NON-COVALENT HALOTAG LIGANDS

Abstract

A first aspect of the invention relates to a non-covalently-HaloTag-binding compound characterized by the general formula D-L-T (I), wherein D is or comprises a functional moiety Z, particularly a fluorescent dye, or D is a linkable moiety (i.e. a moiety that can be coupled to other functional groups), L is a linear linker of 10-15 atoms in length, and T is a moiety selected from the group comprising methylamine, methylsulfonamide, acetamide, or their respective fluorinated analogues, azide, or hydroxyl. Another aspect of the invention relates to a HaloTag variant wherein position D106 of the HaloTag7 sequence is exchanged for a proteinogenic amino acid different from D. The variant has a different binding specificity for HaloTag substrates compared to the non-variant Halotag polypeptide. Yet another aspect of the invention relates to kits comprising polypeptides or nucleic acids and the non-covalently-HaloTag-binding compound according to the invention.

Claims

1. A non-covalently-HaloTag-binding compound characterized by the general formula (I) D-L-T (I), wherein a. D is or comprises a functional moiety Z selected from an organic dye moiety characterized by a molecular mass of between 300 g/mol and 1300 g/mol, particularly a fluorescent organic dye moiety; an affinity purification ligand, a pharmaceutical drug or a pharmaceutical drug candidate, an oligopeptide or a polypeptide, a nanoparticle, a solid surface or a matrix polymer, a nucleic acid oligomer, a carbohydrate, a lipid, a spectroscopic probe, a sensor, particularly a fluorescent sensor, a natural product, and a synthetic ligand binding to a biomolecule with an affinity of at least 100 ?M, or b. D is a linkable moiety selected from an unprotected or protected amine moiety, a sulfonamide moiety, an N.sub.3 moiety, an alkyne moiety, a carboxylic acid or an activated form of a carboxylic acid, particularly an N-hydroxysuccinimide moiety, an ester moiety, an aldehyde, a thiol, and an isothiocyanate; L is a linear linker of 10-15 atoms in length, particularly wherein L is 10-11 atoms in length, wherein L comprises, particularly L essentially consists of, alkyl, trans-alkylene, and/or ether moieties and optionally one or several methyl substituents, particularly L is an unbranched linker that does not comprise methyl substituents; T is a moiety selected from the group comprising methylamine, monofluormethylamine, difluormethylamine, trifluormethylamine, methylsulfonamide, monofluormethylsulfonamide, difluormethylsulfonamide, trifluormethylsulfonamide, azide, acetamide, monofluoracetamide, difluoracetamide, and trifluoracetamide; particularly T is a moiety selected from the group comprising methylamine, trifluormethylsulfonamide and methylsulfonamide.

2. A non-covalently-HaloTag-binding compound characterized by the general formula (I) D-L-T (I), wherein a. D is or comprises a functional moiety Z selected from an organic dye moiety characterized by a molecular mass of between 300 g/mol and 1300 g/mol, particularly a fluorescent organic dye moiety; an affinity purification ligand, a pharmaceutical drug or a pharmaceutical drug candidate, an oligopeptide or a polypeptide, a nanoparticle, a solid surface or a matrix polymer, a nucleic acid oligomer, a carbohydrate, and a lipid, a spectroscopic probe, a fluorescent sensor, a natural product, a synthetic ligand binding to a biomolecule with an affinity of at least 100 ?M, L is a linear linker of 10-15 atoms in length, particularly wherein L is 10-11 atoms in length, wherein L comprises, particularly L essentially consists of, alkyl, trans-alkylene, and/or ether moieties and optionally one or several methyl substituents, particularly L is an unbranched linker that does not comprise methyl substituents; T is a moiety selected from the group comprising methylamine, monofluormethylamine, difluormethylamine, trifluormethylamine, methylsulfonamide, monofluormethylsulfonamide, difluormethylsulfonamide, trifluormethylsulfonamide, azide, acetamide, monofluoracetamide, difluoracetamide, and trifluoracetamide, and hydroxyl, particularly T is a moiety selected from the group comprising methylamine, methylsulfonamide, trifluormethylsulfonamide and hydroxyl.

3. The non-covalently-HaloTag-binding compound according to claim 2, wherein T is hydroxyl.

4. The compound according to claim 1, wherein D comprises a linking moiety X which connects the functional moiety Z to L, particularly wherein X is selected from an amide, a secondary amine, a 1,2,3-triazole, an ester, a sulfonamide, an ether, a thioether, a thiourea, an urea, and a carbamate.

5. The compound according to claim 1, wherein L is a linear unbranched alkyl chain, comprising 1, 2, or 3 moieties independently selected from ether and trans-alkylene.

6. The compound according to claim 1, wherein the compound is characterized by the one of the general formulas (II), (III), (IV), (V) or (VI), particularly of the general formula (II), ##STR00016## wherein n is an integer selected from 1, 2, 3, and 4, particularly n is 1 or 2, more particularly n is 1.

7. The compound according to claim 1, wherein Z is a fluorophore, particularly a fluorophore selected from a rhodamine, a silicon rhodamine, a fluorescein, a Janelia Fluor dye, an olefinic silicon rhodamine derivative with an exocyclic double bond, a MaP 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.

8. The compound according to claim 1, wherein Z is of the general formula (VII) or of the general formula (VIII) ##STR00017## wherein V is selected from C(?O)W, CH.sub.3, and CH.sub.2OH, wherein W is OH or NR.sup.W1R.sup.W2 with R.sup.W1 and R.sup.W2 being independently selected from H, unsubstituted or amino- or hydroxy-substituted C.sub.1-C.sub.8 alkyl, CN, SO.sub.2NR.sup.S1R.sup.S2 and SO.sub.2R.sup.S with R.sup.S being unsubstituted or amino- or hydroxy-substituted C.sub.1-C.sub.6 alkyl, and with R.sup.S1 and R.sup.S2 being independently selected from H and unsubstituted or amino- or hydroxy-substituted C.sub.1-C.sub.6 alkyl; R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are independently selected from H and an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted hydrocarbon moiety selected from C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.4 acyl, C.sub.7-C.sub.12 alkylaryl, an unsubstituted phenyl or a phenyl substituted by any one or several of the following substituents: unsubstituted C.sub.1-C.sub.4 alkyl, halogen, C.sub.1-C.sub.4 oxyalkyl, COOH, COOR.sup.C, CONR.sup.C.sub.2, with R.sup.C being selected from H and unsubstituted or amino- or hydroxy-substituted C.sub.1-C.sub.8 alkyl; or R.sup.1, R.sup.2, R.sup.3, and/or R.sup.4 form a ring structure as described below; R.sup.5 is selected from an amine, carbonyl, ester, amide, sulfonamide, and a hydrocarbon moiety selected from C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.7-C.sub.12 alkylaryl, and phenyl, wherein R.sup.5 is unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted; n is an integer selected from 0, 1, and 2; R.sup.6 is selected from an amine, carbonyl, ester, amide, sulfonamide, and a hydrocarbon moiety selected from C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.7-C.sub.12 alkylaryl, and phenyl, wherein R.sup.6 is unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted; X is selected from O, S, Se, TeO, POR.sup.X, POOR.sup.X, SO.sub.2, NR.sup.X, CR.sup.X.sub.2, SiR.sup.X.sub.2, GeR.sup.X.sub.2, and SnR.sup.X.sub.2, with each R.sup.X being independently selected from H and an unsubstituted or substituted (particularly unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted) moiety selected from C.sub.1-C.sub.12 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, C.sub.7-C.sub.12 alkylaryl, phenyl and 5- or 6-membered heteroaryl, or two RX moieties form a four-, five-, six- or seven-membered unsubstituted or amino-, hydroxy- and/or halogen substituted alkyl ring; particularly X is selected from O, CR.sup.X.sub.2, and SiR.sup.X.sub.2; Y is OH or NR.sup.Y1R.sup.Y2, Z is O or N.sup.+R.sup.Y1R.sup.Y2 with R.sup.Y1, and R.sup.Y2 each independently selected from H, an unsubstituted or hydroxy-, amino-, halogen-, and/or carboxy-substituted hydrocarbon moiety selected from C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.1-C.sub.4 acyl, C.sub.7-C.sub.12 alkylaryl, an unsubstituted phenyl or a phenyl substituted by any one or several of the following substituents: unsubstituted C.sub.1-C.sub.4 alkyl, halogen, C.sub.1-C.sub.4 oxyalkyl, COOH, COOR.sup.YC, CONR.sup.YC.sub.2, with R.sup.YC being selected from H and unsubstituted or amino- or hydroxy-substituted C.sub.1-C.sub.8 alkyl; or R.sup.Y1 and R.sup.Y2 together are a C.sub.3-C.sub.6 unsubstituted or hydroxy-, amino-, halogen-, alkoxy- and/or carboxy-substituted alkyl forming a 4-7-membered ring structure with Y; or one of R.sup.Y1 and R.sup.Y2, or both R.sup.Y1 and R.sup.Y2, together with R.sup.1 and/or R.sup.2, and/or R.sup.3 and/or R.sup.4, respectively, form an unsubstituted or hydroxy-, amino-, halogen-, carboxy- and/or aryl-substituted 4-7-membered alkyl or alkylene ring; wherein Z is connected to L via a substituent selected from R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, Y, Z and V.

9. A kit comprising a. a catalytically functional HaloTag polypeptide, wherein optionally said HaloTag polypeptide is attached to a polypeptide of interest; or an expression vector encoding said catalytically functional HaloTag polypeptide; and the kit further comprising a compound according to claim 1; or b. a D106 mutant HaloTag polypeptide, wherein position D106 of the HaloTag7 sequence (position 103 designated as X in SEQ ID No 001) is exchanged for a proteinogenic amino acid different from D, particularly wherein the mutant HaloTag polypeptide comprises a mutation selected from D106A, D106G, and D106T, more particularly wherein the mutant HaloTag polypeptide comprises a D106A mutation, or a D106 mutant HaloTag polypeptide, wherein, in a HaloTag polypeptide sequence homologous to HaloTag7, at a position homologous to D106 of HaloTag7, D is exchanged for a proteinogenic amino acid different from D, particularly wherein said position is changed to A, G or T, more particularly wherein said position is changed to A, wherein optionally said D106 mutant HaloTag polypeptide is attached to a polypeptide of interest; or an expression vector encoding said D106 mutant HaloTag polypeptide; and the kit further comprising a compound characterized by the general formula (I) D-L-T (I), wherein D is or comprises a functional moiety Z selected from an organic dye moiety characterized by a molecular mass of between 300 g/mol and 1300 g/mol, particularly a fluorescent organic dye moiety; an affinity purification ligand, a pharmaceutical drug or a pharmaceutical drug candidate, an oligopeptide or a polypeptide, a nanoparticle, a solid surface or a matrix polymer, a nucleic acid oligomer, a carbohydrate, and a lipid, a spectroscopic probe, a fluorescent sensor, a natural product, a synthetic ligand binding to a biomolecule with an affinity of at least 100 ?M, L is a linear linker of 10-15 atoms in length, particularly wherein L is 10-11 atoms in length, wherein L comprises, particularly L essentially consists of, alkyl, trans-alkylene, and/or ether moieties and optionally one or several methyl substituents, particularly L is an unbranched linker that does not comprise methyl substituents; T is a moiety selected from the group comprising methylamine, monofluormethylamine, difluormethylamine, trifluormethylamine, methylsulfonamide, monofluormethylsulfonamide, difluormethylsulfonamide, trifluormethylsulfonamide, azide, acetamide, monofluoracetamide, difluoracetamide, and trifluoracetamide, and hydroxyl, particularly T is a hydroxyl.

10. A method for binding a HaloTag polypeptide in a sample, said method comprising the steps of: a. providing an aqueous sample comprising a HaloTag polypeptide; b. contacting the sample with a compound comprising a fluorescent organic dye moiety according to claim 1; c. illuminating the sample with light of an excitation wavelength appropriate for exciting said fluorescent organic dye moiety, and recording light emitted from said sample, particularly at an appropriate emission wavelength 2, more particularly a ? close to a maximum of the emission spectrum of 400-800 nm, d. optionally repeating step c.

11. A method for labelling a HaloTag polypeptide in a sample, said method comprising the steps of: a. providing an aqueous sample comprising a HaloTag polypeptide attached to a luciferase; b. contacting the sample with a compound comprising a fluorescent organic dye moiety according to claim 1; c. contacting the sample with a luciferase substrate and recording light emitted from said sample, particularly at an appropriate emission wavelength ?, more particularly a ? close to a maximum of the emission spectrum of 400-800 nm; d. optionally repeating step c.

12. The method according to claim 10, wherein a first and a second HaloTag polypeptide are specifically labelled, wherein the first HaloTag polypeptide is a wildtype polypeptide and wherein the second HaloTag polypeptide is a HaloTag polypeptide mutant, wherein D106 of HaloTag7 or the analogous D of a different HaloTag, is exchanged for a different proteinogenic amino acid, particularly wherein the second HaloTag polypeptide comprises a mutation selected from D106A, D106G, and D106T, or the analogous mutation of D, more particularly wherein the second HaloTag polypeptide comprises a D106A mutation, or the analogous mutation of D, and wherein a first and a second compound comprising a first fluorescent organic dye moiety and a second fluorescent organic dye, respectively, are employed, wherein for the first compound T is a moiety selected the group comprising of methylamine, methylsulfonamide, monofluormethylsulfonamide, difluormethylsulfonamide, trifluormethylsulfonamide, azide, and acetamide, monofluoracetamide, difluoracetamide, and trifluoracetamide, particularly wherein T is selected from methylamine, methylsulfonamide, and trifluormethylsulfonamide, and wherein for the second compound T is OH, and wherein the emission wavelength of the first fluorescent organic dye moiety is different from the emission wavelength of the second fluorescent organic dye moiety.

13. A D106 mutant HaloTag polypeptide, wherein, in a HaloTag7 polypeptide sequence or a HaloTag polypeptide sequence homologous to HaloTag7, at position D106 of HaloTag7 or at a position homologous to D106 of HaloTag7, D is exchanged for a proteinogenic amino acid different from D, particularly wherein said position is changed to G, A, V, I, L, C, S, T, N, or E, more particularly wherein said position is changed to A, G or T, even more particularly wherein said position is changed to A,

14. The D106 mutant HaloTag polypeptide according to claim 13 comprising or essentially consisting of a sequence selected from SEQ ID NO 001 to SEQ ID NO 007, wherein X is selected from any proteinogenic amino acid except D, particularly wherein X is selected from G, A, V, I, L, C, S, T, N, and E, more particularly wherein X is selected from A, G, and T, most particularly wherein X is A.

15. A nucleic acid sequence encoding the D106 mutant polypeptide according to claim 13.

Description

DESCRIPTION OF FIGURES

[0208] FIG. 1 shows binding affinities of HT7 and TMR-nrHTL initial candidates. A. Chemical structures of nrHTL TMR-28 and TMR-23. B. Comparison of experimental and calculated binding preferences among the nrHTL candidates. C. Titration curves of different TMR-nrHTL with HT7. Binding was measured by fluorescence polarization in techn. triplicates. Avg. data and SD were normalized to lower and higher polarization values and fitted with equation (2). K.sub.D given as concentration at half-maximal fluorescence polarization with a Hill coefficient n=1. Mean K.sub.D-values and SD from at least 3 independent experiments (x.sub.n?3) are presented. MSAmethylsulfonamide, MAmethylamine.

[0209] FIG. 2 shows binding affinities and labeling of TMR- and SiR-nrHTL second candidates. A. Chemical structures of nrHTL2 Dye-24. B. Binding affinities of different nrHTLs. Binding was measured by FP in techn. triplicates. Avg. data were fitted with equation (2) yielding KD as the concentration at half-maximal fluorescence polarization with n=1 (Hill coefficient). Mean KD-values and SD from at least 3 independent experiments (xn?3) are given. C. Reversibility of the nrHTL interaction to HT7. Coomassie-stained SDS-PAGE and in-gel fluorescence of HT7 in excess of ligand (5:20 ?M) after 30 min incubation at 37? C.

[0210] FIG. 3 shows binding affinity of various rhodamine-based dyes modified with nrHTL1 (23). A. Chemical structures of 6-carboxy-Atto565 and -CPy B. Representative selection of titration curves comparing different nrHTL1 Dye-23 binding to HT7. Avg. data and SD from techn. triplicates were normalized to lower and higher polarization values and fitted with equation (2). C. Binding affinities and Hill coefficient determination. KD given as concentration at half-maximal fluorescence polarization from the titration curves presented in B. The Hill coefficient n was extracted as the slope of the regression curves. Mean values and SD from min. three independent experiments (xn?3) are given.

[0211] FIG. 4 shows binding kinetics of nrHTL to HT7. A. Cartoon representation of binding parameters involved in nrHTL binding to HT7 important for PAINT microscopy: ?bbright time, ?ddark time. Lower panel adopted from Jungmann et al. Nat. Meth. (2016). B. Stopped-flow binding kinetics of TMR-24 and ?28 to HT7. FP was tracked over time, data from 10 techn. replicates were averaged, normalized to higher polarization values and fitted with equation (3). Magnification of first second shown in the gray window besides. C. Binding kinetics of TMR- and SiR-23/-24 to HT7 obtain similarly. D. Binding properties of MSA nrHTL to HT7. k1 extracted from kinetic measurements, errors are represented by the regression standard error. k?1 were calculated with eq. (4) from experimental data and errors were determined by standard error propagation.

