BATTISTI UMBERTO MARIA (DK)
POULIE CHRISTIAN BERNARD MATTHIJS (SE)
WO2020242948A1 | 2020-12-03 | |||
WO2012121746A2 | 2012-09-13 | |||
WO2017059397A1 | 2017-04-06 | |||
WO2012121746A2 | 2012-09-13 | |||
WO2017059397A1 | 2017-04-06 | |||
WO2020242948A1 | 2020-12-03 |
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CLAIMS 1. Method for providing a labeled single isomeric chemical entity targeting vector comprising: a) labeling a first chemical entity having inverse electron demand Diels- Alder cycloaddition reactivity and being conjugated to a pharmaceutic agent, an imaging agent, or a therapeutic agent, with a labeling agent; wherein the first chemical entity is selected from the group consisting of a symmetrical substituted diene wherein at least one of the symmetry planes pass through the nitrogen-nitrogen bonds of at least one tetrazine ring, an enantiomerically pure dienophile, and a cis,5,6-disubstituted dienophile; and b) ligating the labeled first chemical entity obtained in step a) with a second chemical entity having complementary inverse electron demand Diels- Alder cycloaddition reactivity and being conjugated to a targeting vector; wherein the second chemical entity is selected from the group consisting of a symmetrical substituted diene wherein at least one of the symmetry planes pass through the nitrogen-nitrogen bonds of at least one tetrazine ring, an enantiomerically pure dienophile, and a cis,5,6-disubstituted dienophile, wherein the reaction kinetics for the inverse electron demand Diels-Alder cycloaddition between the first and second chemical entities has a minimum rate constant of 500 M-1 s-1 in phosphate-buffered saline at 25 °C, determined by stopped-flow spectrophotometry, and wherein the first and second chemical entities having complementary inverse electron demand Diels-Alder cycloaddition reactivity being ligated are selected from one of the following combinations: i) a symmetrical substituted diene, wherein at least one of the symmetry planes pass through the nitrogen-nitrogen bonds of at least one tetrazine ring, and an enantiomerically pure dienophile ii) a symmetrical substituted diene, wherein at least one of the symmetry planes pass through the nitrogen-nitrogen bonds of at least one tetrazine ring, and a cis,5,6-disubstituted dienophile; and c) oxidizing the ligated labeled targeting vector obtained from step b) at a temperature ranging from 15 °C to 50 °C for up to 60 minutes by adding from 1 to 100 equivalents of chloranil, fluoranil, DDQ or NaNO2.. 2. Method according to claim 1 wherein the symmetrical substituted diene is a symmetrical tetrazine and the dienophile is a trans-cycloheptene (TCH), a trans- cyclooctene (TCO) or a trans-cyclononene (TCN) derivative. 3. Method according to claim any of the previous claims wherein the labeling agent in step a) is a radionuclide or a stable isotope of a corresponding element. 4. Method according to claim 3 wherein the labeling agent is selected from 1H, 2H, 3H, 11C, 12C, 13C, 14C, 13N, 14N, 15N, 18F, 19F, 123I, 124I,125I, 127I, 131I, 15O, 16O, 17O, 18O, 43Sc, 44Sc, 45Sc, 45Ti, 46Ti, 47Ti, 48Ti, 49Ti, 50Ti, 55Co, 58mCo, 59Co, 60Cu, 61Cu, 63Cu, 64Cu, 65Cu, 67Cu, 67Ga, 68Ga, 69Ga, 71Ga, 76Br, 77Br, 79Br, 80mBr, 81Br, 72As, 75As, 86Y, 89Y, 90Y, 89Zr, 90Zr, 91Zr, 92Zr, 94Zr, 149Tb, 152Tb, 159Tb, 161Tb, 111In, 113In, 114mIn, 115mIn, 175Lu, 177Lu, 185Re, 186Re, 188Re, 201Tl, 203Tl, 205Tl, 206Pb, 207Pb,208Pb,212Pb, 209Bi, 212Bi, 2 13Bi, 31P, 32P, 33P, 32S, 35S, 45Sc, 47Sc, 84Sr, 86Sr, 87Sr, 88Sr, 89Sr, 165Ho, 166Ho, 156Dy, 158Dy, 160Dy, 161Dy, 162Dy, 163Dy, 164Dy, 165Dy , 227Th, 232Th, 51Cr, 52Cr, 53Cr, 54Cr, 73Se, 74Se, 75Se, 76Se, 77Se, 78Se, 80Se, 82Se, 103Rh,103mRh, 119Sb, 121Sb, 123Sb, 135La, 138La, 139La, 162Er, 164Er, 165Er, 166Er, 167Er, 168Er, 170Er, 193mPt, 195mPt, 192Pt, 194Pt, 195Pt, 196Pt, 198Pt. 5. Method according to any of the previous claims, wherein the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. 6. Method according to any of the previous claims, wherein the oxidant is solid phase supported. 7. Method according to claim 1 step b) wherein the symmetrical substituted diene is a tetrazine with formula Tz1: wherein R and R1 is , wherein the curly sign indicates the link to the tetrazine; and where R2 is -H or (i) an isotope labeling agent directly connected to the aromatic ring; or (ii) an isotope labeling agent connected to the aromatic ring via a linker, said linker being selected from the group consisting of -(CH2)n, - LO(CH2)n, -LNH(CH2)n, -LCONH(CH2)n, -LNHCO(CH2)n, where L is -(CH2)m or - (CH2CH2O)m , where n and m are independently selected from 1-25; or (iii) an isotope labeling agent that is chelated through a chelator selected from: 1,4,7,10- tetraazacyclododecane-N,N',N',N"-tetraacetic acid (DOTA), N,N'-bis(2-hydroxy-5- (carboxyethyl)benzyl)ethylenediamine N,N'-diacetic acid (HBED-CC), 14,7- triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4.7-bis(carboxymethyl)-1,4,7- triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecan-1- yl)pentanedioic acid (DOTAGA), 14,7- triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane-1-methyl(2- carboxyethyl)phosphinic acid-4,7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9,15-tetraazabicyclo9.3.1.pentadeca-1 (15),11,13-triene-3,6,9- triacetic acid (PCTA), N'-(5-acetyl (hydroxy)aminopentyl-N-(5-(4-(5- aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyl- diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10- triazacyclododecane-4,7,10-triacetic acid (OXO-Do3A), p-isothiocyanatobenzyl- DTPA (SCN-BZ-DTPA), 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), 2-(p- isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), and 1-(2)-methyl-4- isocyanatobenzyl-DTPA (MX-DTPA) connected to the aromatic ring through a linker, said linker being selected from the group consisting of -(CH2)n, -LO(CH2)n, - LNH(CH2)n, -LCONH(CH2)n, -LNHCO(CH2)n, where L is -(CH2)m or -(CH2CH2O)m, and n and m are independently selected from 1-25; wherein, when R2 is either (i) or (ii) the isotope labeling agent is selected from the group consisting of: 1H, 2H, 3H, 11C, 12C, 13C, 14C, 13N, 14N, 15N, 18F, 19F, 123I, 124I,125I, 127I, 131I, 15O, 16O, 17O, 18O, 43Sc, 44Sc, 45Sc, 45Ti, 46Ti, 47Ti, 48Ti, 49Ti, 50Ti, 55Co, 58mCo, 59Co, 60Cu, 61Cu, 63Cu, 64Cu, 65Cu, 67Cu, 67Ga, 68Ga, 69Ga, 71Ga, 76Br, 77Br, 79Br, 80mBr, 81Br, 72As, 75As, 86Y, 89Y, 90Y, 89Zr, 90Zr, 91Zr, 92Zr, 94Zr, 149Tb, 152Tb, 159Tb, 161Tb, 111In, 113In, 114mIn, 115mIn, 175Lu, 177Lu, 185Re, 186Re, 188Re, 201Tl, 203Tl, 205Tl, 206Pb, 207Pb,208Pb,212Pb, 209Bi, 212Bi, 2 13Bi, 31P, 32P, 33P, 32S, 35S, 45Sc, 47Sc, 84Sr, 86Sr, 87Sr, 88Sr, 89Sr, 165Ho, 166Ho, 156Dy, 158Dy, 160Dy, 161Dy, 162Dy, 163Dy, 164Dy, 165Dy , 227Th, 232Th, 51Cr, 52Cr, 53Cr, 54Cr, 73Se, 74Se, 75Se, 76Se, 77Se, 78Se, 80Se, 82Se, 103Rh,103mRh, 119Sb, 121Sb, 123Sb, 135La, 138La, 139La, 162Er, 164Er, 165Er, 166Er, 167Er, 168Er, 170Er, 193mPt, 195mPt, 192Pt, 194Pt, 195Pt, 196Pt, 198Pt, and wherein X and Y are independently selected from: -CH and -N- ; and wherein R3 is independently selected from H or a moiety selected from the group consisting of a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, amine, a substituted amine with 1-5 polyethylene glycol unit(s), a −(O−CH2−CH2)n−OCH2-COOH, and n is selected from 1-5; or Methyl, Ethyl, Propyl, optionally substituted heteroaryl, and optionally substituted arylalkyl; wherein optionally substituted in relation to said substituted amine means one or more substituents selected from, a halogen, a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, amine, (C1-C10) alkyl, (C2-C10)alkenyl, (C2-C10)alkynyl, (C1-C10)alkylene, (C1-C10)alkoxy, (C2-C10)dialkylamino, (C1-C10)alkylthio, (C2- C10)heteroalkyl, (C2-C10)heteroalkylene, (C3-30 C10)cycloalkyl, (C3- C10)heterocycloalkyl, (C3-10)cycloalkylene, (C3-C10)heterocycloalkylene, (C1- C10)haloalkyl, (C1-C10)perhaloalkyl, (C2-C10)-alkenyloxy, (C3-C10)-alkynyloxy, aryloxy, arylalkyloxy, heteroaryloxy, heteroarylalkyloxy, (C1-C6)alkyloxy-(C1- C4)alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted arylalkyl; wherein optionally substituted means one or more substituents selected from a halogen, a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, amine, a substituted amine with 1-5 polyethylene glycol unit(s), a −(O−CH2−CH2)n−OCH2-COOH, and n is selected from 1-5; or H, Methyl, Ethyl, Propyl, optionally substituted heteroaryl, and optionally substituted arylalkyl; wherein optionally substituted in relation to said substituted amine means one or more substituents selected from a halogen, a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, and amine; and wherein R and R1 are identical or differs only in the isotope number of the labelling agent. 