative HPLC (9 mg, 69% yield).

[0315] HRMS (El+) m / z: [M+H]+, Calculated for CI5HI5F4N4O2+359.1126, found 359.1126.

[0316] Conjugate 16: Carbamate 35 (10 mg, 0.024 mmol, 1 equiv.) was dissolved in DCM (~5 ml_) and cooled to 0 °C. TFA (3 mL) was added and reaction mixture was stirred at the same temperature for 2 h. Volatiles were removed in flow of nitrogen and crude residue was used for coupling with previously activated 5-Norbornene-2-acetic acid (5.9 mg, 0.024 mmol, 1 equiv.) with PyAOP (4.6 mg, 0.024 mmol, 1 equiv.) in DMF for 3 min in presence of DIPEA as a base (12 mg, 0.96 mmol, 4 equiv.). Reaction mixture was stirred for 8 h. White amorphous product was isolated by preparative HPLC (3 mg, 27% yield).

[0317] HRMS (El+) m / z: [M+H]+, Calculated for C22H23F4N4O2+451.1752, found 451.1750.

[0318] Conjugate 17:

[0319] Carbamate 35 (10 mg, 0.024 mmol, 1 equiv.) was dissolved in DCM (~5 mL) and cooled to 0 °C. TFA (3 mL) was added and reaction mixture was stirred at the same temperature for 2 h. Volatiles were removed in flow of nitrogen and crude residue was used for coupling with active ester rel- ((1 R,8S,9s)-Bicyclo[6.1 ,0]non-4-yn-9-yl)methyl (4-nitrophenyl) carbonate (7.5 mg, 0.024 mmol, 1 equiv.) in DMF in presence of DIPEA as a base (12 mg, 0.96 mmol, 4 equiv.). Reaction mixture was stirred for 8 h. White amorphous product was isolated by preparative HPLC (3 mg, 25% yield).

[0320] HRMS (El+) m / z: [M+H]+, Calculated for C24H25F4N4O3+493.1857, found 493.1861.

[0321] Conjugate 18:

[0322] Carbamate 35 (10 mg, 0.024 mmol, 1 equiv.) was dissolved in DCM (~5 mL) and cooled to 0 °C. TFA (3 mL) was added and reaction mixture was stirred at the same temperature for 2 h. Volatiles were removed in flow of nitrogen and crude residue was used for coupling with previously activated 5-Norbornene-2-acetic acid (3.9 mg, 0.024 mmol, 1 equiv.) with PyAOP (4.6 mg, 0.024 mmol, 1 equiv.) in DMF for 3 min in presence of DIPEA as a base (12 mg, 0.96 mmol, 4 equiv.). Reaction mixture was stirred for 8 h. White amorphous product was isolated by preparative HPLC (6 mg, 54% yield). HRMS (El+) m / z: [M+H]+, Calculated for C2oHi6F4N702+462.1296, found 462.1300. tert-butyl (4-(((2-amino-6-methoxypyrimidin-4-yl)oxy)methyl)benzyl)carbamate VNP_187

[0323] Chloride VNP_186 (50 mg, 0.137 mmol, 1 equiv.) was dissolved in 1 ,4-dioxane (10 mL) and sodium methoxide was added (44 mg, 0.05 mL, 0.2 mmol, 1.5 equiv., 25% in MeOH) at 25 °C.

[0324] Reaction was heated at reflux temperature for 16 h. Remaining unreacted methoxide was quenched with solid NH4AC (~20 mg) and volatiles were removed under reduced pressure. The mixture was purified by automated FCC to provide 39 mg of methyl ether Fehler! Verweisquelle konnte nicht gefunden werden.187 in 79% yield, as a white solid. TLC: Rf- 0.22 (EtOAc / n- Hexane = 3:7).

[0325] 1H NMR1H NMR (400 MHz, DMF-rt7) <5= 7.43 (d, >7.9, 2H), 7.33 (d, >7.8, 3H), 6.74 (s, 2H), 5.43 (s, 1 H), 5.30 (s, 2H), 4.27 (d, >6.3, 2H), 3.81 (s, 3H), 1.42 (s, 9H) ppm.

[0326] 13C{1H} NMR13C NMR (101 MHz, DMF-rtz) 5 = 172.2, 171.6, 163.2, 156.3, 140.4, 135.9, 128.4, 127.2, 78.6, 77.9, 67.0, 53.0, 43.6, 27.9 ppm. HRMS (El+) m / z: [M + H]+, Calculated for C24H25N4O4+361 .1870, found 361.1870.

[0327] Chloride VNP_186 was synthesized by previously reported synthetic route. (Gautier 2021 , ibid)

[0328] Conjugate XX: ### VNP_191

[0329] Conjugate (VNP_191 ) was synthesized like described above for 5. Amorphous solid red product was isolated by preparative HPLC (3 mg, 77% yield).

[0330] HRMS (El+) m / z: [M + H]+, Calculated for CssHszNeOe 673.2769, found 673.2768.

