CARBOHYDRATE DERIVATIVES AND KITS FOR CELL SURFACE LABELING

Abstract

Provided herein are carbohydrate derivatives of general formula (I) bearing a trans-cyclooctene moiety usable in covalently binding active moieties to cell surfaces in vitro and/or in vivo and their use for therapeutic or nontherapeutic purposes. Furthermore, compounds of general formula (II) are used together with the derivatives of general formula (I) in a method of binding active moieties to the cell surface. The compounds may be combined to form a kit for forming a covalent attachment of active moieties to the cell surface.

##STR00001##

Claims

1. A method for binding active moieties to a cell surface comprising the following steps: a) the cells are co-cultured with at least one carbohydrate derivative of general formula I ##STR00017## wherein R.sup.1 is selected from H and C.sub.1-6 linear or branched alkyl, to form trans-cyclooctene presenting cells; b) at least one 1,2,4,5-tetrazine conjugate of general formula II ##STR00018## wherein R.sup.2 is selected from: H, C.sub.1-10 alkyl, C.sub.6-10 aryl, 5-10-membered heteroaryl, C.sub.1-10 alkoxy, C.sub.6-10aryl-C.sub.1-10alkyl, 5-10-membered heteroaryl-C.sub.1-10alkyl; linker is selected from C.sub.1-10 alkylene, C.sub.6-10 arylene, 5-10-membered heteroarylene, C.sub.6-10aryl-C.sub.1-10 alkylene, 5-10-membered heteroaryl-C.sub.1-10alkylene, and polyethylene glycol; R.sup.3 is an active moiety selected from fluorophore, inhibitor, ligand, oligonucleotide, peptide, protein, enzyme, antibody, single-domain antibody, polymer, and nanoparticle; is brought into contact with the trans-cyclooctene presenting cells in order to covalently attach the active moiety to the cell surface.

2. The method according to claim 1, wherein the reaction in step b) between trans-cyclooctene groups on the cell surface of the trans-cyclooctene presenting cells and the tetrazine conjugate of general formula II is carried out at a temperature within the range of room temperature to 37? C. in a cell culturing medium or in a biologically acceptable buffer.

3. The method according to claim 1, wherein the cells to be co-cultured with the carbohydrate derivative of general formula I are selected from cancer cells, epithelial cells, endothelial cells, fibroblasts, stem cells, immune cells, macrophages, blood cells and monocytes.

4. A carbohydrate derivative of general formula I: ##STR00019## wherein R.sup.1 is selected from H and C.sub.1-6 linear or branched alkyl.

5. The carbohydrate derivative according to claim 4, wherein the CO bond between the trans-cyclooctene and the carbamate is axial.

6. A method, comprising covalently binding active moieties to cell surfaces in vitro and/or in vivo for therapeutic purposes using the carbohydrate derivative of general formula I according to claim 4.

7. A method, comprising covalently binding active moieties to cell surfaces in vitro using the carbohydrate derivative of general formula I according to claim 4.

8. A kit containing at least one carbohydrate derivative of general formula I according to claim 4, and at least one 1,2,4,5-tetrazine conjugate of general formula II ##STR00020## wherein R.sup.2 is selected from: H, C.sub.1-10 alkyl, C.sub.6-10 aryl, 5-10-membered heteroaryl, C.sub.1-10 alkoxy, C.sub.6-10aryl-C.sub.1-10alkyl, 5-10-membered heteroaryl-C.sub.1-10alkyl; linker is selected from C.sub.1-10 alkylene, C.sub.6-10 arylene, 5-10-membered heteroarylene, C.sub.6-10aryl-C.sub.1-10alkylene, 5-10-membered heteroaryl-C.sub.1-10alkylene, and polyethylene glycol; R.sup.3 is an active moiety selected from fluorophore, inhibitor, ligand, oligonucleotide, peptide, protein, enzyme, antibody, single-domain antibody, polymer, and nanoparticle.

9. The kit containing at least one carbohydrate derivative of general formula I according to claim 4, cells amenable to modification by the carbohydrate derivative(s), and at least one 1,2,4,5-tetrazine conjugate of general formula II ##STR00021## wherein R.sup.2 is selected from: H, C.sub.1-10 alkyl, C.sub.6-10 aryl, 5-10-membered heteroaryl, C.sub.1-10 alkoxy, C.sub.6-10aryl-C.sub.1-10alkyl, 5-10-membered heteroaryl-C.sub.1-10alkyl; linker is selected from C.sub.1-10 alkylene, C.sub.6-10 arylene, 5-10-membered heteroarylene, C.sub.6-10aryl-C.sub.1-10alkylene, 5-10-membered heteroaryl-C.sub.1-10alkylene, and polyethylene glycol; R.sup.3 is an active moiety selected from fluorophore, inhibitor, ligand, oligonucleotide, peptide, protein, enzyme, antibody, single-domain antibody, polymer, and nanoparticle.

10. The kit according to claim 9, wherein the cells amenable to modification by the carbohydrate derivative(s) are selected from cancer cells, epithelial cells, endothelial cells, fibroblasts, stem cells, immune cells, macrophages, blood cells and monocytes.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0059] FIG. 1: HPLC-MS analysis of sulfonamide-PEG3-diPyTz conjugate. A) PDA total absorbance chromatogram, B) TIC (total ion count) chromatogram, C) extracted m/z from signal at 4.64 min.

[0060] FIG. 2: HPLC-MS analysis of NRP-NBD peptide. A) PDA total absorbance chromatogram, B) TIC (total ion count) chromatogram, C) extracted m/z from signal at 3.81 min.

[0061] FIG. 3: HPLC-MS analysis of NRP-NBD-PEG9-HTz peptide conjugate. A) PDA total absorbance chromatogram, B) TIC (total ion count) chromatogram, C) extracted m/z from signal at 5.37 min.

[0062] FIG. 4: Cell viability of U2OS cells incubated with the indicated concentrations of carbohydrates assessed by crystal violet and resazurin cell viability assay.

[0063] FIG. 5: A) micrograph of U2OS cells acquired using confocal microscope. On the left are cells treated with Cy3-HTz alone, and on the right are cells treated with 1 mM TCO-Sia and Cy3-HTz, the nuclei of the cells were stained with Hoechst nuclear stain. Scale bar 10 ?m. B) Overlay histogram showing the cellular fluorescence intensities of control and carbohydrate-treated cells (flow cytometry analysis).

[0064] FIG. 6: Incorporation of TCO-Sia into surface proteins of various cell lines followed by labeling with Cy3-HTz. Shown is flow cytometry analysis.

[0065] FIG. 7: Persistence of the TCO-Sia on the cell surface in THP-1 and NK-92MI cells detected by labeling with Cy3-HTz.

[0066] FIG. 8: Flow cytometry analysis of A) NK-92MI cells modified with sulfonamide-PEG3-diPyTz conjugate detected by hCAII-Cy3, B) NK-92MI cells modified with duplex Cy3-Oligonucleotide-HTz conjugate, C) NK-92MI cells modified with NRP-NBD-PEG9-HTz peptide conjugate, D) NK-92MI cells modified with Trastuzumab-HTz conjugate.

[0067] FIG. 9: A) micrograph of U2OS cells acquired using confocal microscope. Cells were treated with 1 mM TCO-Sia and eight different Tz-dye conjugates. Control cells are unlabeled cells.

[0068] FIG. 10: A) micrograph of PC3 cells acquired using confocal microscope. Cells were treated with 1 mM TCO-Sia followed by addition of MeTz-PEG3-PEG3-biotin-PEG3-DBCO conjugate, then Cy3-azide and finally with fluorescent streptavidin. The nuclei of the cells were stained with Hoechst nuclear stain.

[0069] FIG. 11: A) micrograph of HeLa cells acquired using confocal microscope. Cells were treated with 1 mM TCO-Sia followed by addition of FLAG-MeTz peptide conjugate or without addition of FLAG-MeTz peptide conjugate as a control. Then, fluorescent anti-FLAG antibody was added. The nuclei of the cells were stained with Hoechst nuclear stain.

[0070] FIG. 12: A) micrograph of HeLa cells acquired using confocal microscope. Cells were treated with 1 mM TCO-Sia followed by addition of Biotin-PEG3-diPy-Tz conjugate or without addition of Biotin-PEG3-diPy-Tz conjugate as a control. Then, fluorescent streptavidin was added. The nuclei of the cells were stained with Hoechst nuclear stain.