8. Method according to claim 7 wherein the symmetrical tetrazine is selected from: 9. Method according to claim 1 ligating combination i) wherein the enantiomerically pure dienophile is a trans-cycloheptene (TCH), trans-cyclooctene (TCO) or a trans-cyclononene (TCN) derivative selected from: wherein X is O, NH, S, or CH2; and wherein the linker is selected from the group comprising: -(CH2)n- (CH2)nNH, (CH2)nCO, (CH2)nO, (CH2CH2O)n (CH2CH2O)nCH2CH2NH, (CH2CH2O)nCH2CH2CO, -CO(CH)2- CO(CH2)nNH, CO(CH2)nCO, CO(CH2)nO, CO(CH2CH2O)n CO(CH2CH2O)nCH2CH2NH, CO(CH2CH2O)nCH2CH2CO, COO(CH)2- COO(CH2)nNH, COO(CH2)nCO, COO(CH2)nO, COO(CH2CH2O)n COO(CH2CH2O)nCH2CH2NH, COO(CH2CH2O)nCH2CH2CO, CONH(CH)2- CONH(CH2)nNH, CONH(CH2)nCO, CONH(CH2)nO, CONH(CH2CH2O)n CONH(CH2CH2O)nCH2CH2NH, CONH(CH2CH2O)nCH2CH2CO, -CONHPhCO, -COOPhCO, -COPhCO, CONHCHMCO, (CH2)nNHCHMCO, (CH2)nOCONHCHMCO, (CH2)nNHCHMCO, (CH2)nNHCOCHMNH, (CH2)OCOCHMNH, (CH2CH2O)nCH2CH2NHCHMCO, (CH2CH2O)nCH2CH2CONHCHMCO, (CH2CH2O)nCH2CH2NHCHMCO, (CH2CH2O)nCH2CH2NHCOCHMNH, (CH2CH2O)nCOCHMNH, where n is 0-25 and where M is a side chain selected from the group consisting of side chains of the natural amino acids: H, CH3, CH2SH, CH2COOH, CH2CH2COOH, CH2C6H5, CH2C3H3N2, CH(CH3)CH2CH3, (CH2)4NH2, CH2CH(CH3)2, CH2CH2SCH3, CH2CONH2, (CH2)4NHCOC4H5NCH3, CH2CH2CH2, CH2CH2CONH2, (CH2)3NH-C(NH)NH2, CH2OH, CH(OH)CH3, CH2SeH, CH(CH3)2, CH2C8H6N, CH2C6H4OH; and where targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. 10. Method according to claim 1 ligating combination ii) wherein the cis,5,6-disubstituted dienophile is trans-cyclooctene (TCO) derivative selected from: Wherein X is -O, NH, S, or CH2; -and wherein the linker is selected from the group comprising: -(CH2)n- (CH2)nNH, (CH2)nCO, (CH2)nO, (CH2CH2O)n (CH2CH2O)nCH2CH2NH, (CH2CH2O)nCH2CH2CO, -CO(CH)2- CO(CH2)nNH, CO(CH2)nCO, CO(CH2)nO, CO(CH2CH2O)n CO(CH2CH2O)nCH2CH2NH, CO(CH2CH2O)nCH2CH2CO, COO(CH)2- COO(CH2)nNH, COO(CH2)nCO, COO(CH2)nO, COO(CH2CH2O)n COO(CH2CH2O)nCH2CH2NH, COO(CH2CH2O)nCH2CH2CO, CONH(CH)2- CONH(CH2)nNH, CONH(CH2)nCO, CONH(CH2)nO, CONH(CH2CH2O)n CONH(CH2CH2O)nCH2CH2NH, CONH(CH2CH2O)nCH2CH2CO, -CONHPhCO, -COOPhCO, -COPhCO, CONHCHMCO, (CH2)nNHCHMCO, (CH2)nOCONHCHMCO, (CH2)nNHCHMCO, (CH2)nNHCOCHMNH, (CH2)OCOCHMNH, (CH2CH2O)nCH2CH2NHCHMCO, (CH2CH2O)nCH2CH2CONHCHMCO, (CH2CH2O)nCH2CH2NHCHMCO, (CH2CH2O)nCH2CH2NHCOCHMNH, (CH2CH2O)nCOCHMNH, where n is 0-25 and where M is a side chain selected from the group consisting of side chains of the natural amino acids: H, CH3, CH2SH, CH2COOH, CH2CH2COOH, CH2C6H5, CH2C3H3N2, CH(CH3)CH2CH3, (CH2)4NH2, CH2CH(CH3)2, CH2CH2SCH3, CH2CONH2, (CH2)4NHCOC4H5NCH3, CH2CH2CH2, CH2CH2CONH2, (CH2)3NH-C(NH)NH2, CH2OH, CH(OH)CH3, CH2SeH, CH(CH3)2, CH2C8H6N, CH2C6H4OH; and where the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. 11. Method according to claim 1, wherein the symmetrical substituted diene is obtained from a precursor selected from: wherein X is CH or N. 12. Method according to claim 1, wherein the enantiomerically pure dienophile targeting vector in ligation combination i) is obtained from a precursor selected from: wherein the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule; 13. Method according to claim 1, wherein the cis,5,6-disubstituted dienophile targeting vector in ligation combination ii) is obtained from a precursor selected from: wherein the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. |
Scheme 2. Explicit example of current state-of-the-art tetrazine ligation. (all isomeric forms are omitted, see Scheme 1 for overview of the isomeric forms. Multiple isomers (at least 6 isomers) can be formed in this reaction, which all have very similar polarities and are accordingly very difficult to separate for instance by HPLC. Herein, we describe a method, which allows for the use of a labeled first chemical entity such as radiolabelled symmetrically tetrazine-based synthons in the radiolabelling of dienophils, such as trans-cycloheptenes (TCH), trans-cyclooctene (TCO) and trans-cyclononene (TCN) functionalized vectors and vice versa, which upon subsequent chemical oxidation yields a single final compound within short time, such as within 0 - 60 minutes (Figure 5). The method comprises two steps: a ligation step followed by an oxidation step.. The final single isomeric form of the radiolabeled diene-dienophile targeting vector which is the outcome of the method can be reached via different alternative starting points for the first step in the method i.e. the ligation step (referred to as combination i), ii), respectively) followed by the second step which is an oxidation step. In a preferred embodiment, the method for providing a labeled single isomeric chemical entity targeting vector comprises: a) labeling a first chemical entity having inverse electron demand Diels-Alder cycloaddition reactivity and being conjugated to a pharmaceutic agent, an imaging agent, or a therapeutic agent, with a labeling agent; wherein the first chemical entity is selected from the group consisting of a symmetrical tetrazine wherein at least one of the symmetry planes pass through the nitrogen- nitrogen bonds of at least one tetrazine ring, an enantiomerically pure trans- cycloheptene (TCH), an enantiomerically pure trans-cyclooctene (TCO), an enantiomerically pure trans-cyclononene (TCN), and a cis,5,6-disubstituted trans-cyclooctene (TCO), b) ligating the labeled first chemical entity obtained in step a) with a second chemical entity having complementary inverse electron demand Diels-Alder cycloaddition reactivity and being conjugated to a targeting vector; wherein the second chemical entity is selected from the group consisting of a symmetrical tetrazine wherein at least one of the symmetry planes pass through the nitrogen-nitrogen bonds of at least one tetrazine ring, an enantiomerically pure trans-cycloheptene (TCH), an enantiomerically pure trans-cyclooctene (TCO), an enantiomerically pure trans-cyclononene (TCN and a cis,5,6-disubstituted trans-cyclooctene (TCO), wherein the reaction kinetics for the inverse electron demand Diels-Alder cycloaddition between the first and second chemical entities has a minimum rate constant of 500 M -1 s -1 in PBS at 25 °C, determined by stopped-flow spectrophotometry, and wherein the first and second chemical entities having complementary inverse electron demand Diels-Alder cycloaddition reactivity being ligated are selected from one of the following combinations: i) a symmetrical tetrazine and an enantiomerically pure TCO, TCN or TCH a symmetrical tetrazine and a cis,5,6-disubstituted TCO c) oxidizing the ligated labeled targeting vector obtained from step b) at a temperature ranging from 20 °C to 30 °C for up to 20 minutes by adding from 1 to 100 equivalents of chloranil, fluoranil, DDQ or NaNO 2 . Ligation combination i): In ligation combination i), the starting entities to be ligated is a symmetrical substituted diene wherein at least one of the symmetry planes pass through the nitrogen-nitrogen bonds of at least one tetrazine ring, such as a symmetrical tetrazine, and an enantiomerically pure dienophile, such as an enantiomerically pure TCH, TCO, or TCN. Using a symmetrical substituted diene, such as a symmetrical tetrazine, in combination with an enantiomerically pure dienophile, such as an enantiomerically pure TCH, TCO, or TCN, reduces the number of formed click-products, by eliminating all enantiomeric products. The formed tautomeric dihydropyridazines will be subsequently oxidized to the corresponding pyridazine, resulting in a single product. The ‘R’ substituents on the diene, such as a tetrazine, employed in this method will be functionalized on one side with 18 F or 123 I, 124 I, 125 I, 131 I and the opposite site with 1 9 F, or 127 I, respectively. Due to the fact that both these isotopes of either fluorine or iodine, respectively, are chemically identical, the single product, formed via this method, is still considered, from a chemical perspective, a single entity. The below scheme 2 is an illustration of a ligation in accordance with ligation combination i) here exemplified in using a symmetrical tetrazine and a TCO conjugated to a targeting vector: Scheme 3. The targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. Ligation combination ii): In ligation combination ii), the starting entities to be ligated is either a symmetrical substituted diene wherein at least one of the symmetry planes pass through the nitrogen-nitrogen bonds of at least one tetrazine ring, such as a symmetrical tetrazine, and a cis,5,6-disubstituted dienophile, such as a cis,5,6-disubstituted TCO. Using a cis,5,6-disubstituted dienophile, such as a cis,5,6-disubstituted TCO, and a symmetrical tetrazine, reduces the number of formed click-products, by eliminating all enantiomeric products. The formed tautomeric entities, such as dihydropyridazines, will be subsequently oxidized to the corresponding single isomeric form, such as a pyridazine, resulting in a single product. The ‘R’ substituents on the dienes employed in this method will typically be functionalized with 18 F, 123 I, 124 I, 125 I, or 131 I. The below scheme 3 is an illustration of a ligation in accordance with ligation combination ii) here exemplified in using an unsymmetrical tetrazine and a TCO conjugated to a targeting vector: Scheme 4. The targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. Symmetrical