[0212] FIG. 5 shows nrHTL ligands bound HT7 by polar interactions with Asp106. A. Schematic representation of proposed nrHTL binding mode. Reversible binding of rhodamine nrHTL and magnification to the HT7 active centre. Chemical structures of the side chain of the amino acids N41, D106 and W107 together with the terminal segment of nrHTL2 TMR-24 are represented. B. Binding affinities of nrHTL1 3 with HT7/dHTD106A as well as TMR-33 with dHTD106A. C. Bar graphs of log KD-values presented in B for nrHTL1-3. Binding was measured by FP in techn. triplicates. Avg. data were fitted with equation (2) whereat KD given as concentration at half-maximal fluorescence polarization with n=1 (Hill coefficient). Mean KD-values and SD from at least 3 independent experiments (xn?3) are given. MSAmethylsulfonamide. MAmethylamine.

[0213] FIG. 6 shows In-silico evaluation of the nrHTL moieties protonation state. A. Representation of the protonation equilibrium of amine and sulfonamide functions leading to the evaluation of pKa1 and pKa2. Less abundant state under physiological conditions is presented in gray. B. In-silico pKa-calculation of different secondary amine and (sulfon)amide residues together with the resulting estimated charge under physiological conditions. Calculations were performed by using Schr?dinger tool Epic. +: positive, o: neutral, ?: negative.

[0214] FIG. 7 shows pH-Dependent binding of TMR-28 to HT7.A. Chemical structure of TMR-28 below pH 10. B. Affinities and binding kinetics measured for TMR 28 and HT7 at different pH. C. pH-Dependent titration of TMR-28 binding to HT7. Fluorescence polarization was measured in techn. triplicates, the data were normalized to lower and higher polarization values and fitted with equation (2). KD given as concentration at half-maximal fluorescence polarization with regression standard error. D. pH-Dependent binding kinetics of TMR-28 to HT7. Data from 3 techn. replicates were averaged, normalized to higher polarization values and fitted with equation (3) yielding k1. Errors are represented as the regression standard error.

[0215] FIG. 8 shows spirolactone equilibrium & HT7-induced SiR fluorescence turn-on of nrHTLs. A. Water/dioxane titration of SIR-23, -28 and -HTL. Normalized absorbance at 646 nm of SiR derivatives in water-dioxane mixtures (v/v, 10/90-80/20) as a function of the dielectric constant ER. Mean values from 3 techn. replicates were fitted with equation (5), error bars presented by the SD. B. Mean D50-values and SD of the different HTLs from 3 independent experiments (xn=3). C. Fluorescence emission spectra of SiR-23 in absence (dashed line) and presence (straight line) of HT7. Avg. spectra from 3 techn. replicates, normalized to SIR-HTL emission spectra in presence of HT7. D. Fluorescence turn-on of SiR 23, 24, 28 and -HTL was extracted from the data shown in C. based on the emission maxima at 670 nm. The errors were determined by standard error propagation. 23C4-MSA, 24C5-MSA, 28C5-MA.

[0216] FIG. 9 shows colocalization of HT7 stained with nrHTL SiR-28 and SNAP-TMR in fixed cells.Sum of 8 confocal z-stacks (z=2 nm) of chemically arrested U-2 OS cells expressing HaloTag7-SNAP-NLS stained with 500 nM SiR-28 and TMR-BG. Images were taken under no-washed conditions with the following excitation lasers: TMR: 560 nm (6.0%), SIR: 633 nm (3.0%). Scale bar: 50 ?m.

[0217] FIG. 10 shows cell permeability of nrHTL probes. Confocal fluorescence imaging of SiR-(A) and TMR-23 (B) staining of HT7-SNAP-NLS in fixed and live U-2 OS cell's nuclei. Images were taken under no-washed conditions with the following excitation lasers: SiR: 633 nm (3.0%), TMR: 560 nm (fixed: 6.0%, live: 12%). Scale bars: 50 ?m. 23C4-MSA.

[0218] FIG. 11 shows improving the fluorogenic effect of nrHTL1 (23). A. General chemical structure of MaP/JF Dyes: X=O (rhodamine), C(CH.sub.3)2 (carbopyronine) or Si(CH.sub.3)2 (silicon rhodamine). Z=NSO2N(CH.sub.3)2. JF525, JF585, JF615: R=R=F. JF635: R=H, R=F. JF656: R =R=H. B. Selection of emission spectra of nrHTL1 Dye-23 in absence (dashed line) and presence (straight line) of HT7. Avg. data from techn. triplicates were normalized to the respective HTL emission spectra. C. Fluorescence turn-on upon binding to HT7 and HT8 shown as bar graphs. It was calculated based on the emission maxima, respectively. Intensity from three techn. replicates were averaged and the errors were determined by standard error propagation. 23: C4-MSA.

[0219] FIG. 12 shows biochemical characterization of the fluorogenic nrHTLs binding to HaloTags. A. Binding kinetics comparison of nrHTL1 SIR-23 to HT7 and HT8, measured by FP under stopped flow-conditions. B. Summarizing table of the biochemical characterization of SiR-, JF585- and JF635-23 with HT7 and HT8. KD given as concentration at half-maximal fluorescence polarization from titration curves. Avg. values from at least three individual experiments (xn?3) are given and errors are represented as SD. Kinetic traces from A. were fitted with equation (3) delivering k1, avg. value and standard regression error from 8 techn. replicates given. k?1 were calculated from experimental data using equation (4) and errors were determined by standard error propagation. 23: C4-MSA.

[0220] FIG. 13 shows in-vitro characterization of the potential nrHTLs 25 and 26. A. Chemical structure of TMR-25 and -26. B. Titration curves of TMR-25 and -26 with HT7 in comparison to TMR-23. Avg. data of techn. triplicates and SD were normalized to higher polarization and fitted with equation (2). C. Stopped-flow binding kinetics of TMR-23 versus TMR-25 to HT7. FP data from 8 techn. replicates were normalized to higher polarization and fitted with equation (3). D. 3D-model of functional groups used as binding motifs for nrHTL 23-26 indicating progressing planarization. Adopted from Shainyan et al. Chem Rev (2013). E-G. Crystal structure of TMR-25-complexed HT7 in comparison to TMR-23. The protein structure is represented as pale cyan cartoon while ligand and interacting residues are represented as indicated colored sticks, respectively. Straight dashed gray lines illustrate putative hydrogen bonds between protein and ligand (in ?) whereat gray curves show the angle (in ?) between two ligand atoms. All-atom RMSD was calculated manually using the pymol software. H. Summarizing table of the TMR- and SiR-25 and 26 interaction with HT7. Avg. KD-values and SD from at least three individual experiments shown (xn?3). k1 and regression standard error extracted from C. k?1 were calculated from experimental data using eq. (4), errors were determined by standard error propagation. I. Non-covalent binding of TMR-25 to HT7. In-vitro protein labeling at 37? C. followed by SDS-PAGE and in-gel fluorescence imaging in comparison to TMR-HTL.

[0221] FIG. 14 shows that Triflamide nrHTL4 improves various dyes fluorogenicity upon HT binding. A. Comparison of fluorescence emission spectra of SIR-25 and SiR-HTL in presence/absence (straight/dashed lines) of HT7. B. Fluorescence emission increase upon MaP555-, JF585- and SiR-25 binding to HT7 and HT8. Intensity at emission maxima from techn. triplicates were averaged, data are shown as bar graphs and the error bars are represented by standard error propagation. 25: C4-F3MSA.

[0222] FIG. 15 shows Characterization of cpHaloTag combination with nrHTLs. A. Structural representation of the circular permutation sites probed in HaloTag7 (PDB: 6Y7A). HaloTag7 is represented as gray cartoon. The circular permutation sites (i.e. new termini) are highlighted by the ?C atom represented as indicated colored spheres, respectively. The TMR substrate bound to the protein is represented as green sticks. B. Titration curves comparison of TMR-24 with HT7 and cp-candidates. FP data from techn. triplicates were averaged, normalized to lower and higher polarized values and fitted with eq. (2). C. Stopped-flow binding kinetic comparison between HT7 and cpHT7-154/156 to TMR-24. Avg. FP data from 10 techn. replicates were fitted with equation (3). D. Live-cell staining of U-2 OS cells transiently transfected with H2B-cpHaloTag154/156-T2A-eGFP with 500 nM SiR-24 in comparison to SiR-HTL. Images were acquired under no-wash confocal conditions with a 488 nm (8.0%, eGFP) and a 633 nm (1.0%, SiR) laser. Scale bars: 10 ?m. E. Summarizing table of the binding properties of TMR 24 to HT7 and different cpHaloTag variants. Avg. KD-value and SD from three individual FP titration experiments given. k1-value extracted from C. given with regression standard error. k?1 were calculated from experimental data with eq. (4) and errors were calculated by standard error propagation. 24: C5-MSA.

[0223] FIG. 16 shows Heat map representing potential TMR-nrHTL binding affinity to HT7 variants.Binding affinities (KD) were obtained from titration curves done by fluorescence polarization. Avg. data from techn. triplicates were fitted with equation (2). KD yielded from titration curves as concentration at half-maximal fluorescence polarization with a Hill coefficient equals 1. The dataset was colored based on the KD-value between red (highest value) and green (lowest values) with a midpoint of 20% of the population (yellow). 24: C5-MSA, 27: C4-N3, 28: C5-MA, 30: C4-OH and 33: C6.

[0224] FIG. 17 shows Characterization of the dHTL/dHT7D106A system. A. Chemical structure of potential dHTL TMR-29 to -32. B. Heat-map representation of KD-values determined by FP between TMR- or SiR-29 to 31 and HT7 or dHT7D106A. C. Representative titration curves showing binding of TMR-29 to -32 to dHT7D106A. FP data from techn. triplicates were averaged and fitted with eq. (2). D. Stopped-flow kinetic traces for dHT7D106A ligand candidates. Data from 10 techn. replicates were averaged and fitted with eq. (3). E. Fluorescence turn-on of SiR-30 and -31 upon binding to dHT7D106A F. Summary of binding properties for TMR- 30, -31 and SiR-30. Mean KD-value and SD from three individual experiments shown. k1, value extracted from D. and standard regression error given. k1 were calculated from experimental data using eq. (4) and errors were determined by standard error propagation. G. Confocal live-cell, no-wash staining of U-2 OS transiently expressing H2B-dHT7D106A-T2A-eGFP with 500 nM TMR-31 or SiR 30. The following excitation lasers were used: TMR: 550 nm (3.0%), SiR 633 nm (1.0%). Dashed white lines indicate surrounding, untransfected cell's nuclei from bright-field images. Scale bars: 10 ?m. 29: C3-OH, 30: C4OH, 31: C5OH, 32: C4-OMe.

[0225] The following pages give the examples for the present specification:

EXAMPLE 1

Ligand Development and Synthesis

[0226] Non-reactive HaloTag7 Ligands (nrHTLs) were designed based on the chemical structure of TMR modified with the chloroalkane HaloTag7 substrate (TMR-PEG.sub.2-C.sub.6-Cl), usually dubbed as HaloTag Ligand (HTL).

Example 2

Characterization of Non-Reactive HT7 Probes for PAINT Microscopy

[0227] Identification of the Best nrHTL Candidates Through In-Vitro Characterization

[0228] PAINT-microscopy experiments are usually performed with DNA yet more recently protein/protein and protein/ligand-pairs interacting were employed. The latter systems utilize affinities of 0.3 to 1.4 ?M, which turned out to be instrumental for achieving high-resolution images. To prove the concept of nrHTL as PAINT probes the binding affinities (K.sub.D) to HT7 were measured via fluorescence polarization for a selection of identified and synthesized potential nrHTL (FIG. 1).

[0229] The methylsulfonamide(MSA) and methylamine-bearing (MA) compounds (TMR-23 and -28) presented sub-?M binding affinities, while the remaining compounds (TMR-27, -30 and -33) showed K.sub.D in the moderate mM range (FIG. 1B, C). The obtained binding affinities makes it clear, that TMR-23 and -28 (FIG. 1A) can be considered as good nrHTL candidates.

[0230] The two best nrHTLs candidates (TMR-23 and TMR-28) show K.sub.D differences by a factor of 3.3. However, because both the binding moiety (MSA vs. MA) as well as the terminal linker length (C.sub.4 vs. C.sub.5) differ between them, preventing a clear structure-function analysis. Therefore, a comparable TMR-24 (C.sub.5-MSA, FIG. 2A) was synthesized, together with SiR versions of theses three nrHTL candidates. Their binding affinities were characterized by measuring the K.sub.D by FP (FIG. 2B). It was discovered, that the use of a longer C.sub.5-linker (TMR-24) enhanced the binding affinity in comparison to TMR-23 by a factor of ?2. Affinity measurements including SiR compounds confirmed this trend and overall showed higher K.sub.D-values.

[0231] The non-covalent nature of the potential nrHTLs was verified by protein labeling followed by SDS-PAGE and fluorescence scan of the gel (FIG. 2C). Labeling for 30 minutes and 37? C. did not lead to a covalent linkage between TMR- or SiR-23, 24 nor 28 in contrast to the HTL substrates that showed intense fluorescence signals received from the respective protein band. Long-term experiments (>48 h) at room-temperature were carried out and exhibit the same results. From here on, fluorophore-conjugated ligand 23, 24 and 28 are also titled as non-reactive HaloTag Ligands 1 to 3 (nrHTL.sub.1 to nrHTL.sub.3).

[0232] Observing the beneficial influence of SiR in contrast to TMR on the nrHTL affinity questions the effect of the fluorophore on the binding process. Therefore, dissociation constants K.sub.D were measured for nrHTL.sub.1 (23) coupled to CPy and Atto565 fluorophores (FIG. 3A), revealing similar K.sub.D-values for SiR and CPy, likely due to their strong structural and electronical similarities (FIG. 3B, C). On the other hand, a lower binding affinity was measured with Atto565-23 which could be imputed to the large modification of the xanthene ring, possibly inducing steric hindrances with the HT protein, together with a more electron-rich central atom.

[0233] Besides the effect on the binding affinity, also a significant difference in the Hill coefficient between different dyes was recognized: rhodamine-derived dyes (X=O) exhibit the expected Hill coefficient of 1, matching the non-cooperative, one-site binding mode expected for HT7. In contrast, the data obtained for nrHTL.sub.1 modified CPy and SiR dyes is more accurately represented by n=1.3 (FIG. 3C). The underlying mechanism causing this difference needs further assessment, however it does not strongly affect the K.sub.D-value determination.

Kinetics of nrHTL Binding to HT7

[0234] PAINT imaging is allowed by an adequate time presence of one fluorophore on a given cellular structure. Within this time span it is required to collect sufficient photons to precisely locate a single molecule at a reasonable frame-rate. Conversely, the maximal imaging speed is limited by the (un-)binding kinetic parameters. Affinities (K.sub.D) are characterized by kinetic constants of binding k.sub.1 (on-rate) and unbinding k.sub.?1 (off-rate) such that the quotient of both result in K.sub.D. In PAINT-microscopy, k.sub.?1 is particularly important because it defines the time presence of the fluorophore on the target (bright-time ?=1/k.sub.?1). Above that, k.sub.1 largely influences the imaging speed at a given probe concentration c (dark-time ?.sub.d=k.sub.1.Math.c, FIG. 4A).

[0235] Therefore, the binding kinetics of nrHTL TMR-24 and TMR-28 to HT7 were measured by fluorescence polarization under stopped-flow conditions (see FIG. 4B). Surprisingly, a significant difference in the binding kinetics between the two classes of binding moieties (methylsulfonamides vs. methylamines) was observed. TMR-28 (MA) shows a binding kinetics to HT7 completed in the 3 to 5 min range, whereas the methylsulfonamide ligand

[0236] TMR-24 binding reached completion within 0.5 s under similar conditions. This resulted in a significant difference of about 4 orders of magnitude in binding speed compared to TMR-28, whilst having similar K.sub.D-values. Furthermore, the impact of the linker length (C.sub.4 VS. C.sub.5) and the fluorophore (TMR vs. SiR) on the binding kinetics of MSA ligands was investigated (FIG. 4C) with the goal to elucidate the most adequate PAINT probes. In combination with the dissociation constants K.sub.D determined in the previous chapter, the off-rates k.sub.?1 were calculated (FIG. 4D).

[0237] C.sub.4-MSA nrHTLs present an overall lower affinity for HT7 as compared to their corresponding C.sub.5-MSA derivatives. Noticeably, this difference could be mostly attributed to an off-rate difference of overall two-fold whereat both ligands yield almost equivalent on-rates. Those findings are consistent for TMR and SiR probes. For the purpose of PAINT-microscopy, the optimal k.sub.?1-values were reported to be above 1 s.sup.?1 allowing high camera frame-rates without compromising S/N. Besides, the dark-time ?.sub.d is of utmost importance for fast image acquisition at a preferable low probe concentration, wherefore k.sub.1-values in the range of 10.sup.6 to 10.sup.7 M.sup.?1 s.sup.?1 are favourable. Concludingly, nrHTL.sub.1 23 in combination with both dyes tested represents the most promising PAINT probe.

[0238] In contrast, nrHTL.sub.3 28 revealed binding kinetics that are not suitable for this particular purpose. Nevertheless, the striking difference in binding speed observed will be the object of the following investigations aiming to identify the binding mode of the neo-developed nrHTL.

Functional and Structural Investigations on the nrHTL Binding Mode

[0239] The virtual docking screen aimed for polar interactions between potential nrHTL and the catalytic amino acid D106 of HT7 (FIG. 5A). To prove such binding mode, the affinity of the different nrHTL discovered (TMR-23, -24, and -28) to a HaloTag dead-mutant, where Asp106 was replaced by an alanine residue (dHT.sup.D106A), was measured. It was found, that all tested nrHTL offer significantly less affinity to dHT.sup.D106A in comparison to the regular HT7 (FIG. 5B). In detail, the difference obtained are about two orders of magnitude (FIG. 5C).