[0071] FIG. 13: A) micrograph of U2OS cells acquired using confocal microscope. A) cells were treated with 1 mM TCO-Sia and labeled with three different phycoerythrin-Tz conjugates. B) histograms of the same cells acquired by flow cytometry analysis.

[0072] FIG. 14: Schematic illustration of the presented procedure for the modification of cell surfaces with various active molecules. The cells are shown as oval shape and the black sphere represents cellular nucleus.

LIST OF ABBREVIATIONS

[0073] TCO=trans-cyclooctene, Tz=1,2,4,5-tetrazine, PEG=polyethyleneglycol, DNA=deoxyribonucleic acid, RNA=ribonucleic acid, PNA=peptide nucleic acid, U2OS=human bone osteosarcoma epithelial cells, HeLa=human cervical cancer cells, THP1=human acute monocytic leukemia cells, Raji=human Burkitt's lymphoblast cells, Jurkat=human T cell leukemia cells, MDCK=Madin-Darby Canine Kidney cells, NK=natural killer cells, PBMC=human peripheral blood mononuclear cells, ATCC=American Type Culture Collection, HPLC=high-performance liquid chromatography, MS=mass spectrometry, FACS=fluorescence-activated cell sorting, NMR=nuclear magnetic resonance, RP=reversed phase, h=hours, MeOH=methanol, MeOD=deuterated methanol, EtOH=ethanol, iPrOH=isopropanol, DCM=dichloromethane, DMF=N,N-dimethylformamide, DMSO=dimethylsulfoxide, CH.sub.3CN=acetonitrile, HCOOH=formic acid, TFA=trifluoroacetic acid, DIPEA=N,N-diisopropylethylamine, HCl=hydrochloric acid, NaOH=sodium hydroxide, CO.sub.2=carbon dioxide, Fmoc=fluorenylmethyloxycarbonyl, H.sub.2O=water, NBD=7-nitrobenzo-2-oxa-1,3-diazole, NRP=neuropilin, hCA=human carbonic anhydrase, FAP=fibroblast activation protein alpha, HER2=human epidermal growth factor receptor 2, EGFR=epidermal growth factor receptor, PE=phycoerythrin, PBS=phosphate-buffered saline, FBS=fetal bovine serum, HEPES=4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, DMEM=Dulbecco's modified eagle medium, RPMI=Roswell Park Memorial Institute medium.

EXAMPLES

I. Synthesis of Carbohydrates

[0074] The chemicals were obtained from Sigma Aldrich, Alfa Aesar, Acros Organics, ABCR, Flouorochem, Iris Biochem, Carbosynth or VWR unless otherwise stated, and were used without further purification. Solutions were concentrated in a rotary evaporator from Heidolph equipped with a PC3001 VARIOpro pump from Vacuubrand. Column chromatography was carried out on silica gel 60 ? (particle size: 40-60 m) from Acros Organics. p.a.-quality solvents from Lach-Ner and Penta were used for elution. A CombiFlash? Rf+ from Teledyne ISCO was used for flash column chromatography. .sup.1H- and .sup.13C-NMR spectra were measured in a Bruker Avance III? HD 400 MHz NMR system equipped with a Prodigy cryo-probe or in a Bruker Avance III? HD 500 MHz Cryo. Analytical HPLC-MS measurements were performed either in an LCMS-2020 system from Shimadzu equipped with a Luna? C18(2) column (3 ?m, 100 A, 100?4.6 mm), CORTECS column (C18 from Waters) or in an HPLC-MS Infinity 1260 system equipped with a 6120 Quadrupole LC/MS detector from Agilent Technologies and either a Luna? 5 ?m C18 preparative column (2), 100 ?, 250?21.2 mm (Phenomenex) or Poroshell 120 analytical column, EC-C18 4 ?m, 4.6?100 mm (Agilent Technologies). Automated peptide synthesis was done in a PS3? Peptide Synthesizer, Protein Technologies, Inc.

Example 1: Preparation of methyl (4S,6R)-5-acetamido-6-((1R,2R)-3-(((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)amino)-1,2-dihydroxypropyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylate (TCO-SiaOMe)

[0075] ##STR00004## [0076] 1. Freshly prepared Dowex 50 W?8 in H.sup.+ cycle (2 g) was added to sialic acid (1) (5 g) dissolved in dry MeOH (100 mL), and the mixture was stirred under an argon atmosphere for 30 hours. The resulting clear solution of the product was concentrated under vacuum to yield the methyl ester of sialic acid as an off-white solid. The methyl ester of sialic acid was dissolved in dry pyridine (50 mL), and the mixture was cooled down on ice before paratoluenesulfonyl chloride (3.2 g) was added to the solution in portions. After stirring for 18 hours, the solvent was removed under vacuum and the residue was further dried under high vacuum. The resulting crude product was purified by silica gel column chromatography (using a gradient of MeOH in DCM) to obtain the product (2) as a white solid, which was used without further purification in the next step. [0077] 2. Sodium azide (0.41 g) was added to compound (2) (0.75 g) dissolved in acetone/water mixture (3/1, 12 mL), and the resulting mixture was stirred at 70? C. for 15 hours. After cooling down to room temperature, the solvents were removed under vacuum and the residue was co-evaporated with EtOH and further dried under high vacuum. The crude reaction product was purified by C18 flash column chromatography (using a gradient of CH.sub.3CN in water, both containing 0.05% HCOOH). The product (3) was isolated as a beige solid after lyophilization, and was used without further purification in the next step. [0078] 3. Pd/C (20 mg) was added to compound (3) (0.26 g) dissolved in water (8 mL), and the flask was purged with H.sub.2 gas. The mixture was stirred under an H.sub.2 atmosphere at room temperature for 6 hours. The crude reaction mixture was passed through a pad of celite to remove Pd/C, and the resulting aqueous solution was lyophilized to yield product 4 as a beige solid, which was used without further purification in the next step. 4. TCO-NHS active ester (TCO-N-hydroxysuccinimide, 5, from SiChem, SC-8070) (105 mg) at 0? C. was added to compound (4) (0.15 g) dissolved in dry dimethylformamide (4.5 mL), followed by DIPEA (diisopropylethylamine) (340 ?L). The reaction mixture was stirred at room temperature for 5 hours. The reaction was quenched by the addition of water (0.5 mL), and the reaction mixture was stirred at room temperature for 30 minutes. The solvents were evaporated under reduced pressure, and the residue was purified by C18 flash column chromatography (using a gradient of CH.sub.3CN in water, both containing 0.05% HCOOH). The product TCO-Sia (6) was obtained as a colorless solid after lyophilization (mixture of anomers). .sup.1H NMR (600.1 MHz, MeOD): 0.83-0.91 (m, 1H); 1.13-1.20 (m, 1H); 1.46-1.53 (m, 1H); 1.60-1.68 (m, 1H); 1.69-1.76 (m, 1H); 1.82 (dd, 1H, J.sub.gem=12.8, J.sub.3b,4=11.6); 1.83-1.90 (m, 1H); 1.95-2.07 (s, 6H, CH.sub.3CO); 2.21 (dd, 1H, J.sub.gem=12.0, J.sub.3a,4=4.9); 2.42-2.48 (m, 1H); 3.15-3.32 (m, 1H); 3.39 (bd, 1H, J.sub.7,8=9.5); 3.546, 3.552 (2?dd, 2?1H, J.sub.gem=14.2, J.sub.9a,8=5.3); 3.71-3.76 (m, 1H); 3.84 (t, 1H, J.sub.5,4=J.sub.5,6=10.4); 4.012, 4.014 (2?dd, 2?1H, J.sub.6,5=10.4, J.sub.6,7=1.3); 4.03 (ddd, 1H, J.sub.4,3=11.6, 4.9, J.sub.4,5=10.4); 5.23-5.27 (m, 1H); 5.55 (dd, 1H, J.sub.2,3=16.4, J.sub.2,1=2.2); 5.82-5.89 (m, 1H). .sup.13C NMR (150.9 MHz, CD.sub.3OD): 22.70 (CH.sub.3CO); 25.20, 25.21 (CH.sub.2-7-cyclooctene); 30.09 (CH.sub.2-6-cyclooctene); 36.79 (CH.sub.2-4-cyclooctene); 37.03 (CH.sub.2-5-cyclooctene); 40.99, 41.00 (CH.sub.2-3); 41.63, 41.66 (CH.sub.2-8-cyclooctene); 45.40, 45.46 (CH.sub.2-9); 54.24 (CH-5); 67.89 (CH-4); 70.84, 70.89 (CH-8); 71.49, 71.54 (CH-7); 72.08, 72.10 (CH-6); 75.30, 75.31 (CH-1-cyclooctene); 96.61 (C-2); 132.61, 132.64 (CH-3-cyclooctene); 132.81, 132.85 (CH-2-cyclooctene); 159.08 (OCON); 173.46 (CH.sub.3CO); 174.93 (C-1). [0079] 5. A solution of 1-Hydroxybenzotriazole (HOBt) (8.8 mg in 140 ?L MeOH) and a solution of N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) (12.5 mg in 140 ?L MeOH) at 0? C. were added to compound (6) (20 mg) dissolved in MeOH (140 ?L), and the reaction was stirred at room temperature for 20 min. The crude reaction mixture was diluted with water (1.5 mL), filtered and loaded into a C18 HPLC column, where it was purified (using a gradient of CH.sub.3CN in water, both containing 0.05% HCOOH). The methyl ester of TCO-Sia (TCO-SiaOMe, 7) was isolated as a colorless solid after lyophilization (mixture of anomers). .sup.1H NMR (MeOD, 400 MHz): ?=0.89 (ddd, 1H, J=3.8, 10.4, 17.9, H-6b-cyclooctene), 1.12-1.25 (m, 1H, H-7b-cyclooctene), 1.46-1.59 (m, 1H, H-5b-cyclooctene), 1.62-1.73 (m, 1H, H-7a-cyclooctene), 1.77 (ddd, 1H, J=1.7, 3.4, 13.0, H-8b-cyclooctene), 1.83-1.96 (m, 1H, H-6a-cyclooctene), 1.96-2.16 (m, 5H, CH3CO, H-4b,5a,8a-cyclooctene), 2.24 (dd, 1H, J=4.9, 12.9 Hz, H-3a), 2.47 (d, 1H, J=10.5 Hz, H-4a-cyclooctene), 3.20 (dt, 1H, J=6.6, 13.5, 1H, H-9b), 3.41 (dd, 1H, J=1.4, 8.8 Hz, H-7), 3.58 (dt, 1H, J=3.6, 14.0, H-9a), 3.70-3.84 (m, 1H, H-8), 3.80 (s, 3H, OCH.sub.3), 3.81-3.88 (m, 1H, H-5), 3.99-4.11 (m, 2H, H-4, H-6), 4.59 (s, 1H), 5.27 (s, 1H, H-1-cyclooctene), 5.49-5.62 (m, 1H, H-2-cyclooctene), 5.88 (t, 1H, J=13.5, H-3-cyclooctene). .sup.13C NMR (101 MHz, MeOD) ?=22.70 (CH.sub.3CO); 25.20, (CH.sub.2-7-cyclooctene); 30.09 (CH.sub.2-6-cyclooctene); 36.79 (CH.sub.2-4-cyclooctene); 37.02 (CH.sub.2-5-cyclooctene); 40.68 (CH.sub.2-3); 41.66 (CH.sub.2-8-cyclooctene); 45.38 (CH.sub.2-9); 53.17 (OCH.sub.3); 54.30 (CH-5); 67.89 (CH-4); 70.92, 70.98 (CH-8); 71.52 (CH-7); 72.00, 72.02 (CH-6); 75.30 (CH-1-cyclooctene); 96.68 (C-2); 132.63 (CH-3-cyclooctene); 132.81 (CH-2-cyclooctene); 159.06 (OCON); 171.76 (CH.sub.3CO); 174.99 (C-1).