tetrazines of formula Tz1 are examples of preferred dienes suitable for both ligation combination i) and ii), respectively: wherein R and R 1 is wherein the curly sign indicates the link to the tetrazine; and where R 2 is -H or (i) an isotope labeling agent directly connected to the aromatic ring; or (ii) an isotope labeling agent connected to the aromatic ring via a linker, said linker being selected from the group consisting of (CH 2 ) n , -LO(CH 2 ) n , -LNH(CH 2 ) n , - LCONH(CH 2 ) n , -LNHCO(CH 2 ) n , where L is -(CH 2 ) m or -O(CH 2 CH 2 O) m , where n and m are independently selected from 1-25; or (iii) an isotope labeling agent that is chelated through a chelator selected from: 1,4,7,10-tetraazacyclododecane- N,N',N',N"-tetraacetic acid (DOTA), N,N'-bis(2-hydroxy-5- (carboxyethyl)benzyl)ethylenediamine N,N'-diacetic acid (HBED-CC), 14,7- triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4.7-bis(carboxymethyl)-1,4,7- triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10- tetraazacyclododecan-1- yl)pentanedioic acid (DOTAGA), 14,7-triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane-1-methyl(2-carboxyethyl)phosphinic acid-4,7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9,15- tetraazabicyclo9.3.1.pentadeca-1 (15),11,13-triene-3,6,9- triacetic acid (PCTA), N'- (5-acetyl (hydroxy)aminopentyl-N-(5-(4-(5- aminopentyl)(hydroxy)amino-4- oxobutanoyl)amino)pentyl-N- hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyl- diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazacyclododecane- 4,7,10-triacetic acid (OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1- (p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), 2-(p-isothiocyanatobenzyl)-4- methyl-DTPA (1M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA) connected to the aromatic ring through a linker, said linker being selected from the group consisting of (CH 2 ) n , -LO(CH 2 ) n , -LNH(CH 2 ) n , -LCONH(CH 2 ) n , -LNHCO(CH 2 ) n , where L is -(CH 2 ) m or -O(CH 2 CH 2 O) m, and n and m are independently selected from 1-25; wherein, when R 2 is either (i) or (ii) the isotope labeling agent is selected from the group consisting of: 1 H, 2 H, 3 H, 11 C, 12 C, 13 C, 13 N, 14 N, 15 N 18 F, 19 F, 123 I, 124 I, 125 I, 127 I, 131 I, 211 At, 15 O, 16 O, 17 O, 18 O, 32 S, 35 S, 43 Sc, 44 Sc, 45 Sc, 45 Ti, 46 Ti, 47 Ti, 48 Ti, 49 Ti, 50 Ti, 55 Co, 58 mCo, 59 Co, 60 Cu, 61 Cu, 63 Cu, 64 Cu, 65 Cu, 67 Cu, 67 Ga, 68 Ga, 69 Ga, 71 Ga, 76 Br, 77 Br, 79 Br, 80 mBr, 81 Br, 72 As, 75 As, 86 Y, 89 Y, 90 Y, 89 Zr, 90 Zr, 91 Zr, 92 Zr, 94 Zr, 149 Tb, 152 Tb, 159 Tb, 161 Tb, 111 In, 113 In, 114 mIn, 115 mIn, 175 Lu, 177 Lu, 185 Re, 186 Re, 188 Re, 201 Tl, 203 Tl, 205 Tl, 206 Pb, 207 Pb, 208 Pb, 212 Pb, 209 Bi, 212 Bi, 2 13 Bi, 31 P, 32 P, 33 P, 45 Sc, 47 Sc, 84 Sr, 86 Sr, 87 Sr, 88 Sr, 89 Sr, 165 Ho, 166 Ho, 156 Dy, 158 Dy, 160 Dy, 161 Dy, 162 Dy, 163 Dy, 164 Dy, 165 Dy , 227 Th, 232 Th, 51 Cr, 52 Cr, 53 Cr, 54 Cr, 73 Se, 74 Se, 75 Se, 76 Se, 77 Se, 78 Se, 80 Se, 82 Se, 94 Tc, 99m Tc, 103 Rh, 103 mRh, 119 Sb, 121 Sb, 123 Sb, 135 La, 138 La, 139 La, 162 Er, 164 Er, 165 Er, 166 Er, 167 Er, 168 Er, 170 Er, 193 mPt, 195 mPt, 192 Pt, 194 Pt, 195 Pt, 196 Pt, 198 Pt, and wherein X and Y are independently selected from: -CH and -N- ; and wherein R 3 is independently selected from H or a moiety selected from the group consisting of a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, amine, a substituted amine with 1-5 polyethylene glycol unit(s), a −(O−CH 2 −CH 2 ) 1- 5 −OCH 2 -COOH, Methyl, Ethyl, Propyl, optionally substituted heteroaryl, and optionally substituted arylalkyl; wherein optionally substituted in relation to said substituted amine means one or more substituents selected from, a halogen, a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, amine, (C1-C10) alkyl, (C2- C10)alkenyl, (C2-C10)alkynyl, (C1-C10)alkylene, (C1-C10)alkoxy, (C2- C10)dialkylamino, (C1-C10)alkylthio, (C2-C10)heteroalkyl, (C2-C10)heteroalkylene, (C3-30 C10)cycloalkyl, (C3-C10)heterocycloalkyl, (C3-10)cycloalkylene, (C3- C10)heterocycloalkylene, (C1-C10)haloalkyl, (C1-C10)perhaloalkyl, (C2-C10)- alkenyloxy, (C3-C10)-alkynyloxy, aryloxy, arylalkyloxy, heteroaryloxy, heteroarylalkyloxy, (C1-C6)alkyloxy-(C1-C4)alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted arylalkyl; wherein optionally substituted means one or more substituents selected from a halogen, a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, amine, a substituted amine with 1-5 polyethylene glycol unit(s), a −(O−CH 2 −CH 2 ) 1-5 −OCH 2 -COOH, H, Methyl, Ethyl, Propyl, optionally substituted heteroaryl, and optionally substituted arylalkyl; wherein optionally substituted in relation to said substituted amine means one or more substituents selected from a halogen, a hydroxy group, a sulfonamide, a carboxyl group, a sulfonyl group, and amine; and wherein R and R 1 are identical or differs only in the isotope number of the labelling agent. The below tetrazines are preferred tetrazines of Formula Tz1 for use in ligation combinations i) and ii) in step b) of the method for providing a labeled single isomeric chemical entity targeting vector:
The below trans-cycloheptenes (TCH’s), trans-cyclooctenes (TCO’s), and a trans- cyclononenes (TCNs) are preferred enantiomerically pure dienophiles for use in ligation combination i) in step b) of the method for providing a labeled single isomeric chemical entity targeting vector: Wherein X is O, NH, S, or CH 2 ; and wherein the linker is selected from the group comprising: -(CH 2 ) n - (CH 2 ) n NH, (CH 2 ) n CO, (CH 2 ) n O, (CH 2 CH 2 O) n (CH 2 CH 2 O) n CH 2 CH 2 NH, (CH 2 CH 2 O) n CH 2 CH 2 CO, -CO(CH) 2 - CO(CH 2 ) n NH, CO(CH 2 ) n CO, CO(CH 2 ) n O, CO(CH 2 CH 2 O) n CO(CH 2 CH 2 O) n CH 2 CH 2 NH, CO(CH 2 CH 2 O) n CH 2 CH 2 CO, COO(CH) 2 - COO(CH 2 ) n NH, COO(CH 2 ) n CO, COO(CH 2 ) n O, COO(CH 2 CH 2 O) n COO(CH 2 CH 2 O) n CH 2 CH 2 NH, COO(CH 2 CH 2 O) n CH 2 CH 2 CO, CONH(CH) 2 - CONH(CH 2 ) n NH, CONH(CH 2 ) n CO, CONH(CH 2 ) n O, CONH(CH 2 CH2O) n CONH(CH 2 CH 2 O) n CH 2 CH 2 NH, CONH(CH 2 CH 2 O)nCH 2 CH 2 CO, -CONHPhCO, -COOPhCO, -COPhCO, CONHCHMCO, (CH 2 ) n NHCHMCO, (CH 2 ) n OCONHCHMCO, (CH 2 ) n NHCHMCO, (CH 2 ) n NHCOCHMNH, (CH 2 )OCOCHMNH, (CH 2 CH 2 O) n CH 2 CH 2 NHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 CONHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 NHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 NHCOCHMNH, (CH 2 CH 2 O) n COCHMNH, where n is 0-25 and where M is a side chain selected from the group consisting of side chains of the natural amino acids: H, CH 3 , CH 2 SH, CH 2 COOH, CH 2 CH 2 COOH, CH 2 C 6 H 5 , CH 2 C 3 H 3 N 2 , CH(CH 3 )CH 2 CH 3 , (CH 2 ) 4 NH 2 , CH 2 CH(CH 3 ) 2 , CH 2 CH 2 SCH 3 , CH 2 CONH 2 , (CH 2 ) 4 NHCOC 4 H 5 NCH 3 , CH 2 CH 2 CH 2 , CH 2 CH 2 CONH 2 , (CH 2 ) 3 NH-C(NH)NH 2 , CH 2 OH, CH(OH)CH 3 , CH 2 SeH, CH(CH 3 ) 2 , CH 2 C 8 H 6 N, CH 2 C 6 H 4 OH; and where targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. The below trans-cyclooctenes (TCO’s) are preferred cis,5,6-disubstituted dienophiles for use in ligating combination ii) in step b) of the method for providing a labeled single isomeric chemical entity targeting vector: Wherein X is -O, NH, S, or CH2; -and wherein the linker is selected from the group comprising: -(CH 2 ) n - (CH 2 ) n NH, (CH 2 ) n CO, (CH 2 ) n O, (CH 2 CH 2 O) n (CH 2 CH 2 O) n CH 2 CH 2 NH, (CH 2 CH 2 O) n CH 2 CH 2 CO, -CO(CH) 2 - CO(CH 2 ) n NH, CO(CH 2 ) n CO, CO(CH 2 ) n O, CO(CH 2 CH 2 O) n CO(CH 2 CH 2 O) n CH 2 CH 2 NH, CO(CH 2 CH 2 O) n CH 2 CH 2 CO, COO(CH) 2 - COO(CH 2 ) n NH, COO(CH 2 ) n CO, COO(CH 2 ) n O, COO(CH 2 CH 2 O) n COO(CH 2 CH 2 O) n CH 2 CH 2 NH, COO(CH 2 CH 2 O) n CH 2 CH 2 CO, CONH(CH) 2 - CONH(CH 2 ) n NH, CONH(CH 2 ) n CO, CONH(CH 2 ) n O, CONH(CH 2 CH2O) n CONH(CH 2 CH 2 O) n CH 2 CH 2 NH, CONH(CH 2 CH 2 O)nCH 2 CH 2 CO, -CONHPhCO, -COOPhCO, -COPhCO, CONHCHMCO, (CH 2 ) n NHCHMCO, (CH 2 ) n OCONHCHMCO, (CH 2 ) n NHCHMCO, (CH 2 ) n NHCOCHMNH, (CH 2 )OCOCHMNH, (CH 2 CH 2 O) n CH 2 CH 2 NHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 CONHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 NHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 NHCOCHMNH, (CH 2 CH 2 O) n COCHMNH, where n is 0-25 and where M is a side chain selected from the group consisting of side chains of the natural amino acids: H, CH 3 , CH 2 SH, CH 2 COOH, CH 2 CH 2 COOH, CH 2 C 6 H 5 , CH 2 C 3 H 3 N 2 , CH(CH 3 )CH 2 CH 3 , (CH 2 ) 4 NH 2 , CH 2 CH(CH 3 ) 2 , CH 2 CH 2 SCH 3 , CH 2 CONH 2 , (CH 2 ) 4 NHCOC 4 H 5 NCH 3 , CH 2 CH 2 CH 2 , CH 2 CH 2 CONH 2 , (CH 2 ) 3 NH-C(NH)NH 2 , CH 2 OH, CH(OH)CH 3 , CH 2 SeH, CH(CH 3 ) 2 , CH 2 C 8 H 6 N, CH 2 C 6 H 4 OH; and where the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. Examples of Enantiopure TCOs TCH, TCN that are suitable for ligation combination Wherein the linker is selected from the group comprising: -(CH 2 ) n - (CH 2 ) n NH, (CH 2 ) n CO, (CH 2 ) n O, (CH 2 CH 2 O) n (CH 2 CH 2 O) n CH 2 CH 2 NH, (CH 2 CH 2 O) n CH 2 CH 2 CO, - CO(CH) 