[0240] The higher K.sub.D-values determined for the nrHTL.sub.1-3 binding to dHT.sup.D106A is consistent to what was reported in a previous chapter: some initially designed nrHTL ligands such as the C.sub.6 with no moiety (TMR-33) lacked the ability to form hydrogen bonds within the HT7 active site. The measured affinity of TMR-33 to HT7 was 40?4 ?M, which is similar to the affinity of nrHTL.sub.1-3 (e.g. TMR-24) with dHT.sup.D106A (FIG. 5B), proving the necessity of the hydrogen bond with D106 for high-affinity binding to HaloTag proteins. However, a moderate mM affinity is offered by rhodamine-dyes modified with any HTL-like linkers in general, indicating an intrinsic rhodamine affinity of HaloTag proteins.

[0241] As demonstrated, the discovered binding can be primarily explained by polar interactions between the D106 residue of HT7 and the secondary amine or sulfonamide moieties of the nrHTL. Presumably, the binding events will depend on the protonation state of the nitrogen atoms: it can serve as a hydrogen donor or expose either a positive or negative charge, resulting in potentially attracting/rejecting the negative charge of the D106 residue. Conclusively, binding events might change with the molecule protonation state depending on the pK.sub.a of the involved chemical functionalities. While the pK.sub.a of amino acids within a protein binding pocket depends on its microenvironment, the pK.sub.a of isolated small molecules in water can be estimated with the Schr?dinger software tool Epic. FIG. 6A visualizes the possible protonation or deprotonation processes of methylamine (MA) and methylsulfonamide (MSA) moieties in a biological relevant pH range. Calculated pK.sub.a-values of different aliphatic secondary amine (pK.sub.a1) and (sulfon)amide (pK.sub.a2) moieties from the nrHTL leads to different overall alkane charge at physiological pH that can be adjusted by introducing electron-withdrawing groups such as methyl fluorides (FIG. 6B).

[0242] These pK.sub.a differences are inherent to the nature of both MA and MSA moieties: while sulfonamides as well as acetamides and carbamates are neutral at physiological pH, secondary amines are mostly protonated and therefore positively charged. This might impact the binding mechanism and the binding kinetics of nrHTL. Indeed, TMR-24 presents fast kinetics and is expected to be mostly neutral at physiological pH, whilest TMR-28 is slow and expected positively charged. Its deprotonation was estimated around pH 10.3?0.7 and it was therefore hypothesized, that a pH increase could promote its deprotonation, render it neutral and concomitantly accelerate its HT7 binding. Hence, the binding kinetics and affinity of TMR-28 were investigated at various pH values (FIG. 7).

[0243] The binding speed of TMR-28 increases significantly between pH 6.0 and 8.0 by a factor of about 7. At the same time, the binding affinity stays comparable over the same pH-range, considering the limits of the measurement inaccuracy. TMR-23 pH-dependent affinities were recorded equally and similar results were obtained: between pH 7.2 and 8.0 constant K.sub.D-values were recorded, only at pH 6.0 a decreased affinity was measured. Thereby, a slight instability of HT7 was detected at mild acidic pH. Despite observing a trend suggesting that an uncharged molecule would bind quicker to HT7, it was not possible to work at a pH allowing TMR-28 to be fully neutral and potentially reach kinetics in the range of TMR-23. Furthermore, the current setup does not allow to distinguish between pH effects on either the protein alone or on the nrHTL. Therefore, it is aimed to cross compare HT7 labeling kinetics with common HT substrates at different pH-values to the nrHTL binding kinetics in order to better interpret the mechanism underlying the fast binding of TMR-23/24 compared to TMR-28. Further experimental assessment of this kinetic interrogation will be provided in chapter 3.3.2 by introducing a triflamide ligand (TMR-25) that is presumably partly negatively charged at a pH around 7 (according to FIG. 6B).

Fluorogenicity of the nrHTL Candidates

[0244] High background signal of unbound fluorophores is a central issue in recent PAINT microscopy approaches. Consequently, prolonged acquisition times and the need of optical sectioning techniques are still restricting PAINT as an universal imaging tool. Lately, fluorogenic DNA-PAINT enabled fast 3D-imaging with a 83-fold fluorescence increase upon hybridization. On the protein site, chromophores exhibiting a fluorescence emission increase upon binding to FAPs, were employed in PAINT-microscopy allowing high-quality and long-term PAINT-microscopy in living cells. The capacity of rhodamine-based dyes (Rho) to switch between a non-fluorescent spirocyclic and a fluorescent zwitterionic form upon binding on HT7 makes of the Rho-nrHTL particularly appealing candidates for PAINT-microscopy applications. In this chapter, the propensity of SIR-23 (MSA) and SiR-28 (MA) to switch from spirolactone to zwitterion upon HT7 binding was therefore evaluated.

[0245] The spirolactone equilibrium can be characterized by measuring the absorbance of the Rho-dyes in different ratio mixture of water/dioxane from which the dielectric constant ?.sub.R is known. The half-absorbance value is known to correspond to a dielectric constant D.sub.50, that allows to compare different rhodamine-based compounds for their open/closed ratio. It was reported, that fluorescent dyes with a D.sub.50-value around 50 are potential candidates for fluorogenic probes whereat SiR highlights a D.sub.50 of 59. Water-dioxane titration with nrHTL SiR-23 and SiR-28 (FIG. 8A) delivered D.sub.50-values (FIG. 8B), indicating the fluorogenic potential of these SiR-probes. In comparison, the common SiR-HTL substrate presents an equilibrium lying more toward the spirolactone form (FIG. 8A) indicating a potentially higher fluorogenicity than offered by the nrHTL equivalents.

[0246] To evaluate the capacity of HT7 to promote the fluorophore to switch to its zwitterionic fluorescent form, the fluorescence emission spectra of SiR-nrHTLs in presence and absence of the HT7 protein were recorded and compared to the common SiR-HTL substrate. From these fluorescence emission spectra (FIG. 8C) the fluorescence turn-on upon HT7 binding observed at the maximal emission wavelength (FIG. 8D) were extracted. As suggested by the D.sub.50-values, SiR-23 shows a higher (background) fluorescence emission in absence of HT7 compared to the SiR-HTL substrate. This indicates that the chemical modification of the linker increases the propensity of SiR to be present in a zwitterionic form. Upon binding to HT7, the fluorescence emission of the SiR-nrHTL increases by a factor of 1.5?0.1 to 1.7?0.1, respectively. In comparison, the covalent labeling of SiR using the respective HTL substrate increases the fluorescence emission by a factor of 7.9?2 under similar experimental conditions.

Cellular Staining Using nrHTLs

[0247] The next chapter demonstrates the applicability of nrHTLs as fluorescent probes for confocal microscopy. The nrHTL cell membrane permeability was assessed by comparing the staining of fixed and live cells expressing HT7 in their nucleus (NLS-tag). Further, the quantification of signal-to-noise ratios (S/N) was used to compare the labeling specificity between the different neo-developed probes under no-wash conditions. Finally, pulse-chase experiments were employed to verify the kinetics of transient (un-)staining of HT7 by nrHTLs, further explored via fluorescence recovery after photobleaching (FRAP) experiments.

[0248] In fixed U-2 OS cells expressing HaloTag7-SNAP-NLS, all nrHTL-modified TMR, CPy and SiR probes delivered a clear fluorescent signal localized at the nuclei and quantitatively comparable to covalent labeling using their respective HTL fluorophore substrates. Only Atto565-23 shows unspecific staining within the whole cell. However, a similar result was obtained from Atto565-HTL substrate, indicating that the dye itself tends to bind unpacifically to some intracellular molecules. Further, a TMR-benzyl guanine (TMR-BG) counterstaining of the fused SNAP protein allowed to demonstrate the proper co-localization of the nrHTL, exemplified by SiR-28 (C.sub.5-MA, FIG. 9).

[0249] The staining comparison between fixed and live U-2 OS cells (HaloTag7-SNAP-NLS) with SiR-23 (FIG. 10A) shows specific and similar nuclear staining under both conditions, with minor intracellular background. In contrast, TMR-23 is not able to stain live cells with intact membranes while chemically arrested cells nuclei are properly stained (FIG. 10B), revealing the difficulty of TMR-23 to pass membranes. This finding also applies to all TMR-nrHTL including -24 and -28, as well as Atto565-23.

EXAMPLE 3

[0250] Fluorescence Increase through Dye Exchange and Engineered HT Variants

[0251] With the scope of finding the most valuable probe for live-cell PAINT imaging, nrHTL.sub.1 23 was coupled to a large panel of both MaP and JF dyes (FIG. 11A). The results described in chapter 3.2.4 suggest, that the modification of the terminal moiety of the common HTL substrate shifts the open/closed equilibrium of the rhodamine dyes toward the zwitterionic form, reducing SiR fluorogenicity upon HT7 binding. Therefore, the hypothesis was made, that fluorophores with a higher propensity to form spirolactons could potentially provide nrHTL with fluorogenicity in the range of SiR-HTL and enhance the cell permeability. Additionally, the recent in-house engineering of HaloTag led to the discovery of a promising

[0252] HaloTag variant (HaloTag8, HT8) characterized for its ability to significantly increase the brightness of rhodamine-based fluorophores upon binding.

[0253] Among all tested Rho-nrHTL.sub.1 (C.sub.4-MSA) combinations, the highest turn-on was obtained for orange to far-red shifted dyes that are known to predominantly adapt to the non-fluorescent spirolactone form such as MaP618 and JF.sub.615 (FIG. 11C). For instance, an intermediate turn-on upon HaloTag7 binding of 29.1?2.3 was measured for MaP618-23. Preferentially, the nrHTL fluorophores were used in combination with HT8 which brought an additional turn-on of roughly two times, as compared to HT7. However, MaP618-HTL is reported to highlight a 1,000? fluorescence turn-on with HT7 in-vitro and side-by-side comparison exhibited that MaP618-nrHTL.sub.1 grasps only 60% of the signal intensity that is reached by covalently labeling of HT7 (FIG. 11B, middle panel).

[0254] On the other hand, the nrHTL modification on a rhodamine scaffold (e.g. MaP555 or JF.sub.525) barely shows any turn-on. The reason here is, that these green to yellow fluorescent nrHTL already exhibit high fluorescence intensity prior to HaloTag binding (FIG. 11B, left panel) corroborating the initial observation made about the overall capacity of the nrHTL moiety to switch the equilibrium of rhodamine-based fluorophore towards their zwitterionic form. In comparison, the turn-on for equivalent HTL substrates was reported of 35? for MaP555. Applying the JF-strategy onto the SiR scaffold switches the open/close equilibrium so much towards the spirolacton form that such a fluorophore-HTL highlights an extreme fluorogenicity upon binding to HT proteins. Despite the fact, that JF.sub.615-23 delivers the highest turn-on (FIG. 11C), it shows less than 4% fluorescence intensity after HT7 binding compared to JF.sub.615-HTL covalently labeled to HaloTag7. Finally, JF.sub.635-23, shows a significant turn-on of 8.4?2.0 (FIG. 11C) and a decent brightness in presence of HT7 (FIG. 11B, right panel), representing the best compromise in between fluorogenicity and overall signal brightness among the SiR-based fluorophores.

[0255] The binding affinities of all fluorogenic variants of the nrHTL.sub.1 23 were measured as previously explained leading to K.sub.D ranging from 120?8 nM (JF.sub.656) to 910?260 nM (MaP555). This stands in a good agreement with the binding affinities reported above for such nrHTLs to HT7, following the trend that rhodamine-derived dyes show weaker binding, than carbopyronine or silicon rhodamine probes.

[0256] Selected as most promising fluorogenic nrHTLs for PAINT microscopy, the binding kinetics of JF.sub.585-23 and JF.sub.635-23 were determined (FIG. 12). In case of JF.sub.585-23 the favourable fluorescence turn-on seems to engender mildly reduced binding kinetics. However, JF.sub.635-23 highlights that a binding speed comparable with SiR-23 can be obtained with fluorogenic nrHTLs (FIG. 12B). That makes it exceptionally beneficial as a potential PAINT probe.

[0257] Overall, the nrHTL fluorophore bound with higher affinity to HT8 than to HT7, however a significantly decreased binding speed to HT8 was detected (FIG. 12A). In combination this yields in a poor off-rate below the aimed 1 s.sup.?1 range.

Introducing Novel Terminal Moieties and Identifying their Binding Behavior

[0258] Based on an initial virtual docking screen, novel non-reactive HaloTag ligands were discovered. During in-cellulo imaging nrHTLs containing methylsulfonamide moieties emerged as powerful probes that offer a high binding affinity, quick binding and precise cell staining, the latter is especially evident in combination with fluorogenic fluorophores. These properties make them appealing for super-resolution imaging applications such as STED and PAINT for which demonstrations were brought during this work. Concretizing the potential such nrHTLs are carrying, the initial design of the terminal nrHTL moiety was revised, aiming for potential better ligands. Moreover, diversifying the toolbox of available ligands is supporting the inventors' mechanistic understanding of the HaloTag-nrHTL interaction since, for instance, the pH-dependence of the binding parameters described in chapter 3.2.3 was not finally conclusive yet.

[0259] Accordingly, two more (sulfon-)amide ligands (25, H.sub.2N-PEG.sub.2-C.sub.4-NHSO.sub.2CF.sub.3 and 26 H.sub.2N-PEG.sub.2-C.sub.4-NAc, FIG. 13A) were synthesized and coupled to TMR and SiR. The novel ligands were fully biochemically characterized as previously explained for 23, 24 and 28 (FIG. 13). Notably, the triflamide 25 shows a high degree of structural similarity to 23 with the difference of the highly electron-withdrawing trifluoromethyl group attached to the sulfonamide. Reducing the electron density also decreases the pK.sub.a2 of the amide as previously displayed (FIG. 6) leaving the ligand partially deprotonated (i.e. negatively charged). On the other hand, 26 represents an uncharged acetamide, offering hydrogen-donor potential though the geometry (planar) and orbital hybridisation (sp.sup.2) of amides is in stark contrast compared to amines and sulfonamides (tetrahedral, sp.sup.3, sees FIG. 13D).

[0260] No binding was observed for TMR-/SiR-26, hinting an incompatibility of amides to either enter or bind to the HT active centre in the same way as demonstrated for methylamines and sulfonamides. In contrast, TMR- and SiR-25 present binding properties (affinity, kinetics) analogous to their corresponding nrHTL 23 (FIG. 13B, C, H). Noticeably, the predicted partial negative charge of 25 does not affect the binding kinetics. The crystal structure of TMR-25 in complex with HT7 highlights similar protein/ligand interactions compared to TMR-23: the binding moiety shows polar contacts with the catalytic site residues N41, D106 and W107 as well as a putative hydrogen bond to T172, placing the xanthene core on the HT7 surface (FIG. 13E, F). However, a 60? rotation of the triflamide group respective to the MSA moiety of TMR-23 was discovered to accommodate the space-demanding trifluoromethane group into the binding site. This results in placing the whole ligand inside HT7 in a slightly shifted conformation respective to TMR-23, illustrated by an all-atom root-mean-square deviation (RMSD) of 0.6 ? (FIG. 13G). Finally, the reversible nature of the binding of -25 to HT7 was demonstrated by SDS-PAGE and in-gel fluorescence imaging (FIG. 131), concluding that fluorophore-25 derivates (further termed as nrHTLA) harbor a great potential as PAINT probes.

[0261] Further, MaP555 and JF.sub.585 were coupled to nrHTL 25 to investigate the impact of the triflamide moiety on their fluorogenicity upon HT7 or HT8 binding (FIG. 14). The fluorogenic effect was improved for both dyes by a factor of 2.5 and 5, respectively. Unexpectedly, the fluorogenicity characterizing the SiR-HTL substrate with HT7 was recovered for SIR-25 in contrast to SiR-23 (FIG. 14A). SiR-25 is relatively non-fluorescent in a buffered solution but exhibit a more then 10-fold turn-on upon HaloTag7 binding. The highest fluorescence turn-on was measured with JF.sub.585-25 in combination with HT8 highlighting a 95.5?11.3 fold increase (FIG. 14B).

[0262] In summary, the fluorescent triflamide probes (25) showed similar binding characteristics to HT7 than methansulfonamide probes (-23, -24), but improved the nrHTL-HaloTag system thanks to a better in-vitro turn-on. A fluorescence increases of up to 92? when used in combination with HT8 was recorded. However, in-cellulo, nuclear staining presents a reduced signal-to-noise ratio because of cytosolic background signal when the probe was used at 500 nM concentration for staining over-night. Nevertheless, staining of H.sub.2B-HT8 with MaP555-, JF.sub.585- and SiR-25 leads to superior brightness, highlighting the suitability of such probe for live-cell confocal microscopy. Herein, the application in concentrations below 500 nM is highly suggested to make perfect use of the fluorogenic potential of those molecules and reduce unwanted background signal.

Circular-Permuted Halo Tag as an Alternative Binding Partner

[0263] Nowadays, resolving distances down to approximately single nanometres is routinely achievable from a technical point of view, as demonstrated recently by DNA-PAINT or MINFLUX (Gwosch, K. C. et al., Nat. Methods 2020, 17, P. 217) fluorescence nanoscopy. Such distances represent the size of proteins, such as the HaloTag, and question the capacity to place a fluorophore close enough to the object of interest via genetic tagging, where the N and C-termini of proteins being rarely close to the fluorescent moiety. Circular permutation (cp) is a protein engineering approach allowing to change the order of the amino acid sequence of a certain protein by linking its initial N and C-termini via a short linker and opening new termini where wished and tolerated. The cp-variant theoretically own similar 3D-structure and functionality. Recently, cpHaloTag7 at position 143 was used in the development of chemigenetic voltage-indicators. Hereby, the cpHalo strategy aimed opening new termini close by the fluorophore binding site. Similar works were undergoing in-house, offering different cp-options to bring the HT7-bound fluorophore in close proximity to a sub-cellular target aiming for a potential resolution increase in SRM.