Example 2: Preparation of ethyl (4S,6R)-5-acetamido-6-((1R,2R)-3-(((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)amino)-1,2-dihydroxypropyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylate (TCO-SiaOEt) and isopropyl (4S,6R)-5-acetamido-6-((1R,2R)-3-(((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)amino)-1,2-dihydroxypropyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylate (TCO-SiaOiPr)

[0080] The ethyl ester (TCO-SiaOEt) and the isopropyl ester (TCO-SiaOiPr) of TCO-Sia were prepared analogously to the procedure described for methyl ester 7 (point 5) but using EtOH or iPrOH as solvents in the reaction.

TCO-SiaOEt (Colorless Solid, 66%)

[0081] .sup.1H NMR (MeOD, 400 MHz): ?=0.83-0.96 (m, 1H, H-6b-cyclooctene), 1.10-1.26 (m, 1H, H-7b cyclooctene), 1.33 (t, 3H, J=7.1 Hz, CH3-ethyl), 1.48-1.59 (m, 1H, H-5b-cyclooctene), 1.60-1.68 (m, OH, H-7a-cyclooctene), 1.68-1.76 (m, 1H, H-8b-cyclooctene), 1.78 (dd, OH, J=1.7, 3.4 Hz, H-3b), 1.83-1.97 (m, 1H, H-6a-cyclooctene), 1.96-2.12 (m, 5H, CH3CO, H-4b,5a,8a-cyclooctene), 2.23 (dd, 1H, J=4.9, 12.9 Hz, H-3a), 2.47 (d, 1H, J=10.5 Hz, H-4a-cyclooctene), 3.20 (dt, 1H, J=7.1, 14.1 Hz, H-9b), 3.42 (dd, 1H, J=1.5, 8.7 Hz, H-7), 3.59 (dt, 1H, J=4.0, 14.2 Hz, H-9a), 3.75 (dt, 1H, J=4.3, 8.3 Hz, H-8), 3.84 (t, 1H, J=10.2 Hz, H-5), 3.99-4.09 (m, 2H, H-4), 4.25 (qd, 2H, J=4.5, 7.1 Hz, H-1a,b-ethyl), 4.59 (s, 1H), 5.25-5.29 (m, 1H, 24 H-1-cyclooctene), 5.57 (dd, 1H, J=2.4, 16.4 Hz, H-2-cyclooctene), 5.82-5.93 (m, 1H, H-3-cyclooctene). .sup.13C NMR (101 MHz, MeOD) ?=14.34 (CH.sub.2CH.sub.3), 22.70 (CH.sub.3CO); 25.20, (CH.sub.2-7-cyclooctene); 30.09 (CH.sub.2-6-cyclooctene); 36.79 (CH.sub.2-4-cyclooctene); 37.03 (CH.sub.2-5-cyclooctene); 40.67 (CH.sub.2-3); 41.66 (CH.sub.2-8-cyclooctene); 45.37 (CH.sub.2-9); 54.32 (CH-5); 62.96 (CH.sub.2CH.sub.3) 67.89 (CH-4); 71.04 (CH-8); 71.53 (CH-7); 72.05, 72.07 (CH-6); 75.30 (CH-1-cyclooctene); 96.60 (C-2); 132.62 (CH-3-cyclooctene); 132.83 (CH-2-cyclooctene); 159.06 (OCON); 171.32 (CH.sub.3CO); 174.99 (C-1).

TCO-SiaOiPr (Colorless Solid, 32%)

[0082] .sup.1H NMR (MeOD, 400 MHz): ?=0.89 (td, J=5.9, 13.5, 1H, H-6b-cyclooctene), 1.12-1.25 (m, 1H, H-7b cyclooctene), 1.23-1.38 (dd, 6H, CH(CH.sub.3).sub.2), 1.45-1.56 (m, 1H, H-5b-cyclooctene), 1.66 (s, 1H, H-7a-cyclooctene), 1.75 (dddd, J=1.8, 3.5, 12.8, 14.8, 1H, H-8b-cyclooctene), 1.89 (td, J=5.4, 11.9, 2H, H-3b, H-6a-cyclooctene), 2.03 (s, 6H, CH3CO, H-4b,5a,8a-cyclooctene), 2.22 (dd, 1H, J=5.0, 12.9 Hz, H-3a), 2.47 (d, 1H, J=10.1 Hz, H-4a-cyclooctene), 3.20 (dt, 1H, J=7.4, 14.4, 1H, H-9b), 3.43 (dd, 1H, J=1.4, 8.3 Hz, H-7), 3.55-3.64 (m, 1H, H-9a), 3.75 (td, 1H, J=3.3, 7.8 Hz, H-8), 3.84 (t, 1H, J=10.3 Hz, H-5), 3.98-4.10 (m, 2H, H-4, H-6), 4.59 (s, 1H), 4.98-5.14 (m, 1H, CH(CH.sub.3).sub.2), 5.27 (s, 1H, H-1-cyclooctene), 5.50-5.62 (m, 1H, H-2-cyclooctene), 5.87 (dd, 1H, J=11.1, 16.2, H-3-cyclooctene). .sup.13C NMR (101 MHz, MeOD) ?=21.77, 21.83 (CH(CH.sub.3).sub.2), 22.69 (CH.sub.3CO); 25.21, (CH.sub.2-7-cyclooctene); 30.10 (CH.sub.2-6-cyclooctene); 36.80 (CH.sub.2-4-cyclooctene); 37.03 (CH.sub.2-5-cyclooctene); 40.65 (CH.sub.2-3); 41.66 (CH.sub.2-8-cyclooctene); 45.34 (CH.sub.2-9); 54.34 (CH-5); 67.95 (CH-4); 70.99 (CH(CH.sub.3).sub.2, 71.26 (CH-8); 71.61 (CH-7); 72.12 (CH-6); 75.30 (CH-1-cyclooctene); 96.56 (C-2); 132.63 (CH-3-cyclooctene); 132.85 (CH-2-cyclooctene); 159.05 (OCON); 171.90 (CH.sub.3CO); 175.02 (C-1).