2 - CO(CH 2 ) n NH, CO(CH 2 ) n CO, CO(CH 2 ) n O, CO(CH 2 CH 2 O) n CO(CH 2 CH 2 O) n CH 2 CH 2 NH, CO(CH 2 CH 2 O) n CH 2 CH 2 CO, COO(CH) 2 - COO(CH 2 ) n NH, COO(CH 2 ) n CO, COO(CH 2 ) n O, COO(CH 2 CH 2 O) n COO(CH 2 CH 2 O) n CH 2 CH 2 NH, COO(CH 2 CH 2 O) n CH 2 CH 2 CO, CONH(CH) 2 - CONH(CH 2 ) n NH, CONH(CH 2 ) n CO, CONH(CH 2 ) n O, CONH(CH 2 CH2O) n CONH(CH 2 CH 2 O) n CH 2 CH 2 NH, CONH(CH 2 CH 2 O)nCH 2 CH 2 CO, -CONHPhCO, -COOPhCO, -COPhCO, CONHCHMCO, (CH 2 ) n NHCHMCO, (CH 2 ) n OCONHCHMCO, (CH 2 ) n NHCHMCO, (CH 2 ) n NHCOCHMNH, (CH 2 )OCOCHMNH, (CH 2 CH 2 O) n CH 2 CH 2 NHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 CONHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 NHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 NHCOCHMNH, (CH 2 CH 2 O) n COCHMNH, where n is 0-25 and where M is a side chain selected from the group consisting of side chains of the natural amino acids: H, CH 3 , CH 2 SH, CH 2 COOH, CH 2 CH 2 COOH, CH 2 C 6 H 5 , CH 2 C 3 H 3 N 2 , CH(CH 3 )CH 2 CH 3 , (CH 2 ) 4 NH 2 , CH 2 CH(CH 3 ) 2 , CH 2 CH 2 SCH 3 , CH 2 CONH 2 , (CH 2 ) 4 NHCOC 4 H 5 NCH 3 , CH 2 CH 2 CH 2 , CH 2 CH 2 CONH 2 , (CH 2 ) 3 NH-C(NH)NH 2 , CH 2 OH, CH(OH)CH 3 , CH 2 SeH, CH(CH 3 ) 2 , CH 2 C 8 H 6 N, CH 2 C 6 H 4 OH; and where the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. Examples of Cis,5,6-disubstituted-TCOs suitable for ligation ii) Wherein X is -O, NH, S, or CH 2; -and wherein the linker is selected from the group comprising: -(CH 2 ) n - (CH 2 ) n NH, (CH 2 ) n CO, (CH 2 ) n O, (CH 2 CH 2 O) n (CH 2 CH 2 O) n CH 2 CH 2 NH, (CH 2 CH 2 O) n CH 2 CH 2 CO, -CO(CH) 2 - CO(CH 2 ) n NH, CO(CH 2 ) n CO, CO(CH 2 ) n O, CO(CH 2 CH 2 O) n CO(CH 2 CH 2 O) n CH 2 CH 2 NH, CO(CH 2 CH 2 O) n CH 2 CH 2 CO, COO(CH) 2 - COO(CH 2 ) n NH, COO(CH 2 ) n CO, COO(CH 2 ) n O, COO(CH 2 CH 2 O) n COO(CH 2 CH 2 O) n CH 2 CH 2 NH, COO(CH 2 CH 2 O) n CH 2 CH 2 CO, CONH(CH) 2 - CONH(CH 2 ) n NH, CONH(CH 2 ) n CO, CONH(CH 2 ) n O, CONH(CH 2 CH2O) n CONH(CH 2 CH 2 O) n CH 2 CH 2 NH, CONH(CH 2 CH 2 O)nCH 2 CH 2 CO, -CONHPhCO, -COOPhCO, -COPhCO, CONHCHMCO, (CH 2 ) n NHCHMCO, (CH 2 ) n OCONHCHMCO, (CH 2 ) n NHCHMCO, (CH 2 ) n NHCOCHMNH, (CH 2 )OCOCHMNH, (CH 2 CH 2 O) n CH 2 CH 2 NHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 CONHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 NHCHMCO, (CH 2 CH 2 O) n CH 2 CH 2 NHCOCHMNH, (CH 2 CH 2 O) n COCHMNH, where n is 0-25 and where M is a side chain selected from the group consisting of side chains of the natural amino acids: H, CH 3 , CH 2 SH, CH 2 COOH, CH 2 CH 2 COOH, CH 2 C 6 H 5 , CH 2 C 3 H 3 N 2 , CH(CH 3 )CH 2 CH 3 , (CH 2 ) 4 NH 2 , CH 2 CH(CH 3 ) 2 , CH 2 CH 2 SCH 3 , CH 2 CONH 2 , (CH 2 ) 4 NHCOC 4 H 5 NCH 3 , CH 2 CH 2 CH 2 , CH 2 CH 2 CONH 2 , (CH 2 ) 3 NH-C(NH)NH 2 , CH 2 OH, CH(OH)CH 3 , CH 2 SeH, CH(CH 3 ) 2 , CH 2 C 8 H 6 N, CH 2 C 6 H 4 OH;; and where the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. Step c) in the method for providing a labeled single isomeric chemical entity targeting vector is an oxidation step. Even though auto-oxidation of the ligated entity targeting vector, such as a pyridazine, obtained in step b) of the method occurs spontaneously, this process is extremely slow and can last from several hours up to several days. Step c) in the method provides a fast way for oxidizing the pyridazine compound wherein only a single isomer form is obtained at least within 60 minutes, such as within 0-20 minutes. In order to facilitate this process, the dihydropyridazines are oxidized by either a standard, or solid-supported oxidant, preferably solid-supported. The oxidizing step can be performed at a temperature ranging from 15 to 50 °C, such as at 20-30 °C, preferably at room temperature, for approximately 10 to 60 minutes, preferably for less than 20 minutes. To facilitate the oxidation adding 1 to 100 equivalents, preferably 1, of an oxidant to the ligated compound obtained from the ligation step. The oxidant needs to be selective for the oxidation of the dihydropyrazine to pyridazine (95% efficiency). The targeting vector must not be chemically modified by the oxidant. The oxidant is a quinone oxidant selected from the group comprising: chloranil, fluoranil, DDQ, NaNO 2. Precursors that are useful in providing some of the dienes and dienophiles suitable for the method for providing a labeled single isomeric chemical entity targeting vector have also been provided herein. The following structures are the preferred precursors of symmetrical substituted dienes for use in the method for providing a labeled single isomeric chemical entity targeting vector for ligating combination i) and ii):
The following structures are the preferred precursors of enantiomerically pure dienophiles for use in the method for providing a labeled single isomeric chemical entity targeting vector for ligating combination i): wherein the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. The following structures are the preferred precursors of cis,5,6-disubstituted dienophile for use in the method for providing a labeled single isomeric chemical entity targeting vector for ligating combination ii): wherein the targeting vector is an antibody, a nanobody, a polymer, a nanomedicine, a cell, a protein, a peptide, or a small molecule. The labeled single isomeric chemical entity targeting vector provided by the method for providing a labeled single isomeric chemical entity targeting vectors can be used in therapy, radiotherapy, theranostics, diagnostics, or imaging, depending on the labeling agent, or the pharmaceutical agent, or imaging agent or therapeutic agent and on the targeting vector. Preferably, the targeting vector is coupled to the linker via a nitrogen on the targeting vector. Alternatively, the targeting vector is preferable coupled to the linker via a carbonyl on the targeting vector. In a preferred embodiment, the labeled single isomeric chemical entity targeting vector provided by the method for providing a labeled single isomeric chemical entity targeting vectors is used in therapy. In another preferred embodiment, the labeled single isomeric chemical entity targeting vector provided by the method for providing a labeled single isomeric chemical entity targeting vectors is used in radiotherapy. In another preferred embodiment, the labeled single isomeric chemical entity targeting vector provided by the method for providing a labeled single isomeric chemical entity targeting vectors is used in theranostics. In another preferred embodiment, the labeled single isomeric chemical entity targeting vector provided by the method for providing a labeled single isomeric chemical entity targeting vectors is used in diagnostics. In another preferred embodiment, the labeled single isomeric chemical entity targeting vector provided by the method for providing a labeled single isomeric chemical entity targeting vectors is used in imaging. The following Examples describes (1) the synthesis of tetrazines and TCOs representative for use in step a) and b) of the present method for providing a labeled single isomeric chemical entity targeting vector and (2) click reactions and oxidations between such compounds, yielding a single isomeric pyridazine. EXAMPLES General All reagents and solvents were dried prior to use according to standard methods. Commercial reagents were used without further purification. Analytical TLC was performed using silica gel 60 F254 (Merck) with detection by UV absorption and/or by charring following immersion in a 7% ethanolic solution of sulfuric acid or KMnO 4 - solution (1.5 g of KMnO 4 , 10 g K 2 CO 3 , and 1.25 mL 10% NaOH in 200 mL water). Purification of compounds was carried out by column chromatography on silica gel (40-60 μm, 60 Å) or employing a CombiFlash NextGen 300+ (Teledyne ISCO). 