[0264] In the following, the binding of nrHTL probes to three different cpHaloTag proteins (141/143, 153/156 and 154/156, FIG. 15A) is characterized in order to verify if these cp-variants retain similar binding characteristics to nrHTLs as HaloTag7. However, investigation of the binding affinity with TMR-24 delivered similar results only for cpHT7-154/156 compared to regular HT7 (FIG. 15B). The following experiments therefore focused on this particular protein: measuring binding kinetics (FIG. 15C) in combination with TMR-24 delivered again comparable results. Moreover, staining of H.sub.2B-cpHaloTag154/156-T2A-eGFP expressed in living U-2 OS cells was demonstrated. Therein, nrHTL SiR-24 exhibits analogous results to covalent labeling using SiR-nrHTL (FIG. 15D). Conclusively, despite a slight reduction of the dissociation constant and the on-rate (FIG. 15E), SiR-24 led to specific nuclear staining making cpHaloTag154/156 an attractive target for nrHTL probes in future SRM applications.

EXAMPLE 4

Development of an Orthogonal Halo Tag Protein/Ligand Systems

[0265] Orthogonal fluorescent staining is of great interest for multi-colour imaging in a biological context. The visualization of several targets at the same time does not only increase the amount of information gathered from fluorescence microscopy, but also enables the temporal study of interactions. Herein, SLPs allow to tag synthetic fluorophores to proteins of interest that offer narrow emission spectra allowing multiplexing in high resolution imaging setups. Up to now, an orthogonal variant of SNAP-tag, dubbed as CLIP-tag, was engineered to allow orthogonal fluorescent labeling. Nevertheless, HaloTag remains the SLP mostly used, notably in animal models, and an orthogonal version of HaloTag would therefore be highly appreciated.

[0266] Considering the progress made with the nrHTL approach and the mechanistic knowledge gained in ligand design, the possibility opened up to engineer an orthogonal HaloTag technology using transiently binding ligands selectively targeting two different HaloTag protein variant with sufficient specificity to allow orthogonal co-staining. Thanks to the reversible binding an orthogonal ligand with the required kinetic properties would be highly desirable for dual-colour HT-PAINT microscopy.

Screening for Potential dHT7 Ligands

[0267] As demonstrated earlier in this work, the single amino acid exchange of the D106 residue of HT7 causes a drop in binding affinity for all neo-discovered nrHTL. Therefore, ten dead variants of HaloTag were generated by site-directed mutagenesis, targeting the catalytic D106 residue mutated into G, A, V, I, L, C, S, T N or E residues (dHT7.sup.D106X), with the goal to find a ligand that bounds with moderate affinity to such mutant but not to the native HT7. The proteins were produced, purified and quality checked prior to characterization.

[0268] The binding affinity of initially designed TMR-nrHTL candidates (TMR-24, -27, -28, -30 and -33) led to measured K.sub.D ranging between 0.2 and >100 ?M (FIG. 16). Herein, the variant HT7.sup.D106C revealed to be an equivalent binder to nrHTL TMR-24 and -28 than the native HT7. Interestingly, the moderate binding affinity of TMR-30 (C.sub.4OH) to some dHT7.sup.D106X variants (e.g. D106G, A and T mutants), while being a poor ligand for the native HT7, makes this ligand a good starting point to develop a dHT7.sup.D106X ligand (termed dHTL) that is orthogonal to HT7. The following chapter focusses on the rational improvement of TMR-30 (C.sub.4OH) to specifically bind dHT7.sup.D106A with significantly higher affinity (further referenced as dHT7) over native HT7. In contrast to the nrHTL system, the transient interaction of the fluorescent probes is ensured on the protein level by the HaloTag dead-mutant.

Improvement and Characterization of Ligands for dHT7 Specific Binding

[0269] In order to optimize a ligand for dHT7, it was hypothesized that the linker length of potential dHTL 30 might not be optimal in the protein active site, in which the D106 was replaced by a residue of a smaller size. To proof such hypothesis, dHTL candidates 29 and 31, presenting .sub.3 and Cs linker length in front of the hydroxy binding moiety (FIG. 17A), were synthesized and coupled to TMR and SiR fluorophores. To understand the ligands binding mode, additionally a methoxy-bearing ligand (32, C.sub.4OCH.sub.3) was produced to mask the hydrogen donor potential of nrHTL 30.

[0270] The following work focused on comparing the neo-synthesized potential dHTL affinity to dHT7.sup.D106A and HT7, with the scope of reaching a high orthogonality (FIG. 17B). The binding affinity of TMR-Cn-OH ligands to dHT7.sup.D106A increases with the linker length, almost a log-scale per additional methylene group (FIG. 17C). As previously described, using SiR over TMR increases the binding. However, a similar trend was also observed for HT7, albeit to a lesser extent. The replacement of the terminal hydroxy group by a methoxy moiety decreases the binding affinity to dHT7.sup.D106A, suggesting once more that a polar interaction might drive the binding process (FIG. 17B). Overall, SiR-30 (dHTL.sub.1) and TMR/SIR-31 (dHTL.sub.2) exhibit sub-micromolar affinity for dHT7.sup.D106A but SiR-31 showed also a decent binding affinity to native HT7. That indicates that SiR-31 most likely cannot be considered as an orthogonal ligand even thought it might be a potent dHTL (and a nrHTL simultaneously).

[0271] The binding kinetics and fluorescence turn-on of the novel dHTL were assessed for their dHT7.sup.D106A target (FIG. 17C and D, respectively). Herein, the binding of TMR-31 and SiR-30 occur even faster than the corresponding nrHTL binding kinetics to HT7 reported previously (FIG. 17D and F). The superior on- and off-rates detected potentially open the doors for dual-colour staining that might be ideal for PAINT-imaging. However, no significant turn-on was reported for these novel SiR-dHTL (FIG. 17E) just as for SiR-nrHTL previously. In conclusion, it was possible to design a novel class of non-covalent HTL offering high affinity and binding speed to dHT7.sup.D106A in-vitro and laying the foundation for an orthogonal nrHTL/dHTL system, whereat further refinement of the fluorescence turn-on will be necessary.

[0272] As an initial proof-of-concept, H.sub.2B-HT7.sup.D106A-T2A-meGFP expressing live cells were stained using dHTL.sub.1 SiR-30 and dHTL.sub.2 TMR-30 and imaged under no-wash conditions (FIG. 17G). Both probes reveal sufficient cell permeability and a specific nuclear staining with almost no background signal. Final evaluation of the mutually orthogonal nrHTL/dHTL system is brought in the following chapter by in-cellulo staining of cells expressing both proteins at different intracellular compartments.

Material and Methods

General Synthesis

[0273] Chemical reagents for synthesis were purchased from commercial suppliers (Acros, Fluka, Merk, Roth, Sigma-Aldrich, TCI, TOCRIS) and used without further purification. Reactions performed under air and moisture exclusion were carried out in heat-dried glassware and under inert argon or N.sub.2 atmosphere using Schlenk techniques. Water-free solvents acetonitrile (MeCN), dichloromethane (DCM), dimethylformamide (DMF), ethanol (EtOH), methanol (MeOH) and tetrahydrofuran (THF) were stored over molecular sieves and used directly from a sealed-bottle. Dimethylsulfoxide-de was taken freshly from 0.75 mL glass-ampoules (Roth), stored in a closed vial and used up within two days. 6-Carboxy modified rhodamine dyes were obtained custom synthesized from Atto-Tec and Spirochrome AG or were kindly provided by Bettina Mathes and Dominik Schmidt.

[0274] Evaporation in vacuo was achieved at 40? C. and 10-850 mbar at a rotary evaporator (Buchi). The compounds purified by high-performance liquid chromatography (HPLC) were flash frozen and lyophilized on a lyophilizer (Christ) equipped with a vacuum pump (Vacuubrand).

Preparative Chromatographic Methods

[0275] Flash column purification was performed using a Biotage (Isolera? One) flash system equipped with pre-packed SiO.sub.2 columns (SiliaSep? Flash Cartridges, 40-63 ?m, 60 ?). Depending on the batch size 12 g, 25 g or 40 g columns with 40, 75 or 100 mL min.sup.?1 flow rate were used, respectively. Typical gradients were 10 to 50% ethyl acetate (EtOAc) in n-hexane (hex) or 1 to 10% MeOH in DCM within 10 column volumes (CV).

[0276] Small-scale preparative reversed-phase high-performance liquid chromatography (RP-HPLC) was carried on an UltiMate 3000 system (Thermo Fisher Scientific). Column: C.sub.18 5 ?m, 21.2?250 mm (Supelco). Buffer A: 0.1% TFA in MiliQ? water (ddH.sub.2O), buffer B: MeCN. Typical gradient was from 20% to 90% B within 45 min with 8 mL min.sup.?1 flow. It was equipped with a 2998 PDA detector for automated product collection based on the absorption wavelength of fluorescent labels (at 280, 550, 620 or 650 nm, respectively).

[0277] Large-scale RP-HPLC-MS purification (>3 mg) was carried-out with a LCMS-2020 unit (Shimadzu) coupled with a prominence LC-20AP UFLC (Shimadzu). Column: C.sub.18 5 ?m, 30?250 mm (Shimadzu). Buffer A: 0.1% FA in ddH.sub.2O, buffer B: MeCN. Typical gradient was from 10% to 90% B within 45 min with 20 mL min.sup.?1 flow. Product visualization was reached on an SPD-M20A UV-VIS photodiode array detector and the desired compounds were collected based on the calc. mass-to-charge ratio (m/z) using a DUIS-2020 dual ion source (Shimadzu).

Analytical Chromatographic Methods

[0278] Reaction progress and chromatography fractions were monitored by analytical thin-layer chromatography (TLC) or liquid chromatography coupled to mass spectrometry (LC-MS). TLC was accomplished on commercially available SiO.sub.2-plates (POLYGRAM? SIL G/UV254, 0.2 mm layer pre-coated polyester sheet, 40?80 mm) and visualized by using 254 nm UV-light or TLC staining solutions (see Table 1) and gentle heating.

TABLE-US-00003 TABLE 1 Recipe of staining solutions for thin-layer chromatography TLC stain Recipe KMnO.sub.4 1.5 g KMnO.sub.4, 10 g K.sub.2CO.sub.3, 1.25 mL 10% NaOH in 200 mL ddH.sub.2O Ninhydrin 5 g Ninhydrin in 100 mL EtOH w. 3% (v/v) AcOH Vanillin 15 g Vanillin in 250 mL EtOH w. 1% (v/v) conc. H.sub.2SO.sub.4

[0279] LC-MS was performed on a LCMS2020 (Shimadzu) connected to a Nexera UHPLC system. Column: C.sub.18 1.7 ?m, 50?2.1 mm (ACQUITY UPLC BEH, Waters). Buffer A: 0.1% FA/ddH.sub.2O, buffer B: MeCN. Typical gradient was from 10% to 90% B within 6 min with 0.5 mL min.sup.?1 flow.

[0280] Analytical RP-HPLC was used to evaluate fluorescent compound purity. Samples were prepared in 5 ?M concentration in 5% H.sub.2O in MeCN with 0.1% (v/v) TFA. It was carried out on Waters e2695 system equipped with a 2998 PDA detector. Column: C.sub.18 4 ?m, 3.9?150 mm, 60 ? (Nova-Pak). Buffer A: 0.1% TFA in ddH.sub.2O, buffer B: MeCN. Gradient: Hold 1.5 min at 20% B and increase within 12.5 min to 80% B, 1.23 mL min.sup.?1 flow. The peaks at the fluorophore-specific absorption wavelength were manually integrated. Compounds with a main peak intensity of ? 95% were considered as suitable for the following experiments.

Biochemical Methods and in-vitro Characterization

UV-Vis Spectroscopy

[0281] The concentration of fluorophore-ligands (HTL and nrHTLs) was determined by measuring the UV-Vis absorption of the dye at their maximum absorption wavelength and using Lambert-Beer's law:

Abs=c.Math.d.Math.?(1)

[0282] Abs: absorption at ?.sub.max [AU], c: concentration [mol/l], d: pathway length [cm], extinction coefficient [l/mol.Math.cm]

[0283] Absorbance were measured with a Nanodrop 2000cTM spectrophotometer using a drop of solution (d=0.1 cm) or a polystyrene cuvette (Sarstedt, 10?4?45 mm). Samples were prepared in PBS pH 7.4 (Gibco), 0.1% SDS in activity buffer (composition see Table 3) or 0.1% TFA in EtOH, depending of the fluorophore properties and according to literature precedents. The previously in-house characterized extinction coefficients & were used to calculate the concentration of neo-synthesized fluorophore ligands (for spectral properties of all used fluorophores see Table 2). Fluorescent molecules for following experiments were prepared as stock solutions in dry DMSO (>1 mM) and diluted such that the DMSO concentration did not exceed 1% (v/v).

TABLE-US-00004 TABLE 2 Spectral properties of fluorophores used for UV/Vis spectroscopy Fluorophore ?.sub.abs [nm] ? [M.sup.?1 .Math. cm.sup.?1] in solvent JF.sub.525 526 138,000 0.1% SDS TMR 555 89,000 PBS MaP555 555 142,000 0.1% SDS Atto565 564 120,000 PBS JF.sub.585 586 52,500 0.1% SDS CPy 609 152,000 0.1% SDS MaP618 613 5,500 0.1% SDS JF.sub.615 615 7,000 0.1% TFA in EtOH JF.sub.635 638 17,000 0.1% SDS SiR 645 140,000 0.1% SDS JF.sub.656 649 106,000 0.1% SDS

Plasmid Generation

[0284] Plasmids were obtained by molecular cloning using the Gibson Assembly (GA) (Gibson, D. G. et al, Nat. Methods 2009, 6, P. 343) method or a site-directed mutagenesis kit (NEB). All DNA-primers were designed using the Geneious software (Biomatters) or online-resources from NEB and further custom-synthesized (Merck). For protein production in Escherichia coli a modified pET-51b(+) plasmid (Novagen) was employed in order to fuse a Histidine tag (10?) and a Tobacco Etch Virus (TEV) protease cleavage site in N-terminal of the protein of interest (POI). For mammalian cells protein expression, the template plasmid pcDNA5/FRT/TO (ThermoFisher Scientific) was employed. All template plasmids were kindly provided by Dr. Julien Hiblot or Dr. Michelle Frei.

[0285] Site-directed mutagenesis were performed to create HT7.sup.D106X variants (X=G, V, I, E or T) of pET-51b(+) His10x-TEV-HT7 and to introduce the HT7.sup.D106A mutation into pcDNA5/FRT/TO H.sub.2B-HT7-T2A-eGFP and IgKchL-HA-HT7-myc-PDGFRtmb constructs according to the manufacturer protocol (NEB). In short, DNA amplified by PCR was submitted to parental template DNA Dpnl digestion, phosphorylation and ligation (KDL treatment). ?200 ng of plasmid DNA was transformed into chemically-competent E. coli cells (Q5? Site-Directed Mutagenesis Kit) by heat-shock (40? C., 45 s) prior to recovery (Super Optimal broth with Catabolite repression, SOC) and selection on Luria-Bertani broth medium containing 100 ?g ml.sup.?1 ampicillin agar plates (LB.sup.Amp) at 37? C. over-night.

[0286] Gibson Assembly was employed to replace the eGFP DNA fragments in a pcDNA5/FRT/TO H.sub.2B-HT7.sup.D106A-T2A-eGFP plasmids by a N-terminally HT7-tagged LaminB gene. PCRs were performed as previously explained. After amplification verification, remaining template DNA was eliminated by enzymatic digestion (DpnI FastDigest, Thermo Fisher Scientific) and the desired PCR fragments were purified using minElute PCR purification kit (Qiagen). GA reaction was performed according to the published protocol by incubation of 1 h at 50? C. Transformation were performed by electroporation of electrocompetent E.cloni? (Lucigene) using Gene Pulser? cuvettes (Bio-Rad) and an Eporator? (Eppendorf, 2200 V). Cells were grown on LB.sup.Amp agar plates at 37? C. over-night.

[0287] Single bacterial colonies were picked and grown in 5 mL sterile LB.sup.Amp at 37? C. over-night shaking at 220 rpm in a 24-deep-well plate. The desired plasmids were purified using the QIAPrep spin miniprep kit (Qiagen) according to the manufacturers protocol. All sequences were verified by Sanger sequencing (Eurofins Scientific) assisted by the Geneious software.

Protein Production and Purification

[0288] The pET51b(+) His 10x-TEV-POI plasmids were transformed as previously described in electrocompetent E. coli strain BL21(DE3)-pLysS and grown on LB-agar.sup.Amp at 37? C. over-night. 5-10 colonies were picked to guarantee an equal expression level and grown in 3 mL sterile LB.sup.Amp pre-culture at 37? C. over-night shaking at 220 rpm. On the next day, 1 L LB.sup.Amp was inoculated with 1 mL pre-culture and grown at 37 ? C. and 220 rpm until an optical density at 600 nm (OD.sub.600) of 0.6 was reached. Then, the temperature was reduced to 18? C. and protein production was induced with 0.5 mM isopropyl ?-thiogalacopyranoside (IPTG). After overnight expression, the cells were harvested by centrifugation (4000? g, 15 min, 4? C.), resuspended in 30 mL ice-cold extraction buffer (composition see Table 3) including 1 mM phenylmethylsulphonyl fluoride (PMFS) and 0.25 mg/mL lysozyme. Cell lysis was carried out by sonication (SONOPLUS, 7 min, 50% on/off cycles, 70% amplitude) at 4? C. The lysate was cleared from the cell debris by centrifugation (20 min, 50,000? g, 4? C.) and cautiously collected in fresh 50 mL Falcon tubes. The desired protein was purified by immobilized metal affinity chromatography (IMAC) on an AEKTAPure M fast protein liquid chromatography (FPLC) system (GE-healthcare). Therefore, a FF-HisTrap column (GE-healthcare) was equilibrated with His wash buffer (composition see Table 3). After binding the crude lysate to the column and extensive wash (6 CV), the desired protein was eluted using His elution buffer (composition see Table 3) and the desired fraction was collected based on UV/Vis absorbance in a ?30 mL fraction. Further the buffer was change on a HiTrap? 26/10 Desalting Column (GE-Healthcare) to activity buffer on the same instrument. The protein solution was concentrated with Amicon? Ultra 15 mL Centrifugal Filters MWKO: 10,000 kDa (5-20 min, 4,500 rpm, 4? C.) to ?500 ?M, flash frozen in liquid nitrogen as 100 ?L aliquot and stored at ?80? C.