II. Synthesis of Tetrazine Conjugates

Example 3: Preparation of (S)N.SUP.1.-(18-methyl-15-oxo-16-(4-(4-sulfamoylphenyl)-1H-1,2,3-triazol-1-yl)-4,7,10-trioxa-14-azanonadecyl)-N.SUP.4.-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)succinamide (sulfonamide-PEG3-diPyTz Conjugate)

[0083] ##STR00005## [0084] 1. The starting sulfonamide carboxylic acid (Sulfonamide COOH) (prepared according to: Mocharla et al. Angewandte Chemie International Edition 44 (1): 116, 2005) (6 mg) and 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU, 7.4 mg) were dissolved in dry DMF (0.5 mL), and the solution was cooled down in an ice/water bath. Dipyridyl tetrazine-PEG3-amine (diPyTz-PEG3-NH.sub.2) (prepared according to: Siegl et al. Chemistry a European Journal, 24 (10): 2426, 2018) dissolved in dry DMF (0.25 mL) was then added dropwise to the reagents. The vial with diPyTz-PEG3-NH.sub.2 was washed with additional dry DMF (0.25 mL), and the solution was added to the reagents. Finally, diisopropylethylamine (15 ?L) was added to the reaction mixture. After one hour water (1 mL) was added to quench the reaction, and the solvent was evaporated under reduced pressure. The residue was purified by RP C18 flash column chromatography (using a gradient of CH.sub.3CN in water, both containing 0.05% HCOOH) to yield the product sulfonamide-PEG3-diPyTz conjugate as a reddish solid after lyophilization (10.3 mg). The product was characterized by RP HPLC-MS (FIG. 1).

Example 5: Preparation of Biotin-PEG3-diPy-Tz Conjugate

[0085] ##STR00006##

[0086] To a solution of 4-oxo-4-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)butanoic acid (300 mg, 0.854 mmol) and N-Boc-4,7,10-trioxa-1,13-tridecanediamine (342 mg, 1.07 mmol) in anhydrous DMF (10 mL) was under argon at 0? C. added HATU (406 mg, 1.07 mmol) followed by DIPEA (446 ?L, 0.0793 mmol). The solution was stirred under argon at room temperature for 1 h, concentrated in vacuo and purified by silica gel column chromatography (DCM/MeOH 10:1) to provide the Boc-protected intermediate as colorless oil (530 mg, 80%) which was used directly in the next step.

[0087] Deprotection of the Boc-protected intermediate (150 mg, 0.229 mmol) was carried out in DCM (5 mL) by adding dropwise TFA (0.25 mL) and stirring the mixture at room temperature for 1 h. The deprotected intermediate was concentrated in vacuo, dried under high vacuum and used in the next step.

[0088] The deprotected intermediate was dissolved together with biotin (56.1 mg, 0.229 mmol) in anhydrous DMF (2.5 mL). Then HATU (96.0 mg, 0.252 mmol) and DIPEA (200 ?L, 1.15 mmol) were added under argon at 5? C. and the solution was stirred at room temperature for 20 h. MeOH was added and the mixture was concentrated in vacuo and dried under high vacuum. The crude product was purified by silica gel column chromatography (DCM/MeOH 5:1). Remaining impurities were removed by reverse phase flash column chromatography (CH.sub.3CN/H.sub.2O mixture) to provide Biotin-PEG3-diPy-Tz conjugate as red semisolid (85 mg, 45%).

[0089] .sup.1H NMR (401 MHz, DMSO-d.sub.6): ? 10.63 (s, 1H), 9.05 (dd, J=2.6, 0.7 Hz, 1H), 8.93 (ddd, J=4.7, 1.8, 0.9 Hz, 1H), 8.62 (dd, J=8.7, 0.6 Hz, 1H), 8.59 (dt, J=8.0, 1.1 Hz, 1H), 8.41 (dd, J=8.7, 2.5 Hz, 1H), 8.15 (td, J=7.8, 1.8 Hz, 1H), 7.90 (t, J=5.6 Hz, 1H), 7.75-7.69 (m, 2H), 6.42-6.35 (m, 2H), 4.30 (ddt, J=7.6, 5.2, 1.1 Hz, 1H), 4.12 (ddd, J=7.5, 4.4, 2.0 Hz, 1H), 3.52-3.45 (m, 8H), 3.39 (dt, J=8.6, 6.4 Hz, 4H), 3.12-3.03 (m, 5H), 2.81 (dd, J=12.4, 5.1 Hz, 1H), 2.67 (t, J=7.0 Hz, 2H), 2.57 (d, J=12.4 Hz, 1H), 2.46 (t, J=7.0 Hz, 2H), 2.04 (t, J=7.4 Hz, 2H), 1.64-1.57 (m, 4H), 1.52-1.42 (m, 2H), 1.35-1.22 (m, 4H).

[0090] .sup.13C NMR (101 MHz, DMSO-d.sub.6): ? 171.9, 171.8, 170.9, 163.0, 162.8, 162.7, 150.6, 150.2, 143.7, 141.2, 138.6, 137.8, 126.6, 126.0, 124.9, 124.2, 69.8, 69.5, 68.10, 68.06, 61.1, 59.2, 55.4, 40.1, 35.9, 35.7, 35.2, 31.7, 30.0, 29.41, 29.39, 28.2, 28.0, 25.3.

[0091] HRMS (ESI): m/z calcd. for C.sub.36H.sub.50N.sub.11O.sub.7S [MH].sup.+780.3610, found 780.3613 and m/z calcd. for C.sub.36H.sub.49NaN.sub.11O.sub.7S [M+Na].sup.+802.3429, found 802.3432.

Example 6: Preparation of FITC-MeTz Conjugate

[0092] ##STR00007##

[0093] To a solution of 6-methyl-3-benzyl-tetrazine-amine (TFA salt, 10 mg, 0.0317 mmol) in anhydrous DMF (2.5 mL) was added DIPEA (13.8 ?L, 0.0793 mmol) followed by fluorescein-5-isothiocyanate (12.4 mg, 0.0317 mmol). The reaction mixture was stirred in the dark at room temperature until the starting materials disappeared (TLC in DCM/MeOH 9:1) and then concentrated in vacuo. The crude product was purified by silica gel column chromatography (EtOAc.fwdarw.EtOAc/MeOH 20:1) to provide FITC-MeTz conjugate as red viscous oil which was re-dissolved in CH.sub.3CN/H.sub.2O and lyophilized to give the title compound as red-orange solid (16 mg, 85%).

[0094] The formation of the product was verified by HPLC-MS and HRMS.

[0095] HRMS (ESI): m/z calcd. for C.sub.31H.sub.23N.sub.6O.sub.5S [MH].sup.+591.1445, found 591.1446.

Example 7: Preparation of BDP-MeTz Conjugate

[0096] ##STR00008##

[0097] To a solution of 6-methyl-3-benzyl-tetrazine-amine (TFA salt, 22 mg, 0.070 mmol) in anhydrous DMF (2 mL) was added bodipy NHS active ester (15 mg, 0,035 mmol, prepared according to Journal of Organic Chemistry 2006, 71, 1718) and DIPEA (61 ?L, 0.351 mmol, 5 equiv.) at 0? C. The reaction mixture was stirred in the dark at room temperature until the starting materials disappeared (ca. 2 hours, verified by TLC in AcOEt/Hex/DCM 3:1:1). The reaction mixture was diluted with AcOEt (50 mL), washed with H.sub.2O and brine. The organic phase was dried over MgSO.sub.4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (AcOEt/Hex/DCM 3:1:1) to provide BDP-MeTz conjugate as orange solid (10 mg, 56%).