1 H and 13 C NMR spectra were recorded on Brucker (400 and 600 MHz instruments), using Chloroform-d, Methanol-d 4 or DMSO-d 6 as deuterated solvent and with the residual solvent as the internal reference. For all NMR experiences the deuterated solvent signal was used as the internal lock. Chemical shifts are reported in δ parts per million (ppm). Coupling constants (J values) are given in Hertz (Hz). Multiplicities of 1 H NMR signals are reported as follows: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublets of doublets; dt, doublet of triplets; t, triplet; q, quartet; m, multiplet; br, broad signal. NMR spectra of all compounds are reprocessed in MestReNova software (version 12.0.22023) from original FID’s files. Mass spectra analysis was performed using MS-Acquity-A: Waters Acquity UPLC with QDa- detector. Purification by preparative HPLC was performed on Agilent 1260 infinity system, column SymmetryPrep-C18, 17 mL/min H 2 O-MeCN gradient 50-100% 15 min with 0.1% trifluoroacetic acid. All final compounds were >95% pure as determined by analytical HPLC. Analytical HPLC method: (Thermo Fisher® UltiMate 3000) with a C- 18 column (Luna® 5u C18(2) 100Å, 150 x 4.6 mm), eluents: A: H2O with 0.1% TFA, B: MeCN with 0.1% TFA. Gradient from 100% A -> 100% B over 15minutes, back to 100% A over 4 minutes, flow rate 1.5 mL/min. Detection by UV-absorption at λ = 254 nm on a UVD 170U detector. Example 1 Synthesis of symmetrical tetrazines and their precursors Compound I and XXIV Figure 1. shows the synthesis of symmetrical tetrazines. Reagents and conditions: i) NH 2 (CH 2 ) 2 R, MeCN, 12 h, rt; ii) Boc 2 O, Et 3 N, DCM, 12 h, rt; iii) Zn(OTf) 2, NH 2 NH 2 , . H 2 O, EtOH, 65 ºC, 24 h; iv) HCl, dioxane, rt, 4 h; v) t-Butyl bromoacetate, Et 3 N, DMF, 50 ˚C, 12 h; vi) TFA, DCM, rt, 2 h; vii) MsCl, Et 3 N, DMAP, DCM, rt, 12 h. Figure 2. Shows an alternative synthesis of symmetrical tetrazines. Reagents and conditions: i) NH 2 (CH 2 ) 2 OH, MeCN, 12 h, rt; ii) Boc 2 O, Et 3 N, DCM, 12 h, rt; iii) Zn(OTf) 2, NH 2 NH 2 . H 2 O, EtOH, 65 ºC, 24 h; iv) HCl, dioxane, rt, 4 h; v) t-Butyl bromoacetate, Et 3 N, DMF, 50 ˚C, 12 h; vi) DAST, DCM, -78 ºC to rt, 4 h; vii) TFA, DCM, rt, 2 h; viii) MsCl, Et 3 N, DMAP, DCM, rt, 12 h. Synthesis of 4-(((2-hydroxyethyl)amino)methyl)benzonitrile (3) To a solution of ethanolamine in DCM (60 mL) was added dropwise a solution of 4- (bromomethyl)benzonitrile (4 gr, 20.20 mmol) in DCM (20 mL). The reaction was stirred at rt for 1 h. The organic phase was then washed with water (3 x 30 mL), dried and concentrated under reduced pressure to give 3.55 g (99%) of the desired product as a white solid. Rf = 0.21 (DCM/MeOH 95/5); 1 H NMR (400 MHz, CDCl 3 ) δ 8.01 – 7.52 (m, 2H), 7.47 – 7.30 (m, 2), 4.05 – 3.76 (m, 2H), 3.74 – 3.43 (m, 2H), 2.73 (dtd, J = 10.7, 6.0, 1.7 Hz, 2H), 2.47 (s, 1H); 13 C NMR (101 MHz, CDCl 3 ) δ 145.76, 132.22, 128.67, 118.88, 110.71, 60.91, 53.07, 50.78. Synthesis of tert-butyl (4-cyanobenzyl)(2-hydroxyethyl)carbamate (4) To a solution of 4-(((2-hydroxyethyl)amino)methyl)benzonitrile (1.62 g, 9.19 mmol) and Et 3N (2.56 mL, 18.39 mmol) in DCM (30 mL) was added Boc2O (2.10 gr, 9.65 mmol). The reaction was stirred at room temperature for 12 h. The solution was then washed with water (50 mL) and K 2 CO 3 saturated solution (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford 2.36 g (93%) of the desired product as a crude (mixture of rotamers). Rf. = 0.4 (heptane/EtOAc 50/50); 1 H NMR (600 MHz, CDCl 3 ) δ 7.63 (d, J = 7.9 Hz, 2H), 4.55 – 4.52 (m, 2H), 3.74 (s, 2H), 3.51 – 2.94 (m, 2H), 2.69 (s, 1H), 1.76 – 0.53 (m, 9H); 13 C NMR (151 MHz, CDCl 3 ) δ 156.91, 144.11, 132.43, 128.02, 127.50, 118.72, 111.19, 81.02, 62.17, 61.46, 52.10, 51.17, 50.37, 49.51, 28.32. Synthesis of 4-(((2-fluoroethyl)amino)methyl)benzonitrile (5) To a solution of 4-(bromomethyl)benzonitrile (0.78 g, 4.00 mmol) in CH 3 CN (40 mL) was added K 2 CO 3 (0.33 g, 24.0 mmol) and 2-fluoroethylamine hydrochloride (0.16 g, 16.0 mmol). The mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure, and the residue was diluted with water (20 mL), extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO 4 , filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography using EtOAc (Heptane/EtOAc 50/50) in heptane to afford 0.54 g (76%) of the desired product as a colorless oil. Rf = 0.24 (Heptane/EtOAc 40/60). 1 H NMR (400 MHz, CDCl 3 ) δ 7.55 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 4.63 – 4.48 (m, 1H), 4.47 – 4.37 (m, 1H), 3.84 (s, 2H), 2.93 – 2.84 (m, 1H), 2.84 – 2.72 (m, 1H), 1.65 (s, 1H). 13 C NMR (101 MHz, CDCl 3 ) δ 145.6, 132.3, 128.6, 118.9, 110.9, 83.5 (d, J = 165.5 Hz), 53.1, 49.1 (d, J = 19.7 Hz). Synthesis of tert-butyl 4-cyanobenzyl(2-fluoroethyl)carbamate (6) To a solution of 4-(((2-fluoroethyl)amino)methyl)benzonitrile (540 mg, 3.03 mmol) and Et 3 N (1.27 mL, 9.09 mmol) in CH 2 Cl 2 (40 mL) was added Boc 2 O (790 mg, 3.63 mmol) and the mixture was stirred at room temperature for 12 h. The solution was washed with water and saturated K 2 CO 3 solution, dried over Na 2 SO 4, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography using (Heptane/EtOAc 70/30) to afford 0.710 g (84%) of the desired product as a colorless oil (mixture of rotamers). Rf = 0.42 (Heptane/EtOAc 80/20). 1 H NMR (400 MHz, CDCl 3 ) δ 7.55 (d, J = 7.8 Hz, 2H), 7.27 (d, J = 7.8 Hz, 2H), 4.79 – 4.10 (m, 4H), 3.62 – 3.28 (m, 2H), 1.96 – 1.05 (m, 9H). 13 C NMR (101 MHz, CDCl 3 ) δ 155.4, 144.2, 143.8, 132.4, 128.1, 127.5, 118.7, 111.1, 83.2 (d, J = 168.2 Hz), 82.7 (d, J = 170.5 Hz), 52.1, 51.2, 47.7, 28.3. Synthesis of di-tert-butyl (((1,2,4,5-tetrazine-3,6-diyl)bis(4,1- phenylene))bis(methylene))bis((2-fluoroethyl)carbamate) (7) and tert-butyl (4- (6-(4-(((tert-butoxycarbonyl)(2-fluoroethyl)amino)methyl)phe nyl)-1,2,4,5- tetrazin-3-yl)benzyl)(2-hydroxyethyl)carbamate (8) To a suspension of tert-butyl 4-cyanobenzyl(2-fluoroethyl)carbamate (1.1 gr, 3.95 mmol), tert-butyl (4-cyanobenzyl)(2-hydroxyethyl)carbamate (0.27 gr, 0.99 mmol) and Zn(OTf) 2 (0.72 gr, 1.98 mmol) in EtOH (30 mL) was added hydrazine monohydrate (3.83 mL, 79 mmol). The mixture was allowed to stir at 70 ºC for 22 hours, and when the reaction is completed, is cooled at room temperature. The volatiles were removed under reduced pressure and the residue solubilized in EtOH (40 mL). A solution of NaNO 2 (5.52 g, 80.00 mmol ) in water (20 mL) was added to the crude reaction followed by dropwise addition of HCl (2M) until gas evolution ceased and a pH of 2-3 was achieved producing a red mixture. The crude reaction was extracted with DCM (3 x 40 mL) and washed with brine (3 x 20 mL). The organic phase was collected, dried over MgSO 4 , filtered and concentrated under reduced pressure. Purification by flash chromatography (DCM/MeOH 98/2) afforded 0.300 g (26%) of di-tert-butyl (((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methyle ne))bis((2- fluoroethyl)carbamate) as a red oil (mixture of rotamers). Rf = 0.45 (DCM/MeOH 98/2); 1 H NMR (600 MHz, CDCl 3 ) δ 8.63 (d, J = 8.0 Hz, 4H), 7.50 (d, J = 7.0 Hz, 4H), 5.11 – 4.38 (m, 8H), 3.97 – 3.26 (m, 4H), 1.95 – 0.57 (m, 18H); 13 C NMR (151 MHz, CDCl 3 ) δ 163.77, 155.61, 155.57, 143.70, 130.81, 128.51, 128.21, 127.87, 83.18 (d, J = 167.9 Hz), 82.61 (d, J = 169.3 Hz), 72.44, 65.78, 52.09, 51.09, 47.67 (d, J = 19.9 Hz), 47.19 (d, J = 21.0 Hz), 28.38. 0.08 g (7%) of a second more polar fraction corresponding to (4-(6-(4-(((tert- butoxycarbonyl)(2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-t etrazin-3-yl)benzyl)(2- hydroxyethyl)carbamate was isolated as a red solid (mixture of rotamers). Rf = 0.22 (DCM/MeOH 98/2); 1 H NMR (400 MHz, CDCl 3 ) δ 8.54 (d, J = 7.9 Hz, 4H), 7.40 (d, J = 8.1 Hz, 4H), 4.79 – 4.34 (m, 8H), 3.52 – 3.25 (m, 5H), 1.38 (d, J = 8.8 Hz, 18H). Synthesis of N,N'-(((1,2,4,5-tetrazine-3,6-diyl)bis(4,1- phenylene))bis(methylene))bis(2-fluoroethan-1-amine) (9) Di-tert-butyl (((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(methyle ne))bis((2- fluoroethyl) carbamate) (0.15 gr, 0.25 mmol) was treated with a solution of HCl (4 M) in dioxane (1 mL). A precipitate was formed. Filtration afforded 0.1 gr (83%) of the desired product as hydrochloride salt. 1 H NMR (600 MHz, DMSO) δ 9.70 (s, 4H), 8.61 (d, J = 8.0 Hz, 4H), 7.89 (d, J = 8.0 Hz, 4H), 4.86 (t, J = 4.6 Hz, 2H), 4.78 (t, J = 4.6 Hz, 2H), 4.37 (s, 4H), 3.40 - 3.25 (m, 4H); 13 C NMR (151 MHz, DMSO) δ 163.63, 136.83, 132.81, 131.66, 128.28, 80.09 (d, J = 165.1 Hz), 50.24, 47.21 (d, J = 19.8 Hz). Synthesis of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1- phenylene))bis(methylene))bis((2-fluoroethyl)azanediyl))diac etate (10) To a suspension of the hydrochloride salt of N,N'-(((1,2,4,5-tetrazine-3,6-diyl)bis(4,1- phenylene))bis(methylene))bis(2-fluoroethan-1-amine) (0.09 gr, 0.20 mmol) in anhydrous DMF (3 mL) was added Et 3 N (0.13 mL, 1.00 mmol). The reaction was stirred until all the precipitate went in solution. Tert-butyl bromo acetate (0.12 mL, 0.80 mmol) was added and the reaction stirred for 6 h at 50 ˚C. The mixture was cooled to room temperature and diluted with EtOAc (30 mL). The organic phase was washed with water (2 x 15 mL) and brine (2 x 15 mL). The organic phase was collected, dried over MgSO 4 , filtered and concentrated under reduced pressure to give 0.11 gr (91%) of the desired compound as a red oil. Rf = 0.48 (Heptane/EtOAc 70/30); 1 H NMR (400 MHz, CDCl 3 ) δ 8.62 (d, J = 8.4 Hz, 4H), 7.65 (d, J = 8.2 Hz, 4H), 4.64 (t, J = 5.0 Hz, 2H), 4.53 (t, J = 5.0 Hz, 2H), 4.04 (s, 4H), 3.42 (s, 4H), 3.14 (t, J = 5.0 Hz, 2H), 3.07 (t, J = 5.0 Hz, 2H), 1.51 (s, 18H); 13 C NMR (101 MHz, CDCl 3 ) δ 170.58, 163.82, 144.39, 130.80, 129.63, 128.02, 83.08 (d, J = 167.6 Hz), 81.19, 58.48, 55.69, 53.70 (d, J = 20.0 Hz), 28.22. Synthesis of 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1- phenylene))bis(methylene))bis((2-fluoroethyl)azanediyl))diac etic acid (I) To a solution of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenyl ene))bis(methylene))bis((2-fluoroethyl)azanediyl))diacetate (0.12 g, 0.19 mmol) in 5 mL of CH 2 Cl 2 was added 2 mL of TFA. The mixture was stirred at room temperature for 4 h. The solvent was then removed under reduced pressure. Purification by preparative HPLC afforded 0.70 g (50%) of the desired compound (TFA salt) as a red solid. 