PHOTOACTIVATABLE FLUORESCENT DYES WITH HYDROPHILIC CAGING GROUPS AND THEIR USE
20220064452 · 2022-03-03
Inventors
Cpc classification
C09B11/28
CHEMISTRY; METALLURGY
C12Q1/6818
CHEMISTRY; METALLURGY
International classification
Abstract
Disclosed are photoactivable fluorescent dye compounds of formula I:
##STR00001##
wherein: n=0, 1, 2, 3; X is selected from O, CRR′, SiRR′ and GeRR′, where R and R′ represent independently alkyl, cycloalkyl, alkenyl, alkynyl or aryl; Y is H, SO.sub.3H or SO.sub.3M, with M being a positively charged counterion, in particular selected from NH.sub.4.sup.+ and cations of organic ammonium compounds; R.sup.1 is H, CO.sub.2H, C(O)NH-linker-CO.sub.2H, C(O)O-ligand, C(O)NH-ligand or C(O)NH-linker-ligand; R.sup.2 may represent H, unsubstituted or substituted alkyl (including cycloalkyl); R.sup.3 and R.sup.4 may represent independently H or F; R.sup.5 is H, Me, CO.sub.2H, C(O)NH-linker-CO.sub.2H, C(O)O-ligand, C(O)NH-ligand or C(O)NH-linker-ligand; wherein the ligand moiety at each occurrence represents a reactive group or tag, capable to form a covalent or non-covalent bond or molecular complex with a target chemical entity or substance. Methods of using the compounds in imaging of fixed and living cells are also disclosed.
Claims
1. A compound which is a photoactivable fluorescent dye having the structural formula I: ##STR00055## wherein: n=0, 1, 2, 3; X is selected from O, CRR′, SiRR′ and GeRR′, where R and R′ represent independently alkyl, cycloalkyl, alkenyl, alkynyl or aryl; Y is H, SO.sub.3H or SO.sub.3M, with M being a positively charged counterion; R.sup.1 is H, CO.sub.2H, C(O)NH-linker-CO.sub.2H, C(O)O-ligand, C(O)NH-ligand or C(O)NH-linker-ligand; R.sup.2 is H, unsubstituted alkyl, substituted alkyl, unsubstituted cycloalkyl or substituted cycloalkyl; R.sup.3 and R.sup.4 are independently H or F; R.sup.5 is H, Me, CO.sub.2H, C(O)NH-linker-CO.sub.2H, C(O)O-ligand, C(O)NH-ligand or C(O)NH-linker-ligand; wherein the ligand at each occurrence represents a reactive group or tag, capable to form a covalent or non-covalent bond or molecular complex with a target chemical entity or substance; and wherein ##STR00056##
2. The compound according to claim 1, which is a 3,3-isomer, a 4,4-isomer or a 5,5-isomer of a compound of type I, as shown in the following structural formulae: ##STR00057## wherein n, X, Y, R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are defined as in claim 1 above.
3. The compound according to claim 1, having one of the following structural formulae Ia-Id: ##STR00058## wherein n, Y, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R and R′ are defined as in claim 1 above.
4. The compound according to claim 1, wherein the ligand: (a) comprises or represents a reactive group which is selected from an activated ester, an activated carbonate, an amine, a thiol, an azide, an alkene or alkyne, including a bicyclic and/or strained alkene or alkyne, a maleimide, and a tetrazine group; (b) is selected from the group consisting of a HaloTag ligand, a SNAP-Tag ligand, a CLIP-Tag ligand, a TMPTag ligand and functional analogs thereof; or (c) is selected from the group consisting of biotin, a taxoid moiety, phalloidin, and jasplakinolide.
5. The compound according to claim 1, having one of the following formulae IIa-IIj: ##STR00059## ##STR00060## wherein Y and R.sup.1 are defined as in claim 1 above.
6. The compound according to claim 5, which is a 4′-isomer, a 5′-isomer or a 6′-isomer of a compound of formulae IIa-IIj wherein R.sup.1 represents a substituent group C(O)—Z and Z may represent OH, NH-linker-CO.sub.2H, O-ligand, NH-ligand or NH-linker-ligand, and which has one of the following formulae 4′-IIIa-IIIj, 5′-IIIa-IIIj or 6′-IIIa-IIIj: ##STR00061## ##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069## ##STR00070##
7. The compound according to claim 6, wherein: the substituent Z represents a leaving group; or the substituent Z represents NH-linker-CO.sub.2H or NH-linker-CO—Z′, where the substituent Z′ represents a leaving group, or the substituent Z represents O-ligand, NH-ligand or NH-linker-ligand, wherein the ligand is capable to bind covalently or non-covalently to an intra- or extracellular target entity or substance, and the ligand: (a) comprises or represents a reactive group which is selected from an activated ester, an activated carbonate, an amine, a thiol, an azide, an alkene or alkyne, including a bicyclic and/or strained alkene or alkyne, a maleimide, and a tetrazine group; (b) is selected from the group consisting of a HaloTag ligand, a SNAP-Tag ligand, a CLIP-Tag ligand, a TMPTag ligand and functional analogs thereof; or (c) is selected from the group consisting of biotin, a taxoid moiety, phalloidin, and jasplakinolide.