[0098] HRMS (ESI): m/z calcd. for C.sub.27H.sub.30N.sub.7OBF.sub.2 [M+Na].sup.+540.2465, found 540.2466.

Example 8: Preparation of 7-(Azetidin-1-yl)-4-methyl-3-(5-(6-phenyl-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)-2H-chromen-2-one conjugate (Coumarin-Tz1)

[0099] ##STR00009##

[0100] This compound was prepared following literature procedure (Galeta et al. Chemistry A European Journal 26(44), 9945, 2020).

[0101] .sup.1H NMR (CDCl.sub.3): ? 9.83 (d, J=2.1, 1H), 8.90 (t, J=2.1, 1H), 8.81 (d, J=2.1, 1H), 8.69-8.65 (m, 2H), 7.68-7.60 (m, 3H), 7.51 (d, J=8.8, 1H), 6.37 (dd, J=8.8, 2.3, 1H), 6.28 (d, J=2.3, 1H), 4.05 (t, J=7.4, 4H), 2.52-2.43 (m, 2H), 2.35 (s, 3H).

[0102] .sup.13C NMR (CDCl.sub.3): ? 164.5 (C), 163.1 (C), 161.6 (C), 155.2 (C), 154.9 (CH), 154.2 (C), 150.3 (C), 148.2 (CH), 137.4 (CH), 133.2 (CH), 132.1 (C), 131.7 (C), 129.6 (2?CH), 128.4 (2?CH), 127.7 (C), 126.4 (CH), 117.2 (C), 110.3 (C), 108.2 (CH), 96.9 (CH), 51.8 (2?CH.sub.2), 16.8 (CH.sub.3), 16.6 (CH.sub.2).

[0103] HRMS (ESI): m/z calcd. for C.sub.26H.sub.21N.sub.6O.sub.2 [MH].sup.+449.1721, found: 449.1718.

Example 9: Preparation of 7-(Azetidin-1-yl)-4-methyl-3-(5-(6-pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)-2H-chromen-2-one (Coumarin-Tz2)

[0104] ##STR00010##

[0105] This compound was prepared following literature procedure (Galeta et al. Chemistry A European Journal 26(44): 9945, 2020).

[0106] .sup.1H NMR (CDCl.sub.3): ? 9.90 (d, J=2.1, 1H), 9.14 (d, J=4.9, 2H), 8.96 (t, J=2.1, 1H), 8.84 (d, J=2.1, 1 H), 7.60 (t, J=4.9, 1H), 7.50 (d, J=8.7, 1H), 6.36 (dd, J=8.7, 2.3, 1H), 6.26 (d, J=2.3, 1H), 4.04 (t, J=7.4, 4H), 2.51-2.42 (m, 2H), 2.34 (s, 3H).

[0107] .sup.13C NMR (CDCl.sub.3): ? 163.8 (C), 163.6 (C), 161.5 (C), 159.5 (C), 158.6 (2?CH), 155.5 (CH), 155.2 (C), 154.2 (C), 150.4 (C), 149.0 (CH), 138.1 (CH), 132.2 (C), 127.2 (C), 126.4 (CH), 122.8 (CH), 117.0 (C), 110.2 (C), 108.2 (CH), 96.8 (CH), 51.8 (2?CH.sub.2), 16.8 (CH.sub.3), 16.6 (CH.sub.2).

Example 10: 7-(Azetidin-1-yl)-3-(3-(6-(2-hydroxyethyl)-1,2,4,5-tetrazin-3-yl)phenyl)-4-methyl-2H-chromen-2-one (Coumarin-Tz3)

[0108] ##STR00011##

[0109] This compound was prepared following literature procedure (Galeta et al. Chemistry A European Journal 26(44): 9945, 2020).

[0110] .sup.1H NMR (CDCl.sub.3): ? 8.59-8.55 (m, 1H), 8.52-8.50 (m, 1H), 7.68-7.63 (m, 1H), 7.60-7.56 (m, 1H), 7.47 (d, J=8.7, 1H), 6.34 (dd, J=8.7, 2.3, 1H), 6.26 (d, J=2.3, 1H), 4.27 (t, J=5.9, 2H), 4.01 (t, J=7.3, 4H), 3.60 (t, J=5.9, 2H), 2.77 (bs, 1H), 2.49-2.40 (m, 2H), 2.28 (s, 3H).

[0111] .sup.13C NMR (CDCl.sub.3): ? 168.4 (C), 164.5 (C), 161.8 (C), 154.9 (C), 153.9 (C), 149.3 (C), 136.7 (C), 135.1 (CH), 131.8 (C), 130.3 (CH), 129.5 (CH), 127.4 (CH), 126.2 (CH), 120.7 (C), 110.6 (C), 108.1 (CH), 96.9 (CH), 60.2 (CH.sub.2), 51.9 (2?CH.sub.2), 37.7 (CH.sub.2), 16.7 (CH.sub.3), 16.6 (CH.sub.2).

[0112] HRMS (ESI): m/z calcd. for C.sub.23H.sub.22N.sub.5O.sub.3 [MH].sup.+416.1717, found: 416.1716.

Example 11: Preparation of MeTz-PEG3-PEG3-biotin-PEG3-DBCO Conjugate

[0113] ##STR00012##

[0114] 3,5-bis(prop-2-yn-1-yloxy)benzoic acid (345 mg, 1.5 mmol) and the N-Boc protected diamine (1.2 equiv.) were dissolved in dry DMF and the solution was cooled down in ice/water bath. HATU (1.2 equiv.) and DIPEA (3 equiv.) were added to the solution which was then stirred at room temperature for 1 hour. The reaction mixture was diluted with AcOEt and the organic phase was washed with water and brine. The organic phase was dried over Na.sub.2SO.sub.4, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (gradient of MeOH in DCM from 2 to 4%) to give the desired compound as dense oily solid (763 mg). This intermediate (50 mg) was combined with azido-PEG3-amine (43 mg) and the mixture was dissolved in mixture of tert-butanol/water=2/3 (1 mL). A premixed solution of BTTP ligand (4 mg) and CuSO.sub.4.5H.sub.2O (1.8 mg) in water (100 ?L) was combined with a solution of sodium ascorbate (3.7 mg) in water (100 ?L) and this colorless solution was added to the solution of reactants. Progress of the reaction was followed by LC-MS. After one hour the solvent was removed under reduced pressure and the residue was purified by RP C18 flash column chromatography (using a gradient of CH.sub.3CN in water, both containing 0.05% HCOOH) to yield the bis-amino N-Boc-protected intermediate (50 mg). To solution of this intermediate (45 mg) in dry DMF (1.5 mL) and cooled down in ice/water bath was added biotin-NHS ester (from Merck, 16 mg) dissolved in dry DMF (0.5 mL) followed by DIPEA (10 ?L). The reaction was stirred at room temperature for 30 min after which, another portion of DIPEA (10 ?L) was added followed by the addition of MeTz-NHS ester (from Merck, 15.2 mg). After stirring the reaction for 30 minutes, the solvent was removed under reduced pressure and the residue was purified by RP C18 flash column chromatography (using a gradient of CH.sub.3CN in water, both containing 0.05% HCOOH) to yield the MeTz-PEG3-PEG3-biotin-PEG3-N-Boc amine intermediate (25 mg). To this intermediate (25 mg) dissolved in DCM (2 mL) was added TFA (0.2 mL) and the reaction was stirred at room temperature for 1 hour. The reaction mixture was diluted with DCM (10 mL) and the solvent was removed under reduced pressure and the residue was dried using high vacuum. To this compound (25 mg) dissolved in dry DMF (1.5 mL) cooled down in ice/water bath was added HBTU (7 mg) and DBCO acid (from Conjuprobe, 5.6 mg). The resulting solution was stirred at room temperature under argon atmosphere overnight. The solvent was removed under reduced pressure and the residue was purified by RP C18 flash column chromatography (using a gradient of CH.sub.3CN in water, both containing 0.05% HCOOH) to yield the MeTz-PEG3-PEG3-biotin-PEG3-DBCO as red solid after lyophilization (19 mg). The product was characterized by LC-MS.