1 H NMR (600 MHz, DMSO) δ 8.51 (d, J = 8.2 Hz, 4H), 7.67 (d, J = 7.9 Hz, 4H), 4.60 (t, J = 5.0 Hz, 2H), 4.53 (t, J = 5.0 Hz, 2H), 4.02 (s, 4H), 3.46 (s, 4H), 3.06 (s, 2H), 3.02 (s, 2H); 13 C NMR (151 MHz, DMSO) δ 172.57, 163.68, 144.56, 131.23, 130.11, 128.01, 82.92 (d, J = 165.3 Hz), 58.08, 54.66, 53.64 (d, J = 19.8 Hz). Synthesis of 2-((4-(6-(4-(((2-fluoroethyl)amino)methyl)phenyl)-1,2,4,5-te trazin-3- yl)benzyl)amino)ethan-1-ol (11) (4-(6-(4-(((Tert-butoxycarbonyl)(2-fluoroethyl)amino)methyl) phenyl)-1,2,4,5-tetrazin- 3-yl)benzyl)(2-hydroxyethyl)carbamate (0.06 gr, 0.1 mmol) was treated with a solution of HCl (4 M) in dioxane (1 mL). A precipitate was formed. Filtration afforded 0.035 gr (75%) of the desired product as hydrochloride salt. 1 H NMR (600 MHz, DMSO) δ 9.64 (s, 4H), 8.61 (d, J = 8.3 Hz, 4H), 8.02 – 7.53 (m, 4H), 5.28 (s, 1H), 4.85 (t, J = 4.6 Hz, 1H), 4.77 (t, J = 4.6 Hz, 1H), 4.37 (s, 2H), 4.34 (s, 2H), 3.73 (s, 2H), 3.40 (t, J = 4.8 Hz, 1H), 3.35 (t, J = 4.8 Hz, 1H), 3.04 (s, 2H); 13 C NMR (151 MHz, DMSO) δ 163.63, 137.00, 136.86, 132.81, 132.74, 131.64, 128.29, 128.27, 80.12 (d, J = 165.3 Hz), 56.84, 50.27, 50.01, 49.12, 47.24 (d, J = 19.9 Hz). Synthesis of tert-butyl N-(4-(6-(4-(((2-(tert-butoxy)-2-oxoethyl)(2- fluoroethyl)amino) methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)-N-(2- hydroxyethyl)glycinate (12) To a suspension of the hydrochloride salt of 2-((4-(6-(4-(((2- fluoroethyl)amino)methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzy l)amino)ethan-1-ol (0.03 gr, 0.077 mmol) in anhydrous DMF (3 mL) was added Et 3 N (0.05 mL, 0.38 mmol). The reaction was stirred until all the precipitate went in solution. Tert-butyl bromo acetate (0.04 mL, 0.3 mmol) was added and the reaction stirred for 6 h at 50 ˚C. The mixture was cooled to room temperature and diluted with EtOAc (30 mL). The organic phase was washed with water (2 x 15 mL) and brine (2 x 15 mL). The organic phase was collected, dried over MgSO 4 , filtered and concentrated under reduced pressure to give 0.03 gr (64%) of the desired compound as a red oil. Rf = 0.25 (Heptane/EtOAc 70/30); 1 H NMR (400 MHz, CDCl 3 ) δ 9.44 – 8.40 (m, 2H), 7.61 (dd, J = 9.6, 8.2 Hz, 2H), 4.62 (t, J = 5.0 Hz, 1H), 4.50 (t, J = 5.0 Hz, 1H), 4.02 (s, 2H), 3.96 (s, 2H), 3.63 (s, 2H), 3.39 (s, 2H), 3.29 (s, 2H), 3.23 (s, 1H), 3.11 (t, J = 5.0 Hz, 1H), 3.04 (t, J = 5.0 Hz, 1H), 2.91 (t, J = 5.1 Hz, 2H), 1.48 (s, 9H), 1.46 (s, 9H); 13 C NMR (151 MHz, CDCl 3 ) δ 170.99, 170.60, 163.87, 163.75, 144.47, 143.63, 131.04, 130.76, 129.75, 129.64, 128.15, 128.05, 83.09 (d, J = 167.8 Hz), 81.71, 81.20, 59.12, 58.56, 58.48, 56.99, 55.71, 55.52, 53.70 (d, J = 20.1 Hz), 28.22, 28.12. Synthesis of tert-butyl N-(4-(6-(4-(((2-(tert-butoxy)-2-oxoethyl)(2- chloroethyl)amino) methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)-N-(2- fluoroethyl)glycinate (XXIV) To a solution of compound tert-butyl N-(4-(6-(4-(((2-(tert-butoxy)-2-oxoethyl)(2- fluoroethyl)amino) methyl)phenyl)-1,2,4,5-tetrazin-3-yl)benzyl)-N-(2- hydroxyethyl)glycinate (0.04 g, 0.065 mmol) and DIPEA (0.034 mL, 0.19 mmol) in CH 2 Cl 2 (10 mL) were added mesyl chloride (0.01 g, 0.019 mmol) and DMAP (0.001 g, 0.01 mmol). The reaction was stirred at room temperature for 12 h. The solvent was removed under reduced pressure. Purification by flash chromatography (80/20 Heptane/EtOAc) afforded 0.020 (48%) of the desired product as a red solid. Rf = 0.55 (Heptane/EtOAc 70/30); 1 H NMR (400 MHz, CDCl 3 ) δ 8.60 (d, J = 8.1 Hz, 4H), 7.62 (dd, J = 8.4, 3.0 Hz, 4H), 4.62 (t, J = 5.0 Hz, 1H), 4.50 (t, J = 5.0 Hz, 1H), 4.11 – 3.89 (m, 4H), 3.57 (t, J = 6.8 Hz, 2H), 3.39 (s, 2H), 3.36 (s, 2H), 3.15-3.10 (m, 3H), 3.04 (t, J = 5.0 Hz, 1H), 1.48 (s, 18H); 13 C NMR (101 MHz, CDCl 3 ) δ 170.51, 163.84, 163.81, 130.87, 130.79, 129.64, 129.56, 128.05, 128.03, 83.08 (d, J = 167.8 Hz), 81.35, 81.19, 58.48, 58.12, 55.96, 55.70, 55.50, 53.70 (d, J = 20.0 Hz), 28.23. MS Synthesis of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene)) bis(methylene))bis((2-hydroxyethyl)azanediyl))diacetate (13) To a suspension of tert-butyl (4-cyanobenzyl)(2-hydroxyethyl)carbamate (4 gr, 14.47 mmol) and Zn(OTf) 2 (2.63 gr, 7.23 mmol) in EtOH (40 mL) was added hydrazine monohydrate (14.04 mL, 289 mmol). The mixture was allowed to stir at 70 ºC for 22 hours, and when the reaction is completed, is cooled at room temperature. The volatiles were removed under reduced pressure and the residue solubilized in EtOH (80 mL). A solution of NaNO 2 (19.97 g, 289.00 mmol ) in water (50 mL) was added to the crude reaction followed by dropwise addition of HCl (2M) until gas evolution ceased and a pH of 2-3 was achieved producing a red mixture. The crude reaction was extracted with DCM (3 x 60 mL) and washed with brine (3 x 50 mL). The organic phase was collected, dried over MgSO 4 , filtered and concentrated under reduced pressure. Purification by flash chromatography (DCM/MeOH 95/5) afforded 1.2 g (28%) of the desired product as a red solid (mixture of rotamers). Rf = 0.21 (DCM/MeOH 95/5); 1 H NMR (600 MHz, CDCl 3 ) δ 8.63 (d, J = 7.9 Hz, 4H), 7.49 (d, J = 7.9 Hz, 4H), 4.62 (s, 4H), 3.79 (s, 4H), 3.60 – 3.37 (m, 4H), 3.01 (s, 2H), 1.48 (s, 18H); 13 C NMR (151 MHz, CDCl 3 ) δ 163.74, 157.21, 156.07, 143.55, 130.84, 128.25, 127.88, 80.91, 62.19, 61.44, 52.12, 51.13, 50.28, 49.41, 28.39. Synthesis of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene)) bis(methylene))bis((2-hydroxyethyl)azanediyl))diacetate (14) (4-(6-(4-(((tert-Butoxycarbonyl)(2-fluoroethyl)amino)methyl) phenyl)-1,2,4,5-tetrazin- 3-yl)benzyl)(2-hydroxyethyl)carbamate (0.9 gr, 1.55 mmol) was treated with a solution of HCl (4 M) in dioxane (15 mL). A precipitate was formed. Filtration afforded 0.68 gr (97%) of the desired product as hydrochloride salt. 1 H NMR (400 MHz, DMSO) δ 9.41 (s, 4H), 8.59 (d, J = 8.4 Hz, 4H), 7.89 (d, J = 8.4 Hz, 4H), 5.29 (s, 2H), 4.33 (s, 4H), 3.74 (s, 4H), 3.04 (s, 4H); 13 C NMR (101 MHz, DMSO) δ 163.62, 137.03, 132.71, 131.65, 128.23, 56.84, 49.98, 49.14. Synthesis of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1- phenylene))bis(methylene))bis((2-hydroxyethyl)azanediyl))dia cetate (15) To a suspension of the hydrochloride salt of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6- diyl)bis(4,1-phenylene)) bis(methylene))bis((2-hydroxyethyl)azanediyl))diacetate (0.25 gr, 0.55 mmol) in anhydrous DMF (10 mL) was added Et 3 N (0.38 mL, 2.75 mmol). The reaction was stirred until all the precipitate went in solution. Tert-butyl bromo acetate (0.32 mL, 2.20 mmol) was added and the reaction stirred for 6 h at 50 ˚C. The mixture was cooled to room temperature and diluted with EtOAc (40 mL). The organic phase was washed with water (2 x 15 mL) and brine (2 x 15 mL). The organic phase was collected, dried over MgSO 4 , filtered and concentrated under reduced pressure to give 0.31 gr (91%) of the desired compound as a red oil. Rf = 0.23 (Heptane/EtOAc 60/40); 1 H NMR (400 MHz, CDCl 3 ) δ 8.28 (d, J = 8.0 Hz, 4H), 7.27 (d, J = 8.0 Hz, 4H), 3.62 (s, 4H), 3.31 (t, J = 5.2 Hz, 4H), 2.96 (s, 4H), 2.58 (t, J = 5.1 Hz, 4H), 1.14 (s, 18H); 13 C NMR (101 MHz, CDCl 3 ) δ 171.14, 163.75, 143.96, 130.90, 129.66, 128.11, 81.56, 59.21, 58.57, 56.95, 55.67. Synthesis of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1- phenylene))bis(methylene))bis((2-fluoroethyl)azanediyl))diac etate (10) and di- tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1- phenylene))bis(methylene))bis((2-hydroxyethyl)azanediyl))dia cetate (12) To a solution of di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1- phenylene))bis(methylene))bis((2-hydroxyethyl)azanediyl))dia cetate (0.24 g, 0.39 mmol) in anhydrous THF (15 mL) at -78 ˚C was added DAST (0.055 mL, 0.39 mmol). The resulting mixture was stirred for 1 hour at -78 ˚C and additional 3 hours at room temperature. Subsequently the reaction was quenched with NaHCO 3 saturated solution (10 mL) and stirred for 30 minutes. The reaction mixture was extracted with DCM (3 x 30 mL) and washed with brine (3 x 30 mL). The organic phase was collected, dried over MgSO 4 , filtered and concentrated under reduced pressure. Purification by flash chromatography (DCM/MeOH 95/5) afforded 0.12 g (50%) of di- tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(m ethylene))bis((2- fluoroethyl)azanediyl))diacetate. A more polar fraction corresponding to di-tert-butyl 2,2'-((((1,2,4,5-tetrazine-3,6-diyl)bis(4,1-phenylene))bis(m ethylene))bis((2- hydroxyethyl)azanediyl))diacetate was isolated in 0.04 g (14%). Example 2 Synthesis of enantiopure TCO Scheme 5. Synthesis of isomer-free TCO. i) mCPBA, THF, H 2 O, 0 °C→RT, 17 h, 47%; ii) LiAlH 4 , THF, 0 °C→RT, 12 h, 99%; iii) a) Et 3 N, DMAP, CH 2 Cl 2 , 0 °C→RT, 12 h, 51%; iv) Crystallization from pentane, 41%; v) NaOH, THF, H 2 O, reflux, 2 h, 52%; vi) AgNO 3 , hV, rt, 8 h, 44%. (Z)‐9‐Oxabicyclo[6.1.0]non‐4‐ene (16) Cis,cis-1,5-cyclooctadiene (22.0 g, 203.36 mmol, 1.00 equiv.) and dry CH 2 Cl 2 (300 mL) were added to a 500 mL round-bottom flask. The mixture was cooled to 0 °C with an ice bath and