Protein-Labeling, Electrophoresis and Visualization on Protein Gels

[0289] The purified HT7 protein (5 ?M) was labeled using 4? excess (20 ?M) of the fluorescent non-reactive HaloTag Ligands (nrHTL) in comparison to the corresponding common HTL. Labeling reaction was carried out in activity buffer for 30 min at 37? C.

[0290] Prior to electrophoresis, the protein samples were prepared in a Laemmli sample buffer (Table 3), including 10 mM dithiothreitol (DTT), and fully denatured at 95? C. for 10 min. 5 ?g fluorescently-labeled proteins were loaded onto precast polyacrylamide gels (mini-PROTEAN? TGX?, 4-20%, 10-well, 30 ?L/well, Bio-Rad) in a PROTEAN? cell (Bio-Rad) chamber. For protein purity evaluation, ?50 ?g of isolated protein was applied onto stain-free gels (mini-PROTEAN? TGX? Stain-Free?, 4- 20%, 10-well, 30 ?L/well, Bio-Rad). PrecisionPlus Protein? All Blue (Bio-Rad) pre-stained marker was used as a reference. Electrophoresis was run for ?35 min at 220 V in 1? TGS running buffer (fisher bioreagents).

[0291] Afterwards, in-gel fluorescence was performed at a ChemiDoc XRS+imager (Bio-Rad). Stain-free gels were exposed for 1 min to UV light to reveal unlabeled protein (purity verification) and imaged with ?.sub.ext/em=302 nm/590?110 nm. Results of protein in-vitro labeling with fluorophore-ligands were revealed using the following channel parameters; TMR: ?.sub.ext/em=520-545/605?50 nm, SiR: ?.sub.ext/em=625650/695?55 nm. Finally, these gels were stained in Coomassie Brilliant Blue R-250 staining solution (Bio-Rad) over-night, washed with ddH.sub.2O and imaged by trans-white illumination (?.sub.em=590?110 nm) at the same imager. The stain-free property of the gels can not be used in parallel to fluorescent labeling of the proteins.

Fluorescence Polarization Assay

[0292] The fluorescent nrHTL (10 nM) were titrated using purified HaloTag proteins [0-200 ?M] in activity buffer containing 1% (w/v) Bovine Serum Albumin (BSA, Fraktion V, Roth), in a black flat bottom 380-well plate (Greiner, 20 ?L) and at 37? C. The fluorescence polarization (FP) was measured on a microplate reader (Spark20-Tecan) by exciting the TMR at 535?12.5 nm and recording the emission at 595?17.5 nm. Gain was determined as optimum at 67%. The SiR fluorophore was excited at 610?10 nm and recording the emission at 690?10 nm (optimal gain: 138%).

[0293] The data from three technical replicates per protein concentration were averaged and normalized to the minimum (A) and maximum (B) fluorescence polarization values, errors are given as standard deviations (SD). The data were fitted with the Hill-Langmuir equation (equation 2) where the dissociation constant (K.sub.D) corresponds to the protein concentration [HT] at which half-polarization was measured. The Hill coefficient n was determined from the slope, whereat HaloTag proteins presenting one binding site, the Hill coefficient should be ideally equal to 1 for accurate K.sub.D determination.

[00001]$\begin{array}{cc}\mathrm{FP}=A+\frac{{\left(B-A\right)}^n}{1+{\left(\frac{K_D}{\left[\mathrm{HT}\right]}\right)}^n}& \left(2\right)\end{array}$

[0294] FP: fluorescence polarization [mFP], A: min. FP, B: max. FP, [HT]: HaloTag protein concentration [mol/L], n: Hill coefficient, K.sub.D: dissociation constant [mol/L].

[0295] Statistical analysis of presented K.sub.D-values was aimed by performing the FP assay at least in three individual replicates (x.sub.n?3). Mean K.sub.D-values and the SD are presented.

Kinetics Measurements and pH-Dependency

[0296] Binding kinetics were measured by tracking the fluorescence polarization in a black flat bottom 380-well plate (20 ?L) measured on a microplate reader (Spark20-Tecan) as previously explained for titration. The fluorescent nrHTL (50 nM) were spiked to HT7 protein (0.5 ?M) and the fluorescence polarization was measured in techn. triplicates every 10 s by exciting the TMR at 535?12.5 nm and recording the emission at 595?17.5 nm. Gain was optimum at 56%.

[0297] To assess the pH-dependency of both FP experiments presented, 100 ?L HT7 protein in activity buffer was dialysed in 200 mL SPG-Buffer (Jena Bioscience) at different pH-values ranging from 6.0 to 8.0 in dialysis units (Slide-A-Lyzer? MINI Dialysis Unit, 7,000 MWCO) over-night at 4? C. It was supplemented with 1% (w/v) BSA, the pH-values were evaluated using a SevenCompact pH-meter S220 device and eventually adjusted using 2 N HCl or NaOH.

[0298] Stopped-flow kinetics were measured at a BioLogic SFM-400 instrument (BioLogic Science Instruments). 4 ?M HaloTag protein was mixed in a 1:1 (v/v) ratio with 1 ?M fluorophore-nrHTL in activity buffer. The fluorescence intensity and the fluorescence polarization were measured over 300 s at 0.1 ms timepoints. Background data for fluorophore only were subtracted and at least 8 techn. replicates were averaged.

[0299] All kinetic measurements were performed at 37? C. For both experimental set-ups the time-dependent FP data were normalized to the highest polarization values (B) and fitted with the following non-linear equation yielding k.sub.1 as the binding kinetic constant (on-rate):

[00002]$\begin{array}{cc}\mathrm{FP}=B+\frac{\left(0-\frac{B}{\left[D\right]}\right).Math.\left(\left[D\right].Math.\left(\left[D\right]-\left[\mathrm{HT}\right]\right)\right).Math.e^{\left(\left[D\right]-\left[\mathrm{HT}\right]\right).Math.k_1.Math.t}}{D.Math.e^{\left(\left(\left[D\right]-\left[\mathrm{HT}\right]\right).Math.k_1.Math.t\right)-\left[\mathrm{HT}\right]}}& \left(3\right)\end{array}$

[0300] FP: fluorescence polarization [mFP], B: max. FP, [D]: dye concentration, [HT]: HaloTag protein concentration, t: time [s], k1: on-rate [M.sup.?1 s.sup.?1].

[0301] In combination with experimentally evaluated K.sub.D-values, the unbinding kinetic constants were calculated with the following equation:

k.sub.?1=K.sub.D.Math.k.sub.1 (4)

[0302] K.sub.D: dissociation constant [mol/L], k.sub.1: on-rate [M.sup.?1 s.sup.?1], k.sub.?1: off-rate [s.sup.?1].

Fluorescence Turn-on Assay

[0303] The fluorophore-nrHTL probes (50 nM) were incubated in presence and absence of HT proteins (100 ?M) for 30 min at 37? C. in activity buffer containing 1% (w/v) BSA in a black flat bottom 380 well plate (Greiner, 20 ?L). Fluorescence emission scans were recorded, for example by exciting the SiR fluorophore at 605 +10 nm and measuring the emission intensity between 652 and 800 nm, on a microplate reader (Spark20-Tecan) with an automated gain of 50%. The data from three techn. replicates were averaged and the fluorescence turn-on was calculated by the quotient of fluorescence intensity in presence and absence of HaloTag protein at the emission maxima (e.g. in the case of SiR at 670 nm). The errors were calculated through standard error propagation from replicate's standard deviations. Reference experiments with corresponding fluorophore-HTLs were made in parallel and used to normalize the fluorescence emission spectra to 1.

Water-Dioxane Titration

[0304] Solutions of 5 ?M fluorophore-nrHTL were prepared in 10/90 to 80/20 (v/v) water-dioxane mixtures (dry, Acros) in transparent flat-bottom polypropylene 96-well plates (Greiner, Chimney Well). Absorbance spectra between 400 and 750 nm were recorded in a microplate reader (Spark20-Tecan) with 2 nm step size. The maximal absorbance at 646 nm was blank corrected, normalized to the max. and min. absorbance and plotted against the corresponding dielectric constants ?.sub.R. Data were fitted with the following sigmoidal function delivering D.sub.50-values corresponding to the dielectric constant at half-maximal absorbance.

[00003]$\begin{array}{cc}\mathrm{Abs}=\frac{A+\left(B-A\right)}{\left(1+{10}^{\left(D50-{?}_R\right).Math.n}\right)}& \left(5\right)\end{array}$

[0305] Abs: absorbance [AU], A: min. abs., B: max. abs., ?.sub.R: dielectric constant, n: slope, D.sub.50: ?.sub.R at half-maximal absorbance.

TABLE-US-00005 TABLE 3 Composition of buffers used for in-vitro characterization. Buffer Composition TAE-Buffer 5 mM Na.sub.2EDTA, 40 mM Tris, 20 mM AcOH Activity buffer 50 mM HEPES, 50 mM NaCl, pH 7.3 Extraction buffer 50 mM KH.sub.2PO.sub.4, 300 mM NaCl, 5 mM imidazole, pH 8.0 His wash buffer 50 mM KH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole, pH 7.5 His elution buffer 50 mM KH.sub.2PO.sub.4, 300 mM NaCl, 500 mM imidazole, pH 7.5 Laemmli Buffer 31.5 mM Tris-HCl, pH 6.8, 10% glycerol, 1% SDS, 0.005% Bromophenol Blue Buffers were prepared in Mili-Q? water.

Cell Biology and In-Cellulo Experiments

Cell Culture and Transient Transfection

[0306] U-2 OS cell lines were maintained in T-75 flasks (Greiner) in high-glucose Dulbecco's Modified Eagle Medium (DMEM GlutaMAX?, phenol-red, Gibco). Growth medium was supplemented with 10% (v/v) fetal calf serum (FCS) and cells were stored in a humidified tissue culture incubator at 37? C. and 5% CO.sub.2. They were passaged using phosphate buffered saline (PBS, pH 7.4, Gibco) and TrypLE? Select Enzyme (1?, phenol-red free, Gibco) every 2-3 days and regularly tested for mycoplasma contamination. U-2 OS Flp-In? T-REX? cells were used from commercial sources (ThermoFisherScientific) while the same cell lines stably expressing HaloTag7-SNAP-NLS, H.sub.2B-HaloTag8 and Tomm20-HaloTag8 were generated and kindly provided by Dr. Birgit Koch or Dr. Michelle Frei. For staining and subsequent confocal microscopy imaging, 1.0 to 1.5?10.sup.5 cells per well were seeded into tissue culture treated CellCarrier-96 black plates with an optically clear glass-bottom (PerkinElmer). Cell titter were determined by counting detached cells in a fluidlab R-300 handheld cell counter.

[0307] Transient transfection was performed using Lipofectamine 3000? reagent (ThermoFisherScientific) according to the manufacturer's protocol (amount per 96-well given): To generate DNA-lipoplexes, plasmid DNA (0.1 ?g) and 0.2 ?L P300 reagent were mixed in 10 ?L Reduced Serum Media Opti-MEM? I (Gibco). In parallel, 0.2 ?L Lipofectamine 3000? reagent was diluted in 10 ?L Opti-MEM?. After a short incubation time the two solutions were mixed in a 1:1 ratio and incubated for at least 15 min at rt. Finally, 20 ?L transfection mixture was added on top of the cells, which were seeded 16 to 18 h prior to transfection as described earlier, and mixed gently. The medium was changed 14 h after transfection back to regular growth medium, supplemented with 0.1 mg/mL doxycycline to induce protein expression.

Staining, Fixation and Permeabilization

[0308] Live-cell staining was performed 22 h after seeding or transfection. Fluorophore-nrHTL were applied in imaging medium (DMEM GlutaMAX?, 10% FCS, phenol-red free, Gibco) at 10 nM to 1 ?M concentrations for 16 h at 37? C., keeping the DMSO concentration below 1%.

[0309] After PBS wash, cells were fixed eventually using 4% (v/v) cell-culture grade paraformaldehyde solution (PFA, Electron Microscopy Sciences) in PBS for 20 min at room-temperature. The cells were subsequently washed with PBS and permeablize with 0.5% (v/v) Triton X-100 (Roth) in PBS (10 min, rt). Removal of the detergent was accomplished by washing three times with 3% BSA in PBS w/v). Cells were stored in PBS for maximum 1 week at 4? C. and stained with 500 nM fluorescent probes in activity buffer at room-temperature for 2 h prior to imaging.

Confocal Fluorescence Microscopy

[0310] Fixed and live cell images were acquired by fluorescence confocal microscopy imaging on a Leica DMi8 microscope (Leica Microsystems) equipped with a Leica TCS SP8 X scanhead and a SuperK white light laser. A HC PL APO 20?/0.75 dry objective or a HC PL APO 40.0?/1.10 ater objective were used in combination with hybrid detectors (HyD). In case of potential spectral overlap (e.g. JF.sub.585 and SiR), sequential images were taken to avoid cross-talks. Laser intensities between 0.8% and 12% were used while all other settings were kept constant. Living cells were maintained in a CO.sub.2 (5%) and temperature-controlled (37? C.) incubator (Life Imaging Services).

[0311] Pulse-chase experiments were carried out by addition or replacement of the staining solution directly under the microscope and recording of z-stacks over time. Photobleaching was performed using the FRAP module of the Leica DMi8 microscope, a TMP detector and the following bleaching sequence: single nuclei were bleached three times in a row with 100% laser power for 3.4 s in a circular ROI. Afterwards, single-stack images were taken every 10 s for 1 min. This sequence was repeated 10 times consecutively and the fluorescence intensity was quantified using the LAX software.

[0312] In-cellulo signal-to-noise ratios (S/N) were extracted from the acquired images by analyzing them with the ImageJ Fiji software as follows: the mean signal intensity of a circular ROI within a labeled nucleus was divided by the mean signal intensity of a circular ROI adjacent to the nucleus (cytosolic background signal).

Super-Resolution Fluorescence Microscopy

[0313] For STED microscopy U-2 OS CRISPR-Vimentin-HaloTag7 cells seeded on glass coverslips were kindly provided by in-house collaborators from the Optical Microscopy facility (MPImF, Heidelberg). They were incubated in imaging medium that contained 0.5 ?M SiR-nrHTL or SIR-HTL for 30 min at 37? C. Living cells were imaged using an Abberior STED 775/595/RESOLFT QUAD scanning microscope (Abberior Instruments GmbH) equipped with a UPlanSApo 100?/1.4 oil immersion objective lens. SiR was excited at 640 nm (2.0%) and detected at 655 to 700 nm. STED-images were recorded by using a STED-laser at 755 nm (15.0%), a pixel dwell time of 15 ?s and a pixel size of 30 nm. Quantitative analysis of single vimentin fibrils was performed with ImageJ by measuring plot profile scans which are represented as Gaussian distribution.

[0314] HT-PAINT microscopy was carried out by Sebastian Strauss (AG Jungmann, MPI for Biochemistry, Martinsried) as explained in the following: 3?10.sup.5 U-2 OS CRISPR-NUP96-Halo cells per well were seeded into 8-well chambered coverslip (ibidi) and grown overnight. The cells were washed with PBS once and fixed with 2.4% PFA in PBS for 30 min at room-temperature. After fixation, the cells were rinsed three times with PBS, permeabilized with 0.25% Triton-X-100 (5 min) and blocked with BSA-PBS (3% w/v, 30 min). Finally, nrHTL were added at a concentration of 2 nM in PBS pH 7.2.

[0315] Image acquisition was carried out on an inverted microscope (Nikon Instruments, Eclipse Ti2) with the Perfect Focus System, applying an objective-type TIRF configuration equipped with an oil-immersion objective (Nikon Instruments, Apo SR TIRF 100?, NA 1.49, Oil) and 561 nm as well as 642 nm laser-lines (MPB Communications Inc., 2 W, DPSS-system) were used for excitation. The laser beams were passed through cleanup filters (Chroma Technology, ZET561/10, ZET 640/10) and coupled into the microscope objective using a beam splitter (Chroma Technology, ZT561rdc, ZT640rdc). Fluorescence light was spectrally filtered with an emission filter (Chroma Technology, ET600/50m and ET700/75m) and imaged on a sCMOS camera (Andor, Zyla 4.2 Plus) without further magnification, resulting in an effective pixel size of 130 nm (after 2?2 binning). Images were acquired choosing a region of interest with the size of 512?512 pixels. The detailed imaging parameters are described in Table 4.

[0316] The raw data was reconstructed and post-processed using the Picasso software package (Schnitzbauer, J. et al., Nat Protoc 12, 1198-1228, 2017). Drift correction was performed using gold nanoparticles as fiducial markers.