[0115] HRMS (ESI): m/z calcd. for C.sub.79H.sub.103N.sub.17O.sub.17NaS [M+Na].sup.+1616.73308, found 1616.73264.

Example 12: Preparation of NRP-NBD-PEG9-HTz Peptide Conjugate

[0116] The following Neuropilin (NRP)-binding peptide (RPARPAR) (prepared according to: Teesalu et al. Proceedings of the National Academy of Sciences, 106 (38): 16157, 2009) with additional Lysine-Alanine linker was assembled using standard Fmoc solid-phase peptide synthesis on HypoGel-4-Hydroxymethylbenzoylamide resin.

##STR00013## [0117] 1. 40 mg of the resin-bound NRP peptide (ca. 0.0136 mmol) was soaked with DMF and washed with DMF. The Fmoc group was deprotected by incubation of the NRP resin-bound peptide with 20% piperidine in DMF (5 mL, 2?20 min), and the resin was washed with DMF, DCM and DMF. [0118] 2. This step was performed to attach the fluorescent dye (NBD) onto the NRP peptide to detect its presence after the modification of the cells. The Fmoc-deprotected NRP peptide on the resin from the previous step was incubated with 4-Chloro-7-nitrobenzofurazan (NBD-Cl) (7 mg) and diisopropylethylamine (DIPEA, 6 ?L) in DMF (1.5 mL) for 60 minutes. The resin was washed with DMF, DCM and DMF, and this step was repeated two more times using the same amounts of reagents and solvents. The resin was then washed with DMF, DCM, DMF, DCM, and was vacuum dried. [0119] 3. The resin-bound peptide was then incubated with a mixture of trifluoroacetic acid/triisopropylsilane/water=90/2/8 (1.5 mL) for one hour. The resin was washed with DCM and the procedure was repeated two more times. [0120] 4. One half of the resin-bound NRP peptide from the previous step was soaked in a dioxane/water mixture and briefly vacuum dried. The resin-bound peptide was then incubated in dioxane (100 ?L), water (500 ?L) and 1 M NaOH solution in water (60 ?L) for 20 min. The resin was spun down in a plastic vial and the liquid phase was neutralized with 1M HCl in water (60 ?L), then the liquid was pipetted off. This cleavage procedure was repeated once again. The combined cleavage liquids were diluted to ca. 5 mL with water containing 0.1% TFA, filtered, and the residue was purified by C18 RP HPLC. Fractions containing the peptide were combined to obtain the desired NRP-NBD-peptide after lyophilization as an orange solid (26 mg). The product was characterized by RP HPLC-MS (FIG. 2).

##STR00014## [0121] 5. The NRP-NBD peptide (8.5 mg) from the previous step was dissolved in HEPES buffer (pH=8.3, 50 mM), and HTz-PEG9-NHS ester (4 mg) (from Conju-Probe, CP-6026) dissolved in DMSO was added to the peptide solution. After 4 hours, the reaction mixture was diluted with water containing 0.05% HCOOH, the liquid was filtered through a syringe filter (0.2 ?m PTFE), and the residue was purified by C18 RP HPLC to obtain the NRP-NBD-PEG9-HTz peptide conjugate as an orange solid after lyophilization (6 mg). The product was characterized by RP HPLC-MS (FIG. 3).

##STR00015##

Example 13: Preparation of FLAG-MeTz Peptide Conjugate

[0122] ##STR00016##

[0123] This conjugate was prepared as previously described in La-Venia et al. Chemistry A European Journal 27(54), 13632, 2021.

Example 14: Preparation of Cy3 Labeled Duplex Oligonucleotide Tetrazine Conjugate (Cy3-Oligonucleotide-HTz)

[0124] 0.176 ?mol of synthetic commercial DNA oligonucleotide modified at the 3 end with Amine-C7 (sequence 5-TTGAATAAGCTGGTAAT-3-[AmC7], SEQ ID NO. 1) was dissolved in 200 ?L of 20 mM HEPES pH 8.3, and combined with 17.6 ?L of 50 mM HTz-peg5-NHS ester (Conju-Probe, cat. no. CP-6025, 5? molar excess). The reaction was incubated for 4 hours, after which time another portion of 50 mM tetrazine (5? molar excess) was added and the reaction was incubated for another 12 hours. Unreacted tetrazine was removed from the reaction by ethanol precipitation. Briefly, the oligonucleotide was precipitated by adding 1 mL of EtOH/3M sodium acetate (9:1 V/V) solution to the conjugation reaction. After vigorous vortexing, the tube was spun down for 20 min at 25000?g/4? C. The precipitated pellet was washed with 500 ?L of cold 70% EtOH (?20? C.), then centrifuged for 20 min at 25000?g/4? C. The washing step was repeated twice. The pellet was then briefly dried in a speedvac and resuspended in H.sub.2O. The presence of tetrazine modification on the oligonucleotide was confirmed by mass spectrometry. Tz-modified oligonucleotide from the previous step was combined with an equal amount of a synthetic commercial complementary oligonucleotide modified at its 5 end with Cy-3 dye. (sequence [Cy3]-5-ATACCAGCTTATTCAATT-3, SEQ ID NO. 2). The reaction was heated to 50? C. for 5 minutes and then cooled down slowly to room temperature to obtain the duplex Cy3-Oligonucleotide-HTz.

Example 15: Preparation of Trastuzumab-HTz Conjugate

[0125] An aliquot of 100 ?L (3.44 nmol) of the humanized Anti-Human HER2, Antibody (Trastuzumab, Med Chem Express, cat. No. HY-P9907, 5 mg/mL, approximately 34.4 ?M) was buffer-exchanged using a Zeba desalting spin column into 50 mM HEPES, pH 8.3. A 5? molar excess of HTz-Peg5-NHS ester (CP-6025, Conju-Probe) was added to this amount of antibody, and the mixture was incubated for 30 min, after which time another aliquot of HTz-Peg5-NHS ester (5? molar excess) was added, and the mixture was incubated for a further 30 minutes. After this time, unconjugated tetrazine was removed by passing the reaction mixture through a pre-equilibrated (PBS) Zeba desalting column to obtain Trastuzumab-HTz conjugate.

Example 16: Preparation of Cetuximab-HTz Conjugate

[0126] An aliquot of 100 ?L (3.44 nmol) of the humanized Anti-Human EGFR, Antibody (Cetuximab, Med Chem Express, cat. No. HY-P9905, 5 mg/mL, approximately 34.4 ?M) was buffer-exchanged using a Zeba desalting spin column into 50 mM HEPES, pH 8.3. A 5? molar excess of HTz-PEG5-NHS ester (CP-6025, Conju-Probe) was added to this amount of antibody, and the mixture was incubated for 30 min, after which time another aliquot of HTz-PEG5-NHS ester (5? molar excess) was added, and the mixture was incubated for a further 30 minutes. After this time, unconjugated tetrazine was removed by passing the reaction mixture through a pre-equilibrated (PBS) Zeba desalting column to obtain Cetuximab-PEG5-HTz conjugate.

Example 17: Preparation of Phycoerythrin-PEG-Tz Conjugates

[0127] 50 ?L of R-phycoerythrin (precipitate4 mg/mL solution in 60% saturated ammonium sulfate, 50 mM potassium phosphate, pH 7.0) was pelleted at 25 000 g/10 min. Supernatant was removed and the pellet was resuspended in 100 ?L of PBS and buffer-exchanged into 150 mM NaCl, 50 mM HEPES pH8.3 using Zeba desalting columns (Thermo). Active ester of methyltetrazine-PEG4-NHS ester (Click Chemistry Tools, 1069-1) was added to the protein in 5? molar excess. After one hour of incubation, another aliquot of methyltetrazine-PEG4-NHS ester (5? molar excess) was added and the reaction was incubated for another hour. After this time unreacted tetrazine was removed using Zeba desalting column giving Phycoerythrin-PEG4-MeTz conjugate.

[0128] The same procedure using HTz-PEG5-NHS ester (Conjuprobe, CP-6025-25 MG) and HTz-PEG9-NHS ester (Conjuprobe, CP-6026) was used for preparation of Phycoerythrin-PEG5-HTz and Phycoerythrin-PEG9-HTz conjugates.