mCPBA (45.57 g, 203.36 mmol, 1.00 equiv.) was added portion wise to give a white suspension. The mixture was allowed to reach room temperature and left stirring overnight. The mixture was filtered and washed with NaHCO 3 saturated solution (3 x 100 mL) and NaCl saturated solution (1 x 100 mL). The organic layer was collected, dried with MgSO 4 , filtered and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc, 90:10) yielded (Z)‐9‐ oxabicyclo[6.1.0]non‐4‐ene (11.82 g, 95.16 mmol, 47%) as a colorless oil. 1 H NMR (600 MHz, CDCl3) δ 5.69 – 5.48 (m, 2H), 3.15 – 2.91 (m, 2H), 2.55 – 2.35 (m, 2H), 2.21 – 2.08 (m, 2H), 2.08 – 1.86 (m, 4H); 13 C NMR (151 MHz, CDCl 3 ) δ 129.00, 56.87, 28.25, 23.82. (Z)‐Cyclooct‐4‐enol (17) Lithium aluminum hydride tablets (3.26 g, 85.93 mmol, 3.00 equiv.) were added to an oven-dried 500 mL three-necked round-bottom flask. The flask was sealed and flushed with argon. The flask was cooled to 0 °C using an ice-bath and dry THF (120 mL) was added slowly while vigorously stirring to give a grey suspension.1,2-Epoxy- 5-cyclooctene (3.56 g, 28.64 mmol, 1.00 equiv.) in dry THF (10 mL) was added dropwise and the mixture was allowed to reach room temperature and stirred overnight. The mixture was cooled to 0 °C in an ice bath and quenched with EtOAc (120 mL). A saturated solution of Rochelle salt (100 mL) was added, and the mixture was stirred vigorously for 10 minutes. The mixture was transferred to a separatory funnel and the organic layer was collected. The aqueous layer was extracted with DCM (3 x 150 mL). The combined organic layers were washed with H 2 O (200 mL), dried over MgSO 4 , filtered and concentrated under reduced pressure to give (Z)‐ Cyclooct‐4‐enol (3.49 g, 28.45 mmol, 99%). 1 H NMR (600 MHz, CDCl 3 ) δ 5.75 – 5.63 (m, 1H), 5.61 – 5.52 (m, 1H), 3.86 – 3.75 (m, 1H), 2.36 – 2.24 (m, 1H), 2.20 – 2.04 (m, 3H), 1.97 (s, 1H), 1.93 – 1.88 (m, 1H), 1.86 – 1.81 (m, 1H), 1.75 – 1.68 (m, 1H), 1.67 – 1.59 (m, 1H), 1.56 – 1.46 (m, 2H); 13 C NMR (151 MHz, CDCl 3 ) δ 130.23, 129.63, 72.85, 37.75, 36.36, 25.75, 24.97, 22.88. (Z)-cyclooct-4-en-1-yl (1R,4S)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane- 1-carboxylate (Epimers mixture) (18) (±)-(Z)-Cyclooct-4-enol (4.5 g, 35.65 mmol) was dissolved in dry CH 2 Cl 2 (100 mL), to which was added DMAP (0.87 g, 7.13 mmol) and Et 3 N (14.9 mL, 106.97 mmol). The solution was cooled to 0°C and (1S)-(-)-camphanic chloride (8.5 g, 38.22 mmol) was added portion wise to the mixture. The resulting solution was allowed to stir at room temperature for 17 hours. The mixture was washed with NaHCO 3 saturated solution (3 x 100 mL) and NaCl saturated solution (1 x 100 mL). The organic layer was collected, dried with MgSO 4 , filtered and concentrated under reduced pressure to give 5.63 g (51%) of a mixture of the desired epimers. Recrystallization from pentane afforded 2.31 g (41%) of (Z)-cyclooct-4-en-1-yl (1R,4S)-4,7,7-trimethyl-3-oxo-2- oxabicyclo[2.2.1]heptane-1-carboxylate as crystals (needles). 1 H NMR (400 MHz, CDCl 3 ) δ 5.75 – 5.58 (m, 2H), 5.05 – 4.94 (m, 1H), 2.46 – 2.29 (m, 2H), 2.28 – 2.08 (m, 3H), 2.06 – 1.84 (m, 4H), 1.84 – 1.74 (m, 1H), 1.74 – 1.33 (m, 4H), 1.10 (s, 3H), 1.04 (s, 3H,). 0.95 (s, 3H); 13 C NMR (101 MHz, CDCl 3 ) δ 178.46, 166.97, 130.05, 129.99, 129.59, 129.56, 91.23, 54.96, 54.21, 33.87, 33.85, 33.81, 30.69, 29.12, 29.10, 25.67, 25.65, 24.92, 24.88, 22.37, 22.35, 17.01, 16.95, 16.88, 9.84. Figure 3. shows the X-ray crystal structures of (S,Z)-cyclooct-4-en-1-yl (1R,4S)-4,7,7- trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate (18). (S,Z)-cyclooct-4-en-1-ol (19) (S,Z)-cyclooct-4-en-1-yl (1R,4S)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1- carboxylate (0.26 g, 0.85 mmol) was dissolved in THF (10 mL), to which was added a solution of NaOH (0.2 g, 4.24 mmol) in H 2 O (1 mL). The reaction was vigorously stirred at reflux for 2 hours. The mixture was quenched with H 2 O (10 mL) and the aqueous layer was extracted with DCM (3 x 30 mL). The combined organic layers were washed with H 2 O (20 mL), dried over MgSO 4 , filtered and concentrated under reduced pressure to give (S,Z)‐Cyclooct4enol (0.056 g, 52%) as a colorless oil. 1 H NMR (600 MHz, CDCl 3 ) δ 5.75 – 5.63 (m, 1H), 5.61 – 5.52 (m, 1H), 3.86 – 3.75 (m, 1H), 2.36 – 2.24 (m, 1H), 2.20 – 2.04 (m, 3H), 1.97 (s, 1H), 1.93 – 1.88 (m, 1H), 1.86 – 1.81 (m, 1H), 1.75 – 1.68 (m, 1H), 1.67 – 1.59 (m, 1H), 1.56 – 1.46 (m, 2H); 13 C NMR (151 MHz, CDCl 3 ) δ 130.23, 129.63, 72.85, 37.75, 36.36, 25.75, 24.97, 22.88. (S,E)-cyclooct-4-en-1-ol (20) A flash cartridge (220g, screw top, luer lock end fittings, Cat# FCSTLL-220-6) was packed with 8 cm silica (15-40µm) on the bottom and silver nitrate impregnated silica until the top. The column was flushed with 9:1 diethyl ether/n-heptane (500 mL) and the column was protected from light with aluminium foil. The cooling fence and UV lamps were turned on and after 10 minutes no detection of silver leakage was observed. Methyl benzoate (1 mL), (S,Z)-cyclooct-4-en-1-ol (1 g) and an additional 50 mL 9:1 diethyl ether/n-heptane solution were added to a round-bottom flask. The mixture was then added to the quartz flask. The pump was turned on (flowrate = 100 mL/min) and the photoreactor was turned on and photoisomerization was conducted for 8 hours. After 8 hours, the photoreactor was turned off and the column was dried by a stream of air. The silica was removed from the column and washed with 400 mL ammonia and 400 mL DCM. The mixture was stirred for 30 minutes, filtered and the organic layer was collected. The organic layer was washed with brine, dried with MgSO4, filtered and concentrated to give 0.44 g (44%) of the product as a yellowish oil (mixture of axial and equatorial isomers). Major equatorial 1 H NMR (400 MHz, CDCl 3 ) δ 5.57 (ddd, J = 15.3, 10.6, 4.1 Hz, 1H), 5.38 (ddd, J = 15.7, 10.9, 3.6 Hz, 1H), 3.50 – 3.40 (m, 1H), 2.32 (tq, J = 17.3, 5.1 Hz, 3H), 2.01 – 1.87 (m, 4H), 1.74 – 1.50 (m, 3H).Minor axial 1 H NMR (400 MHz, CDCl 3 ) δ 6.02 – 5.19 (m, 2H), 4.48 – 3.85 (m, 1H), 2.37 (dddd, J = 15.2, 10.5, 7.0, 4.8 Hz, 1H), 2.30 – 1.96 (m, 4H), 2.00 – 1.75 (m, 3H), 1.65 (dddd, J = 14.4, 12.8, 5.0, 1.7 Hz, 1H), 1.26 (dddd, J = 13.6, 10.9, 3.5, 0.9 Hz, 1H). Example 3 Ligation combination i) and oxidation Scheme 6. (S)-1,4-di(pyridin-2-yl)-5,6,7,8,9,10-hexahydrocycloocta[d]p yridazin-7-ol. 3,6-di(Pyridin-2-yl)-1,2,4,5-tetrazine was obtained as reported in Polezhaev, A. V.; Maciulis, N. A.; Chen, C.-H.; Pink, M.; Lord, R. L.; Caulton, K. G. Tetrazine Assists Reduction of Water by Phosphines: Application in the Mitsunobu Reaction. Chemistry – A European Journal 2016, 22, 13985-13998. (S,E)-cyclooct-4-en-1-ol (20 mg, 0.18 mmol) was dissolved in mixture of MeCN/H 2 O (4/1 v/v, 5 mL), to which was added 21 (37 mg, 0.18 mmol). The reaction was stirred for 10 minutes at rt. p-Chloranil (80 mg, 0.36 mmol) was added and the reaction was stirred for 10 min. The solution was concentrated under reduced pressure and purified by flash chromatography to give 45 mg (0.13 mmol) of the desired product. 1 H NMR (400 MHz, CDCl 3 ) δ 8.71 (dd, J = 5.0, 1.5 Hz, 2H), 8.25 – 7.70 (m, 4H), 7.60 – 7.32 (m, 2H), 4.06 – 3.61 (m, 1H), 3.30 – 2.92 (m, 4H), 2.19 (ddt, J = 13.5, 8.7, 4.1 Hz, 1H), 2.03 – 1.84 (m, 3H), 1.83 – 1.57 (m, 2H); 13 C NMR (101 MHz, CDCl 3 ) δ 158.77, 158.58, 156.68, 148.53, 148.49, 140.68, 140.39, 136.95, 136.88, 125.13, 125.06, 123.47, 123.41, 71.98, 39.00, 35.98, 26.98, 26.19, 23.91, 23.45. Example 4 Synthesis of cis,5,6-disubstituted-TCOs Scheme 7. Synthesis of Cis-cyclooct-5-ene-1,2-diol i)NMO, OsO 4 , THF, H 2 O, acetone, 0° to rt, 12 h, 93% ii) AgNO 3 , hV, rt, 8 h, 0 °C to RT, 12 h, 51%; Cis-Z-cyclooct-5-ene-1,2-diol (22) To a stirred mixture of 1,5-cyclooctadiene (1 1.22 mL, 12.9 mmol), 4- methylmorpholine N-oxide (1.87 g, 13.75 mmol) and THF: H2 O : acetone (1:1:1) (90 mL) at 0 °C was added osmium tetroxide (12.7 mg, 0.05 mmol). After 12 h at 25 °C the reaction mixture was poured into an aqueous saturated solution of NaHSO 3 (60 mL), extracted with EtOAc (3 x 150 mL), washed with water (2 x 50 mL) and brine (50 mL). Drying (MgSO 4 ) and concentration followed by flash chromatography (silica, 30% EtOAc in heptane) afforded the desired compound 1.70 g (93%). 