[0331] Cited references:

[0332] Correa et al., Substrates for improved live-cell fluorescence labeling of SNAP-tag. Current pharmaceutical design 2013, 19 (30), 5414-5420 Gautier et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 2008, 75 (2), 128-36

[0333] Keppler et al., A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 2003, 27 (1 ), 86-9

[0334] Kim, C. S.; Chung, I. H.; Cha, K. S.; Seo, J. H.; Kim, J. H.; Chung, B. Y., 1 , 3-Dihydro-1 , 3-diacetyl- 2H-benzimidazol-2-one: a new versatile and selective acetylating agent. Heterocycles 2000, 53 (3), 529.

[0335] Stirling et al., CellProfiler 4: improvements in speed, utility and usability. BMC Bioinformatics 2021 , 22 (1 ), 433

[0336] Wilhelm et al., Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag. Biochemistry 2021 , 60 (33), 2560-2575

[0337] All scientific publications and patent documents cited in the present specification are incorporated by reference herein.

[0338] SEQUENCES:

[0339] In the event of discrepancies between the sequences shown in the present specification and those of the enclosed sequence protocol according to WIPO standard ST.26, the sequences shown herein shall prevail.

[0340] SEQ ID NO 001 SNAP:

[0341] MDKDCEMKRT TLDSPLGKLE LSGCEQGLHE I IFLGKGTSA ADAVEVPAPA AVLGGPEPLM QATAWLNAYF HQPEAIEEFP VPALHHPVFQ QESFTRQVLW KLLKWKFGE VISYSHLAAL AGNPAATAAV KTALSGNPVP ILI PCHRWQ GDLDVGGYEG GLAVKEWLLA HEGHRLGKPG LG

[0342] SEQ ID NO 002 CLIP:

[0343] MDKDCEMKRTTLDSPLGKLELSGCEQGLHEI IFLGKGTSAADAVEVPAPAAVLGGPEPLIQATAWLNAYFH

[0344] QPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKWKFGEVISESHLAALVGNPAATAAVNTALDGNPVPIL

[0345] IPCHRWQGDSDVGPYLGGLAVKEWLLAHEGHRLGKPGLGG

[0346] SEQ ID NO 003 SNAP2.0:

[0347] MDKDCEMKRT TYDSPLGKLL LSGCEQGLHR IYFVGNGQGE QGPPGPEPLM QATAWLNAYF

[0348] HQPEAIEEFP VPALHHPVFQ QESFTRQVLW KLLKWKFGE VISYSQLAAL AGNPAATAAV

[0349] KTALRGNPVP ILIPCHRWQ GDGDVGGYEG PLYVKEWLLA HEGHRLGKPG LG

[0350] SEQ ID NO 004 CLIP2.0:

[0351] MDKDCEMKRT TLDSPLGKLE LSGCEQGLHR IYFIGNGQGE QGPPGPEPLI QATAWLNAYF

[0352] HQPEAIEEFP VPALHHPVFQ QESFTRQVLW KLLKWKFGE VISESHLAAL VGNPAATAAV

[0353] NTALDENPVP ILIPCHRWQ GDSDVGPYAA GLAVKEWLLA HEGHRLGKPG LGG

Claims

Claims1 . A molecule of the general formula (I)wherein:- SB is selected from the group consisting of a 2-aminopyrimidinyl moiety, an aminopyridinyl moiety, and a 2-aminopurinyl moiety;- RNis selected from the group consisting of H, methyl (CH3), ethyl, fluorinated methyl and fluorinated ethyl, and- L is an optional linker,- R is selected from a detectable label and a selectively reactive moiety.

2. The molecule according to claim 1 , wherein the molecule is described by the general structure (I A)wherein L, RNand R have the meaning assigned in claim 1 .

3. The molecule according to claim 1 , wherein the molecule is described by the general structure (I B)wherein L, RNand R have the meaning assigned in claim 1 , and wherein X is selected from CF3, Cl, CN, N3, CH3, OCH3and H.

4. The molecule according to claim 1 , wherein the molecule is described by the general structure (I C)wherein L, RNand R have the meaning assigned in claim 1 .

5. The molecule according to any one of the preceding claims, wherein a linker moiety L is present, and L is a chain of 2 to 50 atoms selected from C, O, N and S.

6. The molecule according to claim 10, wherein L is a Ci to C10 substituted alkyl, particularly a Ci to C10 substituted alkyl having one more methylene groups exchanged by a group independently selected from -CO-, -O-, -CONH-, -HNC(O)O-, -SS-, -NH-, -NN-.

7. The molecule according to any one of the preceding claims 1 - 6, wherein a linker moiety L is not present.

8. The molecule according to any one of the preceding claims, wherein R is a fluorescent dye moiety.

9. The molecule according to any one of the preceding claims 1 to 9, wherein R is a click reactive moiety, particularly a moiety selected from the group consisting of: a. an azide moiety, b. an alkyne moiety, particularly a cyclooctyne moiety; c. biotin; d. an unprotected or protected amine moiety; e. a sulfonamide moiety, f. a carboxylic acid or an activated form of a carboxylic acid, particularly an N- hydroxysuccinimide moiety, g. an ester moiety, h. an aldehyde, i. a thiol, and j. an isothiocyanate.

10. A compound according to claim 1 , described by a formula:11 . A precursor compound, selected from the group consisting of: k. the compound of formula 34:I. the compound of formula 35:m. the compound of formula 38:

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