TABLE-US-00006 TABLE 4 Microscope settings used for HT-PAINT imaging ?.sub.ext Laser power # Integration time Dye [nm] [mW] Frames [ms] TMR 561 10 30,000 100 SiR 642 30 15,000 200

6.1.1 General Procedures

[0317] General Procedure A: Alkyl Iodination (mesylation and Finkelstein Reaction)

##STR00005##

[0318] A heat-dried round-bottom flaks was charged with dry DCM (5 mL/mmol alkyl alcohol) and 1.5 eq. triethylamine (TEA). 1 eq. alkyl alcohol was added and the mixture was cooled to 0? C. Afterwards, 1.25 eq. methanesulfonylchloride was added dropwise under continued stirring and the solution was left stirring for 1 h at 0? C. and 3 h at room-temperature. The reaction was monitored by TLC and if necessary carried out for 12 to 16 h over-night.

[0319] After full conversion of the starting material 10 mL/mmol 10% NH.sub.4Cl solution was added and the aq. layer was extracted three times with 4 Veq. DCM. The combined organic phases were washed with 2 Veq. brine and dried over MgSO.sub.4. Filtration and evaporation of the solvent under reduced pressure gave the crude compound. Final purification of the desired alkyl methanesulfonate was performed by flash column chromatography (SiO.sub.2. Typical gradient: 0 to 2% MeOH in DCM for 6 CV).

[0320] 1 eq. Methanesulfonate compound and 10 eq. NaI were mixed in 1.4 mL/mmol acetone in a round-bottom flask. The emulsion was stirred at room-temperature over-night. After 16 to 20 h large quantities of a yellow-brown precipitate were observed.

[0321] The residual solvent was removed under reduced pressure and the remaining solid was dissolved in 10 mL/mmol DCM and H.sub.2O each. The aq. layer was extracted twice with 2 Veq. DCM. Afterwards the org. layers where combined and a pale-purple discolouration was removed upon washing with 100 mL sat. Na.sub.2S.sub.2O.sub.3. Subsequent brine wash, drying over MgSO.sub.4, filtration and concentration in vacuo gave the crude compound. Final purification by flash column chromatography (SiO.sub.2. Typical gradient: 5 to 30% EtOAc in n-hexane in 6 CV) afforded the desired compound.

General Procedure B: Williamson Ether Synthesis

[0322] ##STR00006##

[0323] Ether synthesis reaction was adopted according to Takashima et al. 2019. A reaction tube was charged with 2 mL/mmol of a 2/1 ratio (v/v) of dry THF and DMF under Schlenk conditions. 1 eq. tert-Butyl(2-(2-hydroxyethoxy)ethyl)carbamate (BocNH-PEG.sub.2-OH) was added and dissolved under vigorous stirring. The mixture was cooled to 0? C. and 1.1 eq. NaH was added portion-wise. The evolving gas was released carefully and the mixture was left stirring at 0? C. for 30 min under an inert gas atmosphere. Afterwards, 1.4 eq. alkyl halide was added directly into the suspension. The mixture was warmed to room-temperature and left stirring for 3 h to 18 h while the reaction progress was controlled by TLC.

[0324] Upon full conversion of the alkyl halide species 10 mL/mmol 10% aq. NH.sub.4Cl and EtOAc each were added to stop the reaction. The aq. layer was extracted three times with 10 mL/mmol EtOAc. The org. layers were combined and washed with brine once and three times with 10% LiCl to remove residual DMF. Afterwards, it was dried over MgSO.sub.4, filtered and the solvent was removed under reduced pressure. Purification was performed by flash column chromatography (SiO.sub.2. Typical gradient: 20 to 50% EtOAc in n-hexane in 8 CV) to afford the desired BocNH-PEG.sub.2-C.sub.n-R compounds.

General Procedure C: Boc Protecting Group Cleavage

[0325] ##STR00007##

[0326] Removal of Boc protecting groups was reached under acidic conditions. Therefore, the protected compounds were dissolved in 3 mL/mmol of a 1/1 (v/v) mixture of TFA in dry DCM.

[0327] It was stirred at room-temperature for at least 3 h and the reaction progress was followed by TLC.

[0328] Afterwards the solvent was removed under reduced pressure, co-evaporated three times with 1 Veq. DCM at 700 mbar and 40? C. and finally dried under a stream of N.sub.2 for at least 30 min to collect H.sub.2N-PEG.sub.2-Cn-R compounds, which were used without further purification.

6.1.2 Boc-NH-PEG.sub.2-C.sub.4/.sub.5-Methylsuflonamide/Triflamide/Acetamide (9-12)

##STR00008##

a. 4-Azido-1-butanol (1)/5-azido-1-pentanol (2)

[0329] The diazo-transfer reagent imidazole-1-sulfonyl azide hydrochloride was synthesized following the protocol of Goddard-Borger et al. 2007. NaN.sub.3 (6.5 g, 100 mmol, 1 eq.) was filled into a heat-dried Schlenk-flask and suspended in 100 mL dry MeCN under a flow of argon gas. It was cooled to 0? C. and sulfuryl chloride (8.1 mL, 100 mmol, 1 eq.) was added drop-wise. The mixture was left stirring over-night at room-temperature. Afterwards imidazole (12.9 g, 190 mmol, 1.9 eq.) was added portion-wise at 0? C. The resulting slurry was left stirring at rt for 3 h and diluted with 200 mL EtOAc. The organic layer was washed twice with 200 mL H.sub.2O and sat. NaHCO.sub.3 solution each, dried over MgSO.sub.4 and filtrated. Meanwhile, acetyl chloride (10.7 mL, 150 mmol, 1.5 eq.) was added drop-wise to 37.5 mL ice-cold dry ethanol (EtOH) to obtain 4 M HCl in EtOH. Upon dropwise addition onto the filtrate solution stirring at 0? C. the formation of colourless needles was observed. Filtration and washing with 3?100 mL ice-cold EtOAc delivered 15.4 g (88.9 mmol, 74%) imidazole-1-sulfonyl azide.Math.HCl as a white powder. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?7.47 (s, 1H), 5.97 (s, 1H), 5.64 (s, 1H).

[0330] 4-Amino-1-butanol (1.2 mL, 12.9 mmol, 1 eq.) or 5-amino-1-pentanol (3.1 g, 30 mmol, 1 eq.), 2.25 eq. K.sub.2CO.sub.3 and 1 mol % Cu(II)SO.sub.4.5 H.sub.2O were suspended in a heat-dried Schlenk-flask in 5 mL/mmol dry MeOH under a stream of argon gas. The solution was cooled to 0? C. and 1.2 eq. imidazole-1-sulfonyl azide.Math.HCl was added portion-wise. The mixture was left stirring for 12 h at rt, concentrated under reduced pressure and acidified with some drops of conc. HCl. The remaining solid was taken up in 200 mL EtOAc, washed with 100 mL H.sub.2O and brine each, dried over MgSO.sub.4, filtrated and evaporated to afford the crude products as a pale-yellow oil. 1.5 mg 1 (13 mmol) was collected after flash column chromatography on a 25 g SiO.sub.2 column (20% EtOAc in n-hexane for 14 CV). 3.3 mg 2 (25 mmol) was afforded by flash column chromatography on a 40 g SiO.sub.2 column (5 to 70% EtOAc in n-hexane for 8 CV).

[0331] 4-Azido-1-butanol (1)Yield: 97%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.57 (t, 2H), 3.25 (t, 2H), 1.58 (m, 4H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?61.84, 51.16, 29.48, 25.20.

[0332] 5-Azido-1-pentanol (2)Yield: 99%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.63 (t, 2H), 3.26 (t, 2H), 1.59 (m, 4H), 1.44 (m, 2H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?62.24, 51.38, 32.10, 28.62, 22.98. HRMS (m/z): [M +H]+calcd. for C.sub.5H.sub.12N.sub.3O.sup.+, 130.0975; found, 130.0974.

b. 4-Azido-1-iodobutane (3)/5-azido-1-iodopentane (4)

[0333] 4-Azido-1-butanol 1 (1.5 g, 13 mmol) or 5-azido-1-pentanol 2 (3 g, 23.2 mmol) were treated as described in general procedure A. Almost full consumption of the starting materials was observed after stirring for 1 h at 0? C. and 3 h at room-temperature, respectively. Aq. work-up and purification over a 25 g silica column (0 to 3% MeOH in DCM for 9 CV) delivered 1.8 g 4-azidobutyl-1-methansulfonate (9.4 mmol, 72%) and 4.5 g 5-azidopentyl-1-methansulfonate (22 mmol, 95%) as yellow oils, respectively.

[0334] 4-Azidobutyl-1-methansulfonate.sup.1H NMR (400 MHZ, CDCl.sub.3): ?4.20 (t, 2H), 3.29 (t, 2H), 2.95 (s, 3H), 1.79 (m, 2H), 1.67 (m, 2H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?69.28, 50.37, 37.37, 26.33, 25.06. HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.5H.sub.11N.sub.3O.sub.3SNa.sup.+, 216.0413; found, 216.0413.

[0335] 5-Azidopentyl-1-methansulfonate.sup.1H NMR (400 MHZ, CDCl.sub.3): ?4.22 (t, 2H), 3.29 (t, 2H), 3.00 (s, 3H), 1.78 (dq, 2H), 1.63 (ddt, 4H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?69.70, 51.20, 37.44, 28.77, 28.37, 22.84. HRMS (m/z): [M+H].sup.+ calcd. for C.sub.6H.sub.16N.sub.3O.sub.3S.sup.+, 208.0750; found, 208.0753.

[0336] Subsequent Finkelstein reaction was carried out with 1 eq. 4-azidobutyl-1-methanesulfonate (1.8 g, 9.4 mmol) or 5-azidopentyl-1-methanesulfonate (3.3 g, 16 mmol) according to general procedure A. Aq. work-up and purification on a 25 g SiO.sub.2 column (5 to 30% EtOAc in n-hexane for 6 CV) gave 1.84 g 3 (8.2 mmol, 88%) or 3.45 g 4 (14.5 mmol, 91%) as colourless liquids.

[0337] 4-Azido-1-iodobutane (3)Yield: 63% over two steps. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.31 (q, 2H), 3.19 (q, 2H), 1.90 (m, 2H), 1.70 (tt, 2H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?50.43, 29.81, 30.51, 5.83.

[0338] 5-Azido-1-iodopentane (4)Yield: 89% over two steps. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.29 (t, 2H), 3.19 (t, 2H), 1.85 (p, 2H), 1.54 (m, 4H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?51.31, 33.02, 27.97, 27.79, 6.51.

c. Boc-NH-PEG.sub.2-C.sub.4-N.sub.3 (5)/Boc-NH-PEG.sub.2-C.sub.5-N.sub.3 (6)

[0339] Following general procedure B, 1 eq. Boc-NH-PEG.sub.2-OH was taken up in 2 mL/mmol THF/DMF 2/1 and left reacting with 1.1 eq. NaH at 0? C. for 30 min. Immediately after the addition of 1.4 eq. 4-azido-1-iodobutane (1.84 g, 8.2 mmol) or 5-azido-1-iodopentane (1.63 g, 6.82 mmol) the formation of a white solid was observed. The mixture was warmed to rt and after 3 h stirring the complete conversion of the starting materials was observed. Aq. work-up and purification on a 25 g SiO.sub.2 column (20 to 50% EtOAc in n-hexane in 8 CV) was used to afford the desired products. 500 mg 5 (1.65 mmol) and 650 mg 6 (2.1 mmol) were collected as colourless liquids.

[0340] tert-Butyl N-[2-[2-(4-azidobutyloxy)ethoxy]ethyl]carbamate (Boc-NH-PEG.sub.2-C.sub.4-N.sub.3, 5)Yield: 27%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?4.99 (s, NH), 3.55 (m, 8H), 3.30 (t, 4H), 1.67 (m, 4H), 1.43 (s, 9H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?156.05, 79.24, 70.70, 70.28, 51.32, 40.36, 31.65, 28.50 (3C), 26.81, 25.80, 14.26. HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.13H.sub.26N.sub.4O.sub.4Na.sup.+, 325.1846; found, 325.1848.

[0341] tert-Butyl N-[2-[2-(5-azidopentyloxy)ethoxy]ethyl]carbamate (Boc-NH-PEG.sub.2-C.sub.5-N.sub.3, 6)Yield: 42%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?5.01 (s, NH), 3.56 (m, 6H), 3.45 (d, 2H), 3.27 (m, 4H), 1.61 (m, 4H), 1.43 (s, 9H), 1.42 (m, 2H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?156.10, 79.26, 71.20, 70.33, 70.16, 51.45, 40.40, 29.21, 28.77, 28.51 (3C), 21.17, 14.30. HRMS (m/z): [M+H].sup.+ calcd. for C.sub.14H.sub.28N.sub.4O.sub.4Na.sup.+, 339.2003; found, 339.2004.

d. Boc-NH-PEG.sub.2-C.sub.4-NH.sub.2 (7)/Boc-NH-PEG.sub.2-C.sub.5-NH.sub.2 (8)

[0342] For Staudinger reduction 500 mg alkyl azide compounds 5 (1.65 mmol) or 6 (1.56 mmol) were dissolved in 4 mL/g dry THF in a dry round-bottom flask under an argon atmosphere. 1.5 eq. PPh.sub.3 was added and the mixture was left stirring for 24 h air-excluded. Afterwards, 10 eq. H.sub.2O were added and the mixture was left stirring for 24 h. The solution was concentrated in vacuo and purified on a 12 g silica column with 10% MeOH and 0.5% TEA in DCM for 8 CV. For both reactions 230 mg of a pale-yellow gum were collected as 7 (0.83 mmol) and 8 (0.79 mmol).

[0343] tert-Butyl N-[2-[2-(4-aminobutyloxy)ethoxy]ethyl]carbamate (Boc-NH-PEG.sub.2-C.sub.4-NH.sub.2, 7)Yield: 50%. .sup.1H NMR (400 MHZ, MeOD): ?4.58 (NH.sub.2), 3.61 (s, 4H), 3.52 (dt, 4H), 3.22 (t, 2H), 2.97 (t, 2H), 1.74 (m, 4H), 1.43 (s, 9H). .sup.13C NMR (101 MHZ, MeOD): ?157.30, 78.91, 70.27, 69.92, 69.86, 48.57, 49.92, 39.49, 27.47 (3C), 26.51, 24.85. HRMS (m/z): [M+H].sup.+ calcd. for C.sub.13H.sub.29N.sub.2O.sub.4.sup.+, 277.2122; found, 277.2119.

[0344] tert-Butyl N-[2-[2-(5-aminopentyloxy)ethoxy]ethyl]carbamate (Boc-NH-PEG.sub.2-C.sub.5-NH.sub.2, 8)Yield: 50%. .sup.1H NMR (400 MHZ, MeOD): ?3.58 (s, 4H), 3.54 (m, 4H), 3.22 (t, 2H), 2.67 (t, 2H), 1.58 (m, 2H), 1.44 (s, 9H), 1.46 (m, 4H). .sup.13C NMR (101 MHZ, MeOD): ?157.60, 79.20, 71.39, 70.35, 70.18, 41.53, 40.39, 32.51, 29.63, 27.91 (3C), 23.64. HRMS (m/z): [M+H].sup.+ calcd. for C.sub.14H.sub.31N.sub.2O.sub.4.sup.+, 291.2278; found, 291.2275.

e. Terminal (sulfon)amide Synthesis (9-12)

[0345] 1 eq. alkylamine 7 or 8 was dissolved in 6 mL/mmol dry DCM. Afterwards 1.5 eq. TEA was added and the mixture was cooled to 0? C. and stirred vigorously under an argon atmosphere. 1.25 eq. Methanesulfonyl chloride was added dropwise at 0? C. to the solution of 7 or 8. In similar reactions 1.25 eq. trifluormethanesulfonyl chloride or 1.0 eq. acetyl chloride were added to 7. The mixture was stirred for 1 h at 0? C., warmed to room-temperature and left stirring over-night at room-temperature.

[0346] For aq. work-up the reaction mixture was diluted with 2 Veq. DCM, 1 Veq. 1 N HCl was added and the aq. phase was extracted twice with 1 Veq. DCM. The combined organic layers were washed once with 1 Veq. brine and dried over MgSO.sub.4. Filtration and removal of the solvent under reduced pressure delivered the crude product. Final purification was reached by flash column chromatography (12 g SiO.sub.2) and the desired compounds were afforded in 61 to 99% yields.

TABLE-US-00007 TABLE 5 Purification conditions and yields of nrHTL precursors Product Gradient Yield [%] 9 0 to 10% MeOH in DCM in 13 CV 61 10 20 to 80% EtOAc in n-hexane in 8 CV 64 11 40 to 70% EtOAc in n-hexane in 8 CV 99 12 0 to 6% MeOH in DCM in 8 CV 80

##STR00009##

[0347] tert-Butyl (2-(2-((4-(methylsulfonamino)-butyl)oxy)ethoxy)ethylcarbamate (BocNH-PEG.sub.2-C.sub.4-MSA, 9)Yield: 61%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.55 (m, 8H), 3.30 (t, 2H), 3.15 (t, 2H), 2.93 (s, 3H), 1.69 (m, 4H), 1.44 (s, 9H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?157.06, 78.69, 70.40, 69.84, 69.77, 69.65, 42.45, 38.32, 29.37, 27.36 (3C), 26.55, 26.36. HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.15H.sub.30N.sub.2O.sub.6SNa.sup.+, 377.1717; found, 377.1714.

[0348] tert-Butyl (2-(2-((5-(methylsulfonamino)pentyl)-oxy)ethoxy)ethylcarbamate (BocNH-PEG.sub.2-C.sub.5-MSA, 10)Yield: 64%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.59 (m, 6H), 3.33 (t, 2H), 3.34 (q, 2H), 3.16 (q, 2H), 2.94 (s, 3H), 1.65 (m, 4H), 1,46 (m, 2H), 1.44 (s, 9H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ? 156.06, 79.25, 77.26, 70.98, 70.22, 70.06, 43.15, 40.28, 29.83, 28.91, 28.44 (3C), 23.19, 21.3. HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.16H.sub.32N.sub.2O.sub.6SNa.sup.+, 369.2051; found, 369.2051.