III. Incorporation of TCO-Sia into Cells and Labeling of Cell Surfaces with Tetrazine Conjugates

Cells and Cultivation Conditions:

[0129] All cell lines were obtained from ATCC and cultivated in a humidified atmosphere incubator at 37? C./5% CO.sub.2. Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from the blood of a healthy human donor. All cultivation media were supplemented with antibiotics (Pen-Strep, Sigma, cat. No. P4333). U20S and MDCK cells were incubated in high-glucose DMEM medium (Biowest, Cat. No. L0103) supplemented with 10% fetal bovine serum (Thermo, cat. No. 10270106). Raji and Jurkat cells were cultivated in RPMI 1640 medium (Biowest, Cat. No. L0492, Dutch Modification, with 1 g/L Sodium Bicarbonate and 20 mM HEPES) containing 10% FBS and 1? concentrated Glutamax (Thermo, Cat. No. 35050061). THP-1 cells were cultivated in RPMI 1640 (Dutch modification) containing 10% Ultra low Endotoxin FBS (Biosera, Cat. No. FB-1001) and 1? concentrated Glutamax. NK-92MI cells were cultivated in RPMI 1640 (Dutch modification) containing 12.5% FBS and 12.5% Horse serum (Thermo, cat. No. 26050088) and 1? concentrated Glutamax. For some incubations and microscopy, cells were kept in Leibowitz's L15 medium without phenol red (Thermo, Cat. No. 21083027) containing 10% FBS and antibiotics.

Microscopy and Flow Cytometry:

[0130] Images of live U20S cells were obtained using a Zeiss LSM780 AxioObserver, equipped with a C-Apochromat 40?/1.20 W Korr FCS M27 objective. The excitation wavelength for Cy3 was 561 nm, emission was collected in the 569-630 nm window. The image was exported in grayscale and inverted in ImageJ software. Flow cytometric measurements (FACS) were performed in a BD LSR Fortessa flow cytometer with the following setup, Cy3-excitatory laser 561 nm, mirror 600LP, filter 610/20 nm; NBD-excitatory laser 488 nm, mirror 505LP, filter 530/30 nm. The fluorescence of more than 5000 singlets was recorded each time, the raw data were processed using the software FlowJo (FlowJo, LLC).

Example 18: Incorporation of TCO-Sia and Test of Cell Viability

[0131] The cell viability was evaluated on U20S cells. Cells seeded at a density of 15 000/well were incubated at 37? C./5% CO.sub.2 with TCO-Sia or sialic acid (control) at concentrations ranging from 5 mM to 0 mM for 48 h. The cell metabolic activity was measured by Resazurin/Resorufin assay following the manufacturer's protocol (ThermoFisher Scientific). Briefly: the spent medium was replaced with a fresh one containing a 40 ?M concentration of resazurin, and the cells were incubated for 3 hours at 37? C./5% CO.sub.2. After this time, the fluorescence of the produced resorufin was measured using a Spark well plate reader (TECAN Ltd.) Cell viability was plotted as % of fluorescence of the untreated cells. Total cell mass was then compared using a crystal violet assay. Briefly, the cells used in the resazurin/resorufin assay were fixed with ice-cold methanol for 10 min at ?20? C. Fixed cells were then washed 3? with H.sub.2O and incubated with a 0.1% (w/N) solution of crystal violet for 15 min. Cells were washed after staining 3? with H.sub.2O, and the bound dye was dissolved using methanol. Absorbance was measured at 595 nm using a Thermo Multiscan FC spectrophotometer (FIG. 4).

Example 19: Incorporation of TCO-Sia and Labeling with Fluorescent Cy3-HTz Conjugate for Fluorescence Microscopy and FACS Analysis

[0132] U20S cells were seeded on a 96-well plate (15 000 cells/well). Cells were incubated for 48 h with 1 mM TCO-Sia, after which the cells were washed 3? with DMEM medium (with addition of 10% Fetal bovine serum) and incubated for 30 min with 2.5 ?M Sulfo-Cy3-Tetrazine dye (Cy3-HTz, from BroadPharm, BP-23321) in complete DMEM (10% FBS), washed with L15 medium and photographed under a confocal microscope (FIG. 5A). After that, the same cells were detached from the well plate and brought into suspension by the addition of accutase (Thermo, A1110501), and the cellular fluorescence was measured using a flow cytometer (FIG. 5B).

Example 20: Incorporation of TCO-Sia into Cell Surface of Various Cell Lines and Labeling with Fluorescent Cy3-HTz Conjugate for FACS Analysis

[0133] (Jurkat, Raji, NK92-MI, THP-1 and MDCK) and primary human peripheral blood mononuclear cells (PBMCs). Cells were plated at a concentration of 1 million cells on a 6-well cultivation plate. TCO-Sia was added to a final concentration of 1 mM, and cells were cultivated for 48 h at 37? C./5% CO.sub.2, spun down, and washed twice in 1 mL of L15 medium. After the second wash, cells were resuspended in 1 mL of L15 medium and incubated with 2.5 ?M Cy3-HTz dye (BroadPharm, BP-23321) for 30 min in a rotator at room temperature. The cells were then washed twice in PBS and measured in a flow cytometer (FIG. 6).

Example 21: Incorporation of TCO-Sia and Persistence of the TCO Modification on the Cell Surface

[0134] The experiment was performed with THP1-cells seeded at 800 000/well in a 12-well plate. In the pulse experiment, cells were pulsed with 1 mM TCO-Sia for 24, 48 and 72 h. At each timepoint, the cells were centrifuged at 300 g/3 min, washed once with 1 mL of L15 medium and resuspended in 1 mL of L15 medium containing 2.5 ?M Cy3-HTz (BroadPharm, BP-23321) for 30 min. After this time, cells were washed twice with 1 mL PBS, and a sample of 100 ?L was taken, fixed in 4% formaldehyde, and used in flow cytometry analysis. In the chase experiment, cells were first pulsed for 72 h with 1 mM TCO-Sia, washed and harvested after 24, 48, and 72 hours after the washout of the sugar. Cells from each timepoint were labeled with Cy3-HTz (BroadPharm, BP-23321) as described above, and fluorescence was measured using a flow cytometer (FIG. 7A).

[0135] A similar experiment was performed with NK-92MI cells: 6 million NK-92MI cells were plated in a 6-cm dish in 6 mL of RPMI media (12.5% FBS/12.5% Horse serum) containing 1 mM TCO-Sia and cultivated for 48 h. After this time, cells were washed and split into 6 aliquots and plated on a 12-well plate in fresh medium. After 24, 48, 96 and 120 h, cells were washed once in complete cultivation media and reacted with 2.5 ?M Cy3-HTz (BroadPharm, BP-23321) for 30 min, washed twice with PBS, and fixed in 4% formaldehyde solution. Fluorescence intensity was measured using a flow cytometer (FIG. 7B).

IV. Incorporation of TCO-Sia into NK-92MI Cells and Labeling of their Cell Surface with Tetrazine Conjugates

[0136] In each case, cells were first incubated with 1 mM TCO-Sia for 48 h, washed, and subsequently reacted with the indicated tetrazine conjugate molecule. The presence of the conjugate molecule on the surface of the cells was then detected using various probes (see specific detection method below). The histograms show a clear fluorescence shift in each case when compared to control cells that were incubated with the detection probe, but not with TCO-Sia.

Example 22: Modification of NK-92MI Cells with Sulfonamide-PEG3-diPyTz Conjugate

[0137] 1000 000 NK-92MI cells previously incubated with 1 mM TCO-Sia for 48 h were washed twice with complete cultivation medium and resuspended in 1 mL of complete cultivation medium. Sulfonamide-PEG3-diPyTz conjugate was added to the cell suspension to a final concentration of 2.5 ?M, and cells were incubated for 30 min at room temperature in a rotator. Cells were then washed twice in 1 mL of complete medium and resuspended in PBS. 100 ?L of suspension was taken and incubated with Cy3 modified human carbonic anhydrase II (hCAII-Cy3, 0.3 ?M final concentration; for its preparation, see procedure below) for 15 minutes to verify that the sulfonamide inhibitor was present on the cell surface. Cells were washed twice in 1 mL PBS, fixed with 4% formaldehyde, and the cellular fluorescence was measured using a flow cytometer (FIG. 8A). Control cells were processed in the same way, except that they were not treated with the Sulfonamide-PEG3-diPyTz conjugate.