1 H NMR (400 MHz, CDCl 3 ) δ 5.72 – 5.61 (m, 2H), 4.03 – 3.96 (m, 2H), 2.56 – 2.44 (m, 2H), 2.10 – 1.96 (m, 4H), 1.86 – 1.74 (m, 2H); 13 C NMR (101 MHz, CDCl 3 ) δ 130.09, 75.18, 32.08, 23.11. Cis-E-cyclooct-5-ene-1,2-diol (23) A flash cartridge (220g, screw top, luer lock end fittings, Cat# FCSTLL-220-6) was packed with 8 cm silica (15-40µm) on the bottom and silver nitrate impregnated silica until the top. The column was flushed with 9:1 diethyl ether/n-heptane (500 mL) and the column was protected from light with aluminium foil. The cooling fence and UV lamps were turned on and after 10 minutes no detection of silver leakage was observed. Methyl benzoate (1 mL), cis-Z-cyclooct-5-ene-1,2-diol (1 g) and an additional 50 mL 9:1 diethyl ether/n-heptane solution were added to a round-bottom flask. The mixture was then added to the quartz flask. The pump was turned on (flowrate = 100 mL/min) and the photoreactor was turned on and photoisomerization was conducted for 8 hours. After 8 hours, the photoreactor was turned off and the column was dried by a stream of air. The silica was removed from the column and washed with 400 mL ammonia and 400 mL DCM. The mixture was stirred for 30 minutes, filtered and the organic layer was collected. The organic layer was washed with brine, dried with MgSO4, filtered and concentrated to give 0.51 g (51%) of the product as a colorless oil. Example 5 Ligation iv) between a symmetric tetrazine and cis,5,6-disubstitutedTCOs Scheme 8. (cis)-1,4-di(pyridin-2-yl)-5,6,7,8,9,10-hexahydrocycloocta[d ]pyridazine-7,8-diol (24) 23 (40 mg, 0.28 mmol) was dissolved in mixture of MeCN/H 2 O (4/1 v/v, 5 mL), to which was added 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine (66 mg, 0.28 mmol). The reaction was stirred for 30 minutes at room temperature. p-Chloranil (117 mg, 0.47 mmol) was added, and the reaction was stirred for 10 min. After which, the reaction was diluted with H 2 O (50 mL) and filtered. The solution was concentrated under reduced pressure and purified by flash chromatography to give 80 mg (82%) of the desired product. 1 H NMR (400 MHz, CDCl 3 ) δ 9.47 – 8.40 (m, 2H), 7.92 (d, J = 8.0 Hz, 2H), 7.85 (dd, J = 7.7, 1.8 Hz, 2H), 7.37 (ddd, J = 7.5, 4.8, 1.3 Hz, 2H), 3.40 – 3.25 (m, 2H), 3.01 (q, J = 5.3 Hz, 4H), 2.34 (ddd, J = 11.5, 8.2, 4.7 Hz, 2H), 1.86 (dt, J = 15.8, 8.1 Hz, 2H); 13 C NMR (101 MHz, CDCl 3 ) δ 158.55, 156.40, 148.60, 139.90, 136.96, 125.01, 123.55, 72.91, 35.43, 23.50. Radiolabelling General methods: All reagents and solvents were purchased from ABX, Sigma Aldrich, Fluorochem and VWR and used as received, without further purification, unless stated otherwise. Dry THF and DCM were obtained from a SG Water solvent purification system and dry dimethyl sulfoxide (DMSO), MeCN, pyridine and methanol (MeOH) were purchased from commercial suppliers. Room temperature corresponds to a temperature interval from 18–21 ˚C. Reactions requiring anhydrous conditions were carried out under inert atmosphere (nitrogen) and using oven-dried glassware (152 ˚C). NMR ( 1 H, 13 C) spectra were acquired on a 600 MHz Bruker Avance III HD, a 400 MHz Bruker Avance II or a Bruker AC200. Thin-layer chromatography (TLC) was run on silica plated aluminum sheets (Silica gel 60 F254) from Merck and the spots were visualized by ultraviolet light at 254 nm, by anisaldehyde and/or by potassium permanganate staining. Example 6 18 F Radiolabeling of symmetrical tetrazines Scheme 9. Radiolabeling of symmetrical tetrazines. i) [ 18 F]Bu 4 NF/Bu 4 NOMs PO 4 3- , t- BuOH/DMSO, 100 ºC, 5 min; ii) TFA, CH 3 CN, 80 ºC, 10 min. Symmetrical 18 F-labeled tetrazine [ 18 F]I was prepared as follows: The aqueous [ 18 F]fluoride solution received from the cyclotron was passed through a preconditioned anion exchange resin (Sep-Pak Light QMA cartridge). The QMA was preconditioned by flushing it with 10 mL 0.5 M K 3 PO 4 and washing it with 10 mL H 2 O afterwards. [ 18 F]F- was eluted from the QMA into a 4 mL v-shaped vial with 1 mL Bu 4 NOMs dissolved in MeOH. The eluate was dried at 100 °C for 5 min under N 2 - flow. Precursor XIII (9.3 µmol, 6 mg) was dissolved in 167 µL DMSO and then diluted with 833 µL tBuOH. The solution was added to the dried [18F]fluoride solution and allowed to react for 5 min at 100 °C. The reaction was cooled to 50 °C with air before addition of 3 mL H 2 O. Radiochemical conversion (RCC) determined by radio-HPLC after the first step was 54%. The crude mixture was applied to a Sep-pak plus C18 solid phase extraction (SPE) cartridge that was preconditioned by flushing it with 10 mL EtOH followed by 10 mL of H 2 O. The SPE was flushed with another 5 mL of H 2 O and dried with N 2 . The product was eluted from the SPE with 2 mL MeCN into a 7 mL v-shaped vial containing 600 µL TFA. This mixture was reacted for 10 min at 80 °C. The RCC of [ 18 F]I determined by radio-HPLC was 95% (Figure 4). Radio-HPLC was performed on a Luna 5 µm C18(2) column (150 × 4.6 mm) using a gradient of acetonitrile (CH 3 CN) in water with 0.1% TFA. Gradient conditions: 0 min – 0% CH 3 CN, 0-10 min – linear increase of CH 3 CN content to 100%, 10-12 min – 100% CH 3 CN, 12-13 min - linear decrease of CH 3 CN content to 0%, 13-15 min – 0% CH 3 CN, elution speed 2 mL/min. Figure 4. shows the Radio-HPLC of [ 18 F]1 at end of deprotection. Example 7 18 F Radiolabeling of unsymmetrical tetrazines Unsymmetrical 18 F-labeled tetrazine [ 18 F]X was prepared from the nosyl precursor XXXVI via nucleophilic substitution as disclosed in Battisti, U.M.; Bratteby, K.; Jørgensen, J.T.; Hvass, L.; Shalgunov, V.; Mikula, H.; Kjær, A.; Herth, M.M. Development of the First Aliphatic 18F-Labeled Tetrazine Suitable for Pretargeted PET Imaging—Expanding the Bioorthogonal Tool Box. J. Med. Chem. 2021, 64, 15297–15312 Unsymmetrical 18 F-labeled tetrazine [ 18 F]XII was prepared from a trimethylstannyl precursor XXXVIII as disclosed in García-Vázquez, R.; Battisti, U.M.; Jørgensen, J.T.; Shalgunov, V.; Hvass, L.; Stares, D.L.; Petersen, I.N.; Crestey, F.C.; Löffler, A.; Svatunek, D.; et al. Direct Cu-mediated aromatic 18F-labeling of highly reactive tetrazines for pretargeted bioorthogonal PET imaging. Chem. Sci.2021, 12, 11668– 11675 Example 8 Screening of oxidants for the oxidation of the dihydropyridazines to pyridazines, yielding single end-products Figure 5 shows the reaction between a symmetrical tetrazine and an enantiopure TCO. The cycloaddition is completed within 5 minutes to give several isomers. The oxidants is then added to give the final single isomeric product. Each oxidant (5 equivalents) was added to the mixture and the reaction was analyzed by HPLC-MS after 10 minutes. The results are shown in Figure 5. These screening tests surprisingly showed that not all oxidants could be applied to provide a single isomeric form of the tetrazine-TCO pyridazine. Example 9 Provision of single isomeric tetrazine-TCO pyridazines The following oxidation conditions was used in this example: Tz (1 equiv) was dissolved in a mixture of EtOH to which was added a solution containing TCO-OH (1.5 equiv) in a mixture of EtOH. The reaction was stirred for 5 min, followed by the addition of Oxidant (5 equiv). This reaction was stirred for 10 min and subsequently analysed by analytical HPLC. Figure 6a-b. shows the result of the HPLC-MS analysis after oxidation confirming that only one single product was obtained. Compatibility of targeting vectors with oxidation conditions: In order to test whether the conditions leading to the oxidation of click product will not lead to the degradation of typical targeting vectors, we subjected a series of vectors relevant for theranostic radiopharmaceutical development to oxidation conditions previously shown to result in efficient conversion of dihydropyridazines to single- product pyridazines. Structures of tested vectors are shown in Figure 7. Vector oxidation test procedure: solution of targeting vector (70 µM) and oxidant (350 µM, 5 eq) in EtOH/water mixture (89-94% EtOH v/v) was stirred for 10 min at 25°C and subsequently analysed by analytical HPLC and LC/ESI-MS. The results are shown in Figure 8. None of the tested compounds showed oxidation and/or degradation meaning that they do not react with oxidants. The results showed that these vectors are compatible with the tested oxidants. Example 10 Measurement of second-order rate constants The second-order rate constant of all the click reactions made during the previous examples were measured by stopped-flow spectrometry in phosphate-buffered saline (PBS) at 25 °C in accordance with the method described in Battisti et al. J. Med. Chem. 2021, 64, 20, 15297–15312 (see page 15310 for experimental details and influencing factors). In short, stopped-flow measurements were performed using an SX20-LED stopped-flow spectrophotometer (Applied Photophysics) equipped with a 535 nm LED (optical pathlength 10 mm and full width half-maximum 34 nm) to monitor the characteristic tetrazine visible light absorbance (520−540 nm). The reagent syringes were loaded with a solution of axial-TCO-PEG 4 , and the instrument was primed. The subsequent data were collected in triplicate for each tetrazine. Reactions were conducted at 25 °C in PBS and recorded automatically at the time of acquisition. The data sets were analyzed by fitting an exponential decay using Prism 6 (GraphPad) to calculate the observed pseudo-first-order rate constants that were converted to second-order rate constants by dividing with the concentration of the excess TCO compound. Only reactions that showed a minimum second-order rate constant of 500 M -1 s -1 in phosphate-buffered saline at 25 °C are considered suitable for providing the sufficient speed kinetics and therefore, reactions wherein the reaction kinetics was lower were disregarded for the purpose of the method according to the invention.