[0349] tert-Butyl (2-(2-((4-(trifluoromethylsulfonamino)-butyl)oxy)ethoxy)ethylcarbamate (BocNH-PEG.sub.2-C.sub.4-F.sub.3MSA, 11)Yield: 99%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.56 (m, 4H), 3.52 (m, 4H), 3.30 (m, 4H), 1.74 (m, 4H), 1.43 (s, 9H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?156.16, 121.35 (q, CF.sub.3), 77.26, 70.97, 70.16 (2C), 69.80, 44.16 (2C), 28.37 (3C), 28.99 (2C). .sup.19F NMR (376 MHZ, CDCl.sub.3): ?-77.23 (s, 3F). HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.16H.sub.29N.sub.2O.sub.6SF.sub.3Na.sup.+, 431.1434; found, 431.1431.

[0350] tert-Butyl (2-(2-((4-(acetamidobutyl)oxy)-ethoxy)ethylcarbamate (BocNH-PEG.sub.2-C.sub.4-NAc, 12)Yield: 80%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.54 (m, 6H), 3.44 (t, 2H), 3.25 (p, 2H), 3.19 (t, 2H), 1.92 (s, 3H), 1.57 (tdd, 4H), 1.43 (s, 9H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?170.51, 156.12, 79.85, 70.13, 70.00, 50.58, 40.27, 39.32, 28.38 (3C), 26.89, 26.21(2C), 23.14. HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.15H.sub.30N.sub.2O.sub.5Na.sup.+, 341.2047; found, 341.2042.

6.1.3 Boc-NH-PEG.sub.2-C.sub.5-Methylamine(NBoc) (15)

##STR00010##

a. tert-Butyl (5-hydroxypentyl)(methyl)carbamate (13)

[0351] Reductive amination was carried out in a two-step reaction according to Ji et al. 2019 (US2019192668A1). First, 1 eq. 5-aminopentane-1-ol (5.3 g, 49 mmol) was dissolved in 4 eq. ethyl formate (15.7 mL, 194 mmol). The mixture was heated to 90? C. and stirred under reflux conditions for 6 h, cooled to room-temperature and concentrated in vacuo. The crude compound was purified on a 40 g silica-column with 2 to 8% MeOH in DCM over 14 CV and 4.8 g N-(5-hydroxypentyl) formamide (36.4 mmol, 74%) was collected as a colourless liquid. .sup.1H NMR (400 MHZ, MeOD): ?8.02 (s, 1H), 3.55 (t, 2H), 3.22 (t, 2H), 1.55 (m, 4H), 1.41 (m, 2H). .sup.13C NMR (101 MHZ, MeOD): ?163.72, 62.71, 38.90, 33.18, 30.13, 24,20. HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.6H.sub.12NO.sub.2Na.sup.+, 154.0838; found, 154.0837.

[0352] Next, a flame-dried 250 mL round-bottom flaks was charged with 694 mg LiAlH.sub.4 (18.3 mmol, 1.2 eq.) and 22 mL dry THF. The mixture was cooled to 0? C. 1 eq. N-(5-hydroxypentyl) formamide (2 g, 15.2 mmol) from the previous reaction was dissolved in 5 mL dry THF and added drop-wise under strong stirring and continuous cooling. A gray precipitate formed rapidly. After complete addition of the reagents, the mixture was heated to 80? C. and stirred under reflux conditions for 2 h. The reaction was stopped by the addition of 2 mL 15% NaOH and some drops of water. It was dried over MgSO.sub.4 the precipitated was removed by filtration and washed extensively with THF. 1.8 g crude 5(-methylamino)pentan-1-ol was collected as a colourless oil. A small proportion was purified over 12 g SiO.sub.2 with 6 to 20% MeOH in DCM over 8 CV. Residual silica gel was removed by filtration over a short plug of Celite? and the desired compound was afforded in 97% yield. .sup.1H NMR (400 MHZ, MeOD): ?3.47 (t, 3H), 3.27 (s, 3H), 3.16 (q, 2H), 1.47 (m, 4H), 1.33 (m, 2H). HRMS (m/z): [M+H].sup.+ calcd. for C.sub.6H.sub.12NO, 118.1226; found, 118.1227.

[0353] Finally, 1 eq. purified 5(-methylamino)pentan-1-ol (300 mg, 2.6 mmol) was dissolved in 4 mL dry MeOH and 550 UL di-tert-butyl dicarbonate (2.6 mmol, 1 eq.) were added. The mixture was stirred for 12 h at room-temperature, concentrated and purified by silica flash column chromatography (12 g SiO.sub.2) with 30 to 50% EtOAc in n-hexane over 12 CV. The title compound 13 was collected in 95% yield (650 mg, 2.3 mmol) and appeared as a colourless liquid.

[0354] tert-Butyl (5-hydroxypentyl)(methyl)carbamate (13)Yield: 75% over three steps. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.63 (t, 2H), 3.30 (t, 2H), 2.82 (s, 3H), 1.56 (m, 4H), 1.44 (s, 9H), 1.34 (m, 2H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?156.04, 79.33, 62.87, 34.31, 32.45, 28.59 (4C), 22.85. HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.11H.sub.23NO.sub.3Na.sup.+, 240.1570; found, 240.1569.

b. tert-Butyl (5-iodopentyl)(methyl)carbamate (14)

[0355] Mesylation of 500 mg 13 (2.3 mmol) was carried out as described in general procedure A. After flash column purification (12 g SiO.sub.2, 0 to 5% MeOH in DCM, 15 CV) 640 mg 5-((tert-butoxycarbonyl)(methyl)amino)pentyl methanesulfonate (2.3 mmol, 93%) was received as a pale-yellow oil. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?4.22 (t, 2H), 3.21 (t, 2H), 2.99 (s, 3H), 2.82 (s, 3H), 1.76 (m, 2H), 1.54 (qd, 2H) 1.44 (s, 9H), 1.39 (m, 2H). HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.12H.sub.25NO.sub.5SNa.sup.+, 318.1346; found, 318.1241.

[0356] Further, 300 mg 5-((tert-butoxycarbonyl)(methyl)amino)pentyl methanesulfonate (1 mmol) was used in a Finkelstein reaction as described in general procedure A. Flash column purification with 12 g SiO.sub.2 (5 to 20% EtOAc in n-hexane, 6 CV) was performed to afford 240 mg the title compound 14 (0.74 mmol, 73%) which appears as a pale-orange liquid.

[0357] tert-Butyl (5-iodopentyl)(methyl)carbamate (14)Yield: 90% over two steps. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.10 (q, 4H), 2.83 (s, 3H), 1.84 (p, 2H), 1.52 (m, 2H), 1.45 (s, 9H), 1.39 (m, 2H). HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.11H.sub.22NO.sub.2INa.sup.+, 350.0587; found, 350.0589.

c. tert-Butyl N-[2-[2-(5-N-Boc-(methyl)aminopentyl)ethoxy]ethyl]carbamate (15)

[0358] According to general procedure B, 3 mL of a 2/1 mixture of dry THF and DMF and 500 mg tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate (2.4 mmol, 1 eq.) were mixed by vigorous stirring. The mixture was cooled to 0? C. and 102 mg NaH (2.55 mmol, 1.1 eq.) were added portion-wise. The emulsion was left stirring for 30 min at 0? C. under air-exclusion. Afterwards 860 mg 14 (2.9 mmol, 1.2 eq.) were added quickly and the reaction was left stirring for 18 h. The subsequent aq. work-up and column purification (12 mg SiO.sub.2, 20 to 50% EtOAc/n-hexane, 8 CV) gave the title compound (360 mg, 0.89 mmol, colourless oil) in 37% yield.

[0359] tert-Butyl N-[2-[2-(5-N-Boc-(methyl)aminopentyl)ethoxy]ethyl]carbamate (15)Yield: 37%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.42 (m, 6H), 3.31 (t, 2H), 3.16 (t, 2H), 3.04 (t, 2H), 2.67 (s, 3H), 1.46 (m, 2H), 1.36 (m, 2H), 1.29 (dd, 18H), 1.18 (qd, 2H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?155.12, 155.94, 79.33, 79. 23, 71.45, 70.39, 70.34, 70.15, 48.72, 40.60, 34.21, 29.46, 28.60, 28.54, 27.71, 23.40. HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.20H.sub.40N.sub.2O.sub.6Na.sup.+, 427.2779; found, 427.2779.

6.1.4 Boc-NH-PEG.sub.2-C.sub.3-5-OTBS (18-20)/Boc-NH-PEG.sub.2-C.sub.4-OMe (21)

##STR00011##

a. tert-Butyl (3-bromopropoxy)dimethylsilane (16)/tert-butyl (5-bromopentoxy)-dimethylsilane (17)

[0360] TBS protection of commercially available bromoalkyl alcohols was carried out in DCM with tert-butyl-chlordimethylsilan (TBDMS-Cl). Therefore, 1 eq. 1-bromopropan-3-ol (0.5 mL, 5.76 mmol) or 1-bromopentan-5-ol (1.5 g, 9 mmol) was dissolved in 1.75 mL/mmol dry DCM. 1.5 eq. imidazole was added and the mixture was cooled to 0? C. under vigorous stirring.

[0361] Finally, 1.1 eq. TBDMS-Cl was added portion-wise. The formation of a white precipitate was observed immediately. After 1 h stirring at room-temperature 1 mL/mmol 10% NH.sub.4Cl was added to deactivated excess of TBDMS-Cl. The aq. layer was extracted with 5 mL/mmol DCM, the org. phases were combined and washed with brine once. It was dried over MgSO.sub.4, filtrated and concentrated in vacuo. Purification of the crude product was reached on 25 g silica column with 0 to 30% EtOAc/n-hexane in 8 CV. The title compounds 16 were collected in 97% yield (1.42 g, 5.6 mmol) or 17 in 99% yield (2.5 g, 9.25 mmol) appearing as a colourless oil.

[0362] tert-Butyl (3-bromopropoxy)dimethylsilane (16)Yield: 97%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.73 (t, 2H), 3.51 (t, 2H), 2.03 (ddd, 2H), 0.89 (s, 9H), 0.06 (s, 6H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?60.55, 35.70, 30.78, 26.04 (3C), ?5.24 (2C). HRMS (m/z): [M+H].sup.+ calcd. for C.sub.9H.sub.21BrOSi.sup.+, 253.0618, 255.0598; found, 253.0618, 255.0597.

[0363] tert-Butyl (5-bromopentoxy)dimethylsilane (17)Yield: 99%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.61 (t, 2H), 3.41 (t, 2H), 1.88 (m, 2H), 1.52 (m, 4H), 0.89 (s, 9H), 0.05 (s, 6H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?62.99, 33.96, 32.76, 32.05, 26.01 (3C), 24.73, ?5.15 (2C). HRMS (m/z): [M+H].sup.+ calcd. for C.sub.11H.sub.25BrOSi.sup.+, 281.0931, 283.0911; found, 281.0933, 283.0913.

b. Boc-NH-PEG.sub.2-C.sub.3-5-OTBS (18-20)/Boc-NH-PEG.sub.2-C.sub.4-OMe (21)

[0364] The title compounds were synthesized according to general procedure B. Therefore, 1 mg 16 (3.95 mmol) or 2 g 17 (7.1 mmol) were used. Further, commercially available tert-butyl (3-bromobutoxy)dimethylsilane (2 mL, 7.2 mmol) or 4-methoxy-1-bromobutan (0.8 g, 4.8 mmol) were used. All reactions were completed overnight after 16 to 20 h. Subsequent aq. work-up and flash column purification (25 g SiO.sub.2, 10 to 50% EtOAc in n-hexane, 10 CV) gave the desired compounds in 38 to 43% yield.

[0365] tert-Butyl (2,2,3,3-tetramethyl-4,8,11-trioxa-3-silatridecan-13-yl)carbamate (Boc-NH-PEG.sub.2-C.sub.3-OTBS, 18)Yield: 40%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.68 (t, 2H), 3.55 (m, 8H), 3.20 (q, 2H), 1.78 (p, 2H), 1.78 (s, 9H), 0.87 (s, 9H), 0.03 (s, 6H). HRMS (m/z): [M+H].sup.+ calcd. for C.sub.18H.sub.39NO.sub.5Si.sup.+, 378.2670; found, 378.2667.

[0366] tert-Butyl (2,2,3,3-tetramethyl-4,8,11-trioxa-3-silatetradecan-14-yl)carbamate (Boc-NH-PEG.sub.2-C.sub.4-OTBS, 19)Yield: 38%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.56 (m, 10H), 1.60 (m, 6H), 1.44 (s, 9H), 0.89 (s, 9H), 0.04 (s, 6H). HRMS (m/z): [M+Na].sup.+ (ATBST) calcd. for C.sub.13H.sub.27NO.sub.5Na, 300,1718; found 300, 1718.

[0367] tert-Butyl (2,2,3,3-tetramethyl-4,8,11-trioxa-3-silapentadecan-15-yl)carbamate (Boc-NH-PEG.sub.2-C.sub.5-OTBS, 20)Yield: 46%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.56 (m, 8H), 3.44 (t, 2H), 3.29 (q, 2H), 1.55 (m, 3H), 1.42 (s, 9H), 1.36 (m, 2H), 0.87 (s, 9H), 0.02 (s, 6H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?156.11, 71.57, 50.39, 70.32, 70. 12, 40.46, 37.75, 29.49, 28.58 (3C), 26.08 (3C), 22.45, 18.46, ?5.17 (2C). HRMS (m/z): [M+H].sup.+ calcd. for C.sub.20H.sub.43NO.sub.5Si.sup.+, 406.2983; found, 406.2983.

[0368] tert-Butyl (2-(2-(4-methoxybutoxy)ethoxy)ethyl)carbamate (Boc-NH-PEG.sub.2-C.sub.3-OTBS, 21)Yield: 43%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.54 (m, 6H), 3.45 (m, 2H), 3.35 (m, 2H), 3.28 (s, 3H), 2.26 (m, 2H), 1.61 (tdd, 4H), 1.40 (s, 9H). HRMS (m/z): [M+H].sup.+ calcd. for C.sub.14H.sub.30NO.sub.5.sup.+, 292.2118; found, 292.2114.

6.1.5 Boc-NH-PEG.sub.2-C.sub.6 (22)

##STR00012##

[0369] According to Tang et al. (2017). 6-bromon-hexane (1.02 mg, 7.32 mmol, 1.2 eq.) was used in an Williamson ether synthesis reaction carried out according to general procedure B. The reaction was completed after 16 h. Aq. work-up and flash column purification (25 g SiO.sub.2, 10 to 50% EtOAc in n-hexane, 10 CV) delivered 22 as a colourless oil (1.06 g, 3.66 mmol).

[0370] tert-Butyl N-(2-(2-(6-hexyl)ethoxy)ethyl)carbamate (22)Yield: 71%. .sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.60 (m, 6H), 3.49 (dd, 2H), 3.35 (m, 2H), 1.61 (dq, 2H), 1.47 (s, 9H), 0.94 (t, 3H). HRMS (m/z): [M+Na].sup.+ calcd. for C.sub.15H.sub.31NO.sub.4Na.sup.+, 312.2145; found, 312.2140.

6.1.6 Fluorophore Labeling

[0371] a. Deprotection

##STR00013##

[0372] Primary amines were generated by Boc protecting group cleavage according to general procedure C. The generated compounds were well-dried but used without further purification for fluorophore coupling. All compounds were obtained in quantitative yields.

TABLE-US-00008 TABLE 5 Deprotection of potential nrHTL precursors used in this work Reaction Abbreviation R Full name 9 .fwdarw. 23 C.sub.4-MSA N-(4-(2-(2-aminoethoxy)ethoxy)butyl)meth- anesulfonamide 10 .fwdarw. 24 C.sub.5-MSA N-(5-(2-(2-aminoethoxy)ethoxy)pentyl)meth- anesulfonamide 11 .fwdarw. 25 C.sub.4F.sub.3-MSA N-(4-(2-(2-aminoethoxy)ethoxy)bu- tyl)triflamide 12 .fwdarw. 26 C.sub.4-NAC N-(4-(2-(2-aminoethoxy)ethoxy)bu- tyl)acetamide 5 .fwdarw. 27 C.sub.4N.sub.3 2-(2-(4-azidobutoxy)ethoxy)ethan-1-amine 15 .fwdarw. 28 C.sub.5-MA 5-(2-(2-aminoethoxy)ethoxy)-N-meth- ylpentan-1-amine 18 .fwdarw. 29 C.sub.3OH 5 -(2-(2-aminoethoxy)ethoxy)propan-1-ol 19 .fwdarw. 30 C.sub.4OH 5-(2-(2-aminoethoxy)ethoxy)butan-1-ol 20 .fwdarw. 31 C.sub.5OH 5-(2-(2-aminoethoxy)ethoxy)pentan-1-ol 21 .fwdarw. 32 C.sub.4-OMe 2-(2-(4-methoxybutoxy)ethoxy)ethan-1- amine 22 .fwdarw.33 C.sub.6 2-(2-(hexyloxy)ethoxy)ethan-1-amine Reaction number, nomenclature and full name of nrHTL linkers (H.sub.2N-PEG.sub.2-R) synthesized in this work.

##STR00014##

[0373] N-(4-(2-(2-aminoethoxy)ethoxy)butyl)methanesulfonamide (H.sub.2N-PEG.sub.2-C.sub.4-MSA, 23).sup.1H NMR (400 MHZ, MeOD): ?3.66 (m, 6H), 3.53 (t, 2H), 3.11 (m, 4H), 2.92 (s, 3H), 1.64 (m, 4H). .sup.13C NMR (101 MHZ, MeOD): ?71.20, 70.72, 70.48, 43.17, 40.05, 39.01, 27.32, 27.02. HRMS (m/z): [M+H].sup.+ calcd. for C.sub.9H.sub.22N.sub.2O.sub.4S, 255.1373; found, 255.1370.