[0138] Preparation of human carbonic anhydrase II Cy3 conjugate (hCAII-Cy3): 100 ?L of recombinant human Carbonic anhydrase II (hCAII, 34 ?M, kindly provided by P. Rezacova's group, IOCB, Prague) was transferred into 0.2 M bicarbonate buffer, pH 9, using aZeba spin desalting column (Thermo cat. no. 89882). A 10? (molar) excess of Cy3-NHS ester (Lumiprobe cat. no. 11020) was added to the hCAII solution, and the mixture was incubated for 1 hour at room temperature with constant shaking. After that, the sample was spun down at 11000 RPM/1 minute to pellet any insoluble material. To remove the unreacted dye, the reaction mixture was passed through a Zeba desalting column preconditioned in 1? concentrated phosphate-buffered saline (PBS) to obtain hCAII-Cy3 conjugate.

Example 23: Modification of NK-92MI Cells with Cy3-Oligonucleotide-HTz Conjugate

[0139] 1000 000 NK-92MI cells previously incubated with 1 mM TCO-Sia for 48 h were washed twice with complete cultivation medium and resuspended in 0.5 mL of complete cultivation medium containing 2.5 ?M Cy3-Oligonucleotide-HTz conjugate. Cells were incubated on a rotating wheel at room temperature for 30 minutes, washed twice with PBS, and a 50 ?l aliquot was fixed with 4% formaldehyde. Cellular fluorescence was measured using a flow cytometer (FIG. 8B). The fluorescence intensity of oligonucleotide-modified cells was compared to the control sample processed in the same way except for prior incubation with TCO-Sia.

Example 24: Modification of NK-92MI Cells with Fluorescent NRP-NBD-PEG9-HTz Peptide Conjugate

[0140] 1000 000 NK-92MI cells previously incubated with 1 mM TCO-Sia for 48 h were washed twice with complete cultivation medium to remove the unincorporated TCO, and resuspended in 1 mL of complete cultivation medium containing 2.5 ?M NRP-NBD-PEG9-HTz peptide conjugate. Cells were incubated on a rotating wheel at room temperature for 30 minutes, washed twice with PBS, and a 100 ?l aliquot was fixed with 4% formaldehyde. The cellular fluorescence of the fixed cells was measured using a flow cytometer (FIG. 8C). This conjugate can be detected by fluorescence due to the presence of the NBD dye. The fluorescence intensity of the modified cells was compared to control sample cells processed in the same way except for prior incubation with TCO-Sia.

Example 25: Modification of NK-92MI Cells with Trastuzumab-HTz Conjugate

[0141] 1000 000 NK-92MI cells previously incubated with 1 mM TCO-Sia for 48 h were washed twice with complete cultivation medium to remove the unincorporated TCO-Sia, and cells were resuspended in 0.5 mL of complete cultivation medium containing 0.34 ?M Trastuzumab-HTz conjugate. Cells were incubated on a rotating wheel at room temperature for 30 minutes, and were washed twice with PBS. To detect the antibody immobilized on the surface of NK-92MI cells, a 100 ?l aliquot was incubated with a goat anti-human secondary antibody (Thermo, cat. No. SA5-10135, DyLight 550 conjugate, 0.5 ?g/mL) for 15 min, washed twice, and the cells were fixed with 4% formaldehyde. The cellular fluorescence of the fixed cells was measured using a flow cytometer (FIG. 8D). The fluorescence intensity of antibody-modified cells was compared to the control sample processed in parallel but lacking the TCO-Sia modification.

Example 26: Incorporation of TCO-Sia into Cell Surface and Labeling with Various Fluorophore Tz Conjugates (Microscopy Analysis)

[0142] U2OS cells were seeded on a 96-well plate (15 000 cells/well). Cells were incubated for 48 h with 1 mM TCO-Sia, after which the cells were washed 3? with DMEM medium (with addition of 10% Fetal bovine serum) and incubated for 30 min with or without (control cells) 2.5 ?M tetrazine conjugates in complete DMEM (10% FBS), washed with L15 medium and photographed under a confocal microscope. The following compounds were used: TAMRA-Pyrimidyl-Tz (Jena Bioscience, CLK-097), Cy3-HTz (BroadPharm, BP-23321), FITC-MeTz, ATTO-488-HTz (Jena Bioscience, CLK-010-02), AF-488-HTz (Click Chemistry Tools, 1361-1), BDP-MeTz, Coumarin-Tz2 and Coumarin-Tz1 (FIG. 9).

Example 27: Incorporation of TCO-Sia into Cell Surface and Labeling with MeTz-PEG3-PEG3-Biotin-PEG3-DBCO Conjugate Followed by Double Labeling with Fluorophore and Protein

[0143] PC3 cells were seeded on a 96-well plate cultivation dishes (15000 cells/well) and incubated for 48 h with 1 mM TCO-Sia in high glucose DMEM medium supplemented with 10% FBS (Thermo) and 0.1 mg/mL of penicillin-streptomycin at 37? C./5% CO.sub.2, after which the cells were washed 3? with DMEM medium (with addition of 10% Fetal bovine serum) and incubated for 1 hour with Cy3-azide (Lumiprobe, A1330) to label the DBCO moiety of the MeTz-PEG3-PEG3-biotin-PEG3-DBCO conjugate on the cell surface. Cells were washed with L15 medium, and photographed under a confocal microscope. The, cells were incubated with streptavidin (Thermo 12-4317-87), washed with L15 medium and photographed under a confocal microscope (FIG. 10).

Example 28: Incorporation of TCO-Sia into Cell Surface and Labeling with FLAG-MeTz Peptide Conjugate

[0144] HeLa cells were seeded on a 96-well plate cultivation dishes (2?10.sup.4 cells/well) and incubated for 48 h with 1 mM TCO-Sia in high glucose DMEM medium supplemented with 10% FBS (Thermo) and 0.1 mg/mL of penicillin-streptomycin at 37? C./5% CO.sub.2, after which the cells were washed 3? with DMEM medium (with addition of 10% Fetal bovine serum) and incubated for 1 hour with or without (control cells) 5 ?M FLAG-MeTz peptide conjugate (prepared as previously described in La-Venia et al. Chemistry A European Journal 27(54), 13632, 2021) in complete DMEM (10% FBS), washed with L15 medium, incubated with anti-FLAG antibody (Thermo, MA1-91878-D488) and photographed under a confocal microscope. The nuclei were stained with Hoechst nuclear stain (FIG. 11).

Example 29: Incorporation of TCO-Sia into Cell Surface and Labeling with Biotin-PEG3-diPy-Tz Conjugate

[0145] HeLa cells were seeded on a 96-well plate cultivation dishes (2?10.sup.4 cells/well) and incubated for 48 h with 1 mM TCO-Sia in high glucose DMEM medium supplemented with 10% FBS (Thermo) and 0.1 mg/mL of penicillin-streptomycin at 37? C./5% CO.sub.2, after which the cells were washed 3? with DMEM medium (with addition of 10% Fetal bovine serum) and incubated for 30 min with or without (control cells) 2.5 ?M Biotin-PEG3-diPy-Tz conjugate in complete DMEM (10% FBS). Cells were then washed with L15 medium, incubated with streptavidin (Thermo 12-4317-87) and photographed under a confocal microscope. The nuclei were stained with Hoechst nuclear stain (FIG. 12).

Example 30: Incorporation of TCO-Sia into Cell Surface and Labeling with PE-Tetrazine Conjugates

[0146] U2OS cells were seeded on a 96-well plate (15 000 cells/well). Cells were incubated for 48 h with 1 mM TCO-Sia, after which the cells were washed 3? with DMEM medium (with addition of 10% Fetal bovine serum) and incubated for 2 hours with 5 ?M PE-PEG4-MeTz or 5 ?M PE-PEG5-HTz or 5 ?M PE-PEG9-HTz conjugates or with 5 ?M unconjugated Phycoerythrin as a control in complete DMEM (10% FBS). Cells were then washed with L15 medium and photographed under a confocal microscope (FIG. 13).

INDUSTRIAL APPLICABILITY

[0147] The provided procedure can be used to attach different active moieties to the cell surface of various cell lines. The additional active moiety provides the cells with a new function, property, or alters the cell surface structure. Depending on the intended application, the resulting cell-surface modified cells can be used in bioimaging, for the construction of biomaterials, in regenerative medicine, as drug delivery systems, in immunotherapy and other biomedical applications. The preferred examples include the use of the cell surface-modified cells to direct therapeutic cells to a specific location in the body (e.g. immune cells to cancer), the use of the modified cells as drug delivery carriers, the use of the modified cells as carriers of fluorescent, radiolabel or other biophysical imaging probes, the protection of cells by the active moiety from unwanted interactions, or for promoting cellular interactions by the active moiety.