[0374] N-(5-(2-(2-aminoethoxy)ethoxy)pentyl)methanesulfonamide (H.sub.2N-PEG.sub.2-C.sub.5-MSA, 24).sup.1H NMR (400 MHZ, MeOD): ?3.66 (m, 6H), 3.51 (t, 2H), 3.12 (m, 2H), 3.09 (t, 2H), 2.91 (s, 3H), 1.60 (m, 4H), 1.46 (m, 2H). .sup.13C NMR (101 MHZ, MeOD): ?71.53, 70.68, 70.50, 67.22, 43.26, 40.04, 38.91, 30.26, 29.47, 23.56. HRMS (m/z): [M+H].sup.+ calcd. for C.sub.10H.sub.24N.sub.2O.sub.4S, 269.1530 found, 269.1528.

[0375] N-(4-(2-(2-aminoethoxy)ethoxy)butyl)trifluoromethanesulfonamide (H.sub.2N-PEG.sub.2-C.sub.4-F.sub.3MSA, 25).sup.1H NMR (400 MHZ, CDCl.sub.3): ?3.68 (t, 2H), 3.60 (m, 2H), 3.55 (m, 2H), 3.52 (m, 2H), 3.27 (dq, 4H), 1.71 (td, 4H). .sup.13C NMR (101 MHZ, CDCl.sub.3): ?119.92 (q, 1C), 71.18, 70.20, 44.30, 30.05, 31.08, 27.11, 27.62. 19F NMR (377 MHZ, CDCl.sub.3): ?-77.6 HRMS (m/z): [M+H].sup.+ calcd. for C.sub.9H.sub.19F.sub.3N.sub.2O.sub.4S, 309.1090 found, 309.1089.

[0376] N-(4-(2-(2-aminoethoxy)ethoxy)butyl)acetamide (H.sub.2N-PEG.sub.2-C.sub.4-NAc, 26).sup.1H NMR (400 MHZ, MeOD): ?3.65 (m, 6H), 3.51 (t, 2H), 3.18 (t, 2H), 3.12 (t, 2H), 1.93 (s, 3H), 1.59 (m, 4H). .sup.13C NMR (101 MHZ, MeOD): ?173.32, 71.86, 71.32, 71.09, 67.81, 40.65, 40.20, 27.85, 27.01, 22.48. HRMS (m/z): [M+H].sup.+ calcd. for C.sub.10H.sub.22N.sub.2O.sub.3, 219.1703 found, 219.1701.

[0377] 2-(2-(4-azidobutoxy)ethoxy)ethan-1-amine (27).sup.1H NMR (H.sub.2N-PEG.sub.2-C.sub.4-N.sub.3, 400 MHZ, MeOD): ?3.66 (m, 6H), 3.53 (ddt, 2H), 3.34 (m, 2H), 3.12 (m, 2H), 1.67 (tq, 4H). HRMS (m/z): [M+H].sup.+ calcd. for C.sub.8H.sub.18N.sub.4O.sub.2, 203.1503 found, 203.1503.

[0378] 5-(2-(2-aminoethoxy)ethoxy)-N-methylpentan-1-amine (H.sub.2N-PEG.sub.2-C.sub.5-MA, 28).sup.1H NMR (400 MHZ, MeOD): ?3.51 (q, 4H), 3.42 (m, 4H), 2.70 (t, 2H), 2.49 (t, 2H), 2.30 (s, 3H), 1.49 (m, 4H), 1.32 (m, 2H). .sup.13C NMR (101 MHZ, MeOD): ?73.46, 72.16, 71.26, 71.12, 52.51, 42.09, 35.97, 30.51, 29.96, 24.85. HRMS (m/z): [M+H].sup.+ calcd. for C.sub.10H.sub.24N.sub.2O.sub.2, 205.1911 found, 205.1910

[0379] 5-(2-(2-aminoethoxy)ethoxy)propan-1-ol (H.sub.2N-PEG.sub.2-C.sub.3-OH, 29).sup.1H NMR (400 MHZ, MeOD): ?4.46 (t, 2H), 3.75 (t, 2H), 3.63 (m, 6H), 3.28 (h, 2H), 2.02 (p, 2H). .sup.13C NMR (101 MHZ, MeOD): ?70.10, 67.11, 66.05, 65.19, 40.37, 28.11. HRMS (m/z): [M+H].sup.+ calcd. for C.sub.7H.sub.17NO.sub.3, 164.1281 found, 164.1281.

[0380] 5-(2-(2-aminoethoxy)ethoxy)butan-1-ol (H.sub.2N-PEG.sub.2-C.sub.4-OH, 30).sup.1H NMR (400 MHZ, MeOD): ?3.66 (m, 2H), 3.57 (t, 2H), 3.53 (t, 2H), 3.12 (m, 2H), 1.63 (m, 4H). HRMS (m/z): [M+H].sup.+ calcd. for C.sub.8H.sub.19NO.sub.3, 178.1438 found, 178.1439.

[0381] 5-(2-(2-aminoethoxy)ethoxy)pentan-1-ol (H.sub.2N-PEG.sub.2-C.sub.4-OH, 31).sup.1H NMR (400 MHZ, MeOD): ?4.33 (t, 2H), 3.71 (m, 2H), 3.59 (ddt, 4H), 3.45 (t, 2H), 3.14 (m, 2H), 1.75 (dt, 2H), 1.59 (h, 2H), 1.42 (m, 2H). .sup.13C NMR (101 MHZ, MeOD): ?71.01, 70.31, 69.90, 68.16, 39.70, 28.89, 27.85, 22.18. HRMS (m/z): [M+H].sup.+ calcd. for C.sub.9H.sub.21NO.sub.3, 192.1594 found, 192.1593.

[0382] 2-(2-(4-methoxybutoxy)ethoxy)ethan-1-amine (H.sub.2N-PEG.sub.2-C.sub.4-OMe, 32).sup.1H NMR (400 MHZ, MeOD): ?3.76 (m, 2H), 3.68 (m, 4H), 3.56 (m, 4H), 3.44 (t, 2H), 3.43 (s, 3H), 1.66 (ddp, 4H). HRMS (m/z): [M+H].sup.+ calcd. for C.sub.9H.sub.21NO.sub.3, 192.1594 found, 192.1594.

[0383] 2-(2-(hexyloxy)ethoxy)ethan-1-amine (H.sub.2N-PEG.sub.2-C.sub.6, 33).sup.1H NMR (400 MHZ, MeOD): ?3.60 (m, 6H), 3.49 (t, 2H), 3.07 (t, 2H), 1.58 (dq, 42H), 1.35 (m, 6H), 0.94 (t, 3H). HRMS (m/z): [M+H].sup.+ calcd. for C.sub.10H.sub.23NO.sub.2, 190.1801found, 190.1802

b. Coupling to 6-carboxy rhodamine-based Fluorophores

##STR00015##

[0384] Coupling condition I: The following 6-carboxy modified rhodamine-based fluorescent dyes were coupled to the respective amino linker with the coupling reagent N,N,N,N-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) in dry DMSO: Tetramethyl rhodamine (TMR), carbopyronine (CPy), silicon rhodamine (SiR), 3-(N,N-dimethylaminosulfonamide) tetramethyl rhodamine (MaP555), F.sub.4-bisazetidine rhodamine (F.sub.4-BAR, JF.sub.525), F.sub.4-bisazetidine carbopyronine (F.sub.4-BACPy, JF.sub.585).

[0385] Coupling condition II: The following 6-carboxy modified rhodamine-based fluorescent dyes were coupled to the respective amino linker with the coupling reagent (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) in dry DMSO: (N,N-dimethylaminosulfonamide) carbopyronine (MaP618), bisazetidine silicon rhodamine (BASiR, JF.sub.656), F.sub.2-bisazetidine silicon rhodamine (F.sub.2-BASiR, JF.sub.635), F.sub.4-bisazetidine silicon rhodamine (F.sub.4-BASiR, JF.sub.615), Atto565.

[0386] 1 eq. 6-Carboxy fluorophore was dissolved in ?100 ?L/?mol dry DMSO-d.sub.6. The mixture was added on top of 1.2 eq. coupling reagent (I: TSTU, II: HBTU) and 4-10 eq. diisopropylethylamine (DIPEA) were spiked into the solution. It was left stirring for at least 10 min at rt. The formation of the respective N-hydroxysuccinimid (NHS) or hydroxybenzotriazole (HOBT) intermediate (active ester) was monitored by LC-MS (NHS: M.sub.Dye+98 m/z, HOBT: M.sub.Dye+133 m/z). Meanwhile, the amine linker 23-33 was dissolved in the same amount of dry DMSO-de with 4-10 eq. DIPEA. Both solutions were mixed after full activation of the dye and left stirring for 1-4 h at 35 to 50? C.

[0387] After completion of the reaction excess of MeCN with 20% H.sub.2O and 0.1% acetic acid was added and the mixture was purified by RP-HPLC.

[0388] TMR-23.sup.1H NMR (400 MHZ, CD.sub.3CN): ?8.27 (d, 1H), 8.11 (d, 1H), 7.70 (d, 1H), 7.73 (d, 1H), 7.01 (d, 2H), 6.85 (dd, 2H), 6.78 (d, 2H), 3.53 (m, 8H), 3.38 (m, 2H), 3.19 (s, 12H), 2.97 (m, 2H), 2.82 (s, 3H), 1.51 (m, 4H).

[0389] TMR-24.sup.1H NMR (400 MHZ, CD.sub.3CN): ?8.11 (d, 1H), 8.95 (dd, 1H), 7.58 (d, 1H), 7.32 (t, 1H), 6.78 (d, 2H), 6.62 (d, 4H), 3.52 (m, 4H), 3.45 (m, 4H), 3.32 (t, 2H), 3.05 (s, 12H), 2.95 (q, 2H), 2.82 (s, 3H), 1.44 (h, 4H), 1.28 (m, 2H).

[0390] TMR-25.sup.1H NMR (400 MHZ, CD.sub.3CN): ?8.07 (m, 2H), 7.56 (s, 2H), 7.22 (s, 2H), 6.73 (d, 2H), 6.60 (m, 2H), 3.54 (m, 6H), 3.36 (t, 2H), 3.18 (t, 2H), 3.04 (s, 12H), 1.51 (m, 4H), 1.27 (m, 4H). .sup.19F NMR (377 MHZ, CD.sub.3CN): ?-79.62.

[0391] TMR-26.sup.1H NMR (400 MHZ, CD.sub.3CN): ?9.14 (d, 1H), 8.96 (d, 1H), 8.58 (s, 1H), 8.38 (t, 1H), 7.89 (dd, 2H), 7.72 (d, 2H), 7.64 (s, 2H), 4.38 (m, 8H), 3.21 (td, 2H), 3.05 (s, 12H), 3.87 (q, 2H), 2.61 (s, 3H), 2.27 (dq, 2H).

[0392] TMR-27.sup.1H NMR (400 MHZ, CD.sub.3CN): ?8.25 (d, 1H), 8.09 (dd, 1H), 7.67 (s, 1H), 7.31 (s, 2H), 6.98 (d, 2H), 6.80 (m, 4H), 3.52 (m, 8H), 3.38 (t, 2H), 3.27 (s, 12H), 3.16 (m, 2H), 1.54 (m, 4H).

[0393] TMR-28.sup.1H NMR (400 MHZ, CD.sub.3CN): ?8.28 (dd, 1H), 8.10 (dd, 1H), 7.72 (t, 1H), 7.57 (d, 1H), 7.04 (t, 2H), 6.87 (ddd, 2H), 6.79 (m, 2H), 3.54 (m, 8H), 3.35 (t, 2H), 3.20 (d, 12H), 2.88 (t, 2H), 2.56 (s, 3H), 1.61 (p, 2H), 1.45 (m, 2H) 1.29 (m, 2H).

[0394] TMR-30.sup.1H NMR (400 MHZ, CD.sub.3CN): ?8.32 (d, 1H), 8.14 (dd, 1H), 7.82 (t, 1H), 7.75 (d, 1H), 7.09 (d, 2H), 6.92 (dd, 2H), 6.83 (d, 2H), 3.54 (m, 8H), 3.39 (t, 2H), 3.36 (t, 2H), 3.23 (s, 12H), 1.52 (m, 2H), 1.44 (m, 2H).

[0395] TMR-31.sup.1H NMR (400 MHZ, CD.sub.3CN): ?8.24 (d, 1H), 8.09 (dd, 1H), 7.67 (d, 1H), 7.36 (d, 1H), 6.97 (d, 2H), 6.81 (dd, 2H), 6.75 (d, 2H), 3.51 (m, 7H), 3.42 (t, 2H), 3.35 (t, 2H), 3.17 (s, 12H), 1.43 (tt, 4H), 1.28 (m, 4H).

[0396] TMR-33.sup.1H NMR (400 MHZ, CD.sub.3CN): ?8.32 (d, 1H), 8.14 (dd, 1H), 7.74 (d, 1H), 7.58 (t, 1H), 7.07 (d, 2H), 6.91 (dd, 2H), 6.83 (d, 2H), 3.55 (m, 8H), 3.37 (t, 2H), 3.24 (s, 12H), 1.45 (q, 2H), 1.27 (m, 2H), 0.88 (t, 3H).

TABLE-US-00009 TABLE 6 Derivatization of various rhodamine-based dyes with HTL-like linkers. linker Dye LC [min] Purity [%] Chem. formula [M].sub.calcd. [M].sub.found 23 TMR 6.94 95 C.sub.34H.sub.43N.sub.4O.sub.8S 667.2786 667.2786 SiR 7.57 97 C.sub.36H.sub.49N.sub.4O.sub.7SSi 709.3086 709.3083 CPy 7.12 95 C.sub.40H.sub.51N.sub.4O.sub.8S 747.3422 747.3418 Atto565 9.79 100 C.sub.37H.sub.49N.sub.4O.sub.7S 693.3316 693.3318 MaP555 7.83 95 C.sub.36H.sub.48N.sub.6O.sub.9S.sub.2 773.2997 773.3001 JF.sub.525 6.23 99 C.sub.36H.sub.39F.sub.4N.sub.4O.sub.8S 763.2419 763.2419 JF.sub.585 10.77 95 C.sub.39H.sub.45F.sub.4N.sub.4O.sub.7S 789.2940 789.2936 MaP618 12.08 88 C.sub.39H.sub.54N.sub.6O.sub.8S.sub.2 799.3517 799.3516 JF.sub.615 6.50 95 C.sub.38H.sub.45F.sub.4N.sub.4O.sub.7SSi 805.2709 805.2703 JF.sub.635 10.48 98 C.sub.38H.sub.47F.sub.2N.sub.4O.sub.7SSi 769.2897 769.2894 JF.sub.656 9.84 97 C.sub.38H.sub.49N.sub.4O.sub.7SSi 733.3086 733.3091 24 TMR 6.53 99 C.sub.35H.sub.45N.sub.4O.sub.8S 681.2953 681.2952 SiR 7.93 95 C.sub.37H.sub.51N.sub.4O.sub.7SSi 723.3242 723.3240 CPy 7.87 96 C.sub.38H.sub.51N.sub.4O.sub.7S 707.3473 707.3473 Atto565 N.D. N.D. C.sub.38H.sub.51N.sub.4O.sub.7S 719.1990 719.1989 25 TMR 9.54 97 C.sub.34H.sub.40N.sub.4O.sub.8F.sub.3S 721.2513 721.2513 SiR 10.66 95 C.sub.36H.sub.46N.sub.4O.sub.7F.sub.3SSi 763.2803 763.2805 MaP555 10.31 98 C.sub.36H.sub.45F.sub.3N.sub.6O.sub.9S.sub.2 827.2714 827.2714 JF.sub.585 12.76 96 C.sub.39H.sub.42F.sub.7N.sub.4O.sub.7S 843.2657 843.2657 26 TMR 6.58 99 C.sub.35H.sub.43N.sub.4O.sub.7 631.3126 631.3121 SiR 7.64 98 C.sub.37H.sub.49N.sub.4O.sub.6Si 673.3416 673.3416 27 TMR 8.60 96 C.sub.33H.sub.39N.sub.6O.sub.6 615.2926 615.2926 28 TMR 6.71 100 C.sub.35H.sub.45N.sub.4O.sub.6H.sup.+ 309.1703 309.1703 SiR 8.73 100 C.sub.37H.sub.51N.sub.4O.sub.5SiH.sup.+ 330.1848 330.1845 29 TMR 6.49 99 C.sub.32H.sub.38N.sub.3O.sub.7 576.2704 576.2704 SiR 7.36 98 C.sub.34H.sub.44N.sub.3O.sub.6Si 618.2994 618.2994 30 TMR 6.56 95 C.sub.33H.sub.40N.sub.3O.sub.7Na.sup.+ 444.0431 444.0428 SiR 7.70 99 C.sub.35H.sub.46N.sub.3O.sub.6Si 632.3150 632.3153 31 TMR 6.96 96 C.sub.34H.sub.42N.sub.3O.sub.7 604.3017 604.3016 SiR 8.08 98 C.sub.36H.sub.48N.sub.3O.sub.6Si 646.3307 646.3306 32 TMR 8.02 99 C.sub.34H.sub.42N.sub.3O.sub.7 604.3017 604.3016 SiR 6.21 99 C.sub.36H.sub.48N.sub.3O.sub.6Si 646.3307 646.3307 33 TMR 10.60 95 C.sub.35H.sub.44N.sub.3O.sub.6 602.3225 602.3219