8. The compound according to claim 7, wherein Z and/or Z′ is a leaving group selected from the group consisting of azide, fluoride, N-succinimidyloxy, 3-sulfo-N-succinimidyloxy, N-phthalimidyloxy, N-tetrachlorophthalimidyloxy, pentachlorophenoxy, pentafluorophenoxy, 2,3,5,6-tetrafluorophenoxy, 4-(hydroxysulfonyl)-2,3,5,6-tetrafluorophenoxy, 1-benzotriazolyloxy, and cyanomethoxy as shown in the formulae below: Z= ##STR00071## and/or wherein the linker is selected from an alkyl (polymethylene) chain —(CH.sub.2).sub.n—, where n=an integer from 1-20, or a PEG chain having the structural formula —(CH.sub.2CH.sub.2O).sub.n— or —(CH.sub.2CH.sub.2O).sub.n—CH.sub.2CH.sub.2—, where n=an integer from 1-100.
9. The compound according to claim 1 which is selected from the group of compounds 9, 13, 17, 21, 9-NHS, 13-NHS, 17-NHS, 21-NHS, 17-Halo, 21-Maleimide, 21-BG, 21-Halo and 21-Picolyl azide below: ##STR00072## ##STR00073## ##STR00074##
10. A composition comprising one or more of the compounds of the structures IIa-IIj or the structures 4′-IIIa-IIIj, 5′-IIIa-IIIj or 6′-IIIa-IIIj as their 3,3-, 4,4- or 5,5-isomers, as defined in claim 2, or their salt forms.
11. A method of using the compound according to claim 1 or a composition thereof as a reagent for conjugation or bioconjugation with a chemical entity or substance.
12. A method of using the compound according to claim 1 or a composition thereof with a chemical entity or substance as a photoactivatable fluorescent dye.
13. A method of using the compound according to claim 1, a composition thereof or a conjugate thereof with a chemical entity or substance in an unmasked fluorescent form as a cell membrane-permeant fluorescent dye, capable to penetrate through the membranes of fixed and living cells, or in a masked non-fluorescent form as a cell membrane-impermeant precursor for said cell membrane-permeant fluorescent dye.
14. A method of using the compound according to claim 1, a composition thereof or a conjugate thereof with a chemical entity or substance as such or after photoactivation for tracking and monitoring dynamic processes in a sample or an object, or tracking and monitoring a behavior of single molecules within a sample or an object.
15. The method according to claim 14 wherein changes in a shape, dimensions and/or an intensity of a fluorescence signal obtained after photoactivation of the compound, composition or conjugate correspond to changes of the sample or object or of its environment.
16. A method of using the compound according to claim 1, a composition thereof or a conjugate thereof with a chemical entity or substance as such or after photoactivation as fluorescent tags, analytical reagents and labels in optical microscopy, imaging techniques, protein tracking, nucleic acid labeling, glycan analysis, capillary electrophoresis, flow cytometry or as a component of biosensors, or as analytical tools or reporters in microfluidic devices or nanofluidic circuitry.
17. The method according to claim 16, wherein the compound, composition or conjugate as such or after photoactivation is used as an energy donor or an energy acceptor (reporters) in applications based on a fluorescence energy transfer (FRET) process or as energy acceptors (reporters) in applications based on a bioluminescence resonance energy transfer (BRET) process.
18. The method according to claim 16, wherein the optical microscopy and imaging techniques comprise single molecule switching techniques, photoactivation localization microscopy, stochastic optical reconstruction microscopy, minimal photon fluxes or their parallelized implementations, fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, fluorescence lifetime imaging, ground state depletion with individual molecular return and stimulated emission depletion microscopy.
19. The method according to claim 18, wherein additional color multiplexing is achieved by using the compound according to claim 1, a composition thereof or a conjugate thereof as such or after photoactivation together with any other fluorescent dyes in a single sample or object under study, or wherein the controlled photoactivation of spatiotemporal subpopulations of molecules of the compound, the composition or the conjugate allows imaging with the uncaged fluorophore molecules while protecting the remaining caged fluorophores from photobleaching, or wherein the controlled photoactivation of the compound, the composition or the conjugate is achieved by a two-photon or multiphoton activation process.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0097]
[0098]
[0099]
[0100]
[0101]
[0102] The present invention is further illustrated by the following specific but non-limiting examples.
Example 1
Synthesis of Starting Materials and Photoactivatable Dyes
5-(Methoxycarbonyl)-2-nitrobenzyl 1H-imidazole-1-carboxylate (1)
[0103] ##STR00025##
[0104] Compound 1. 1,1′-Carbonyldiimidazole (CDI; 972 mg, 6 mmol, 1.5 eq) was added portionwise to a stirred solution of methyl 3-(hydroxymethyl)-4-nitrobenzoate (844 mg, 4 mmol) in dry CH.sub.2Cl.sub.2 (20 mL). After stirring for 2 h at rt, sat. aq. NH.sub.4Cl (20 mL) was added and the reaction mixture was extracted with CH.sub.2Cl.sub.2 (3×20 mL). The combined extracts were dried over Na.sub.2SO.sub.4, filtered, evaporated, the residue was dissolved in EtOAc and the solution was passed through a plug of silica gel, washing with EtOAc. The filtrate was evaporated to viscous light yellow oil, which was freeze-dried from 1,4-dioxane to provide 1 as yellowish solid (832 mg, 68%).
[0105] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.31 (dd, J=1.7, 0.8 Hz, 1H), 8.23 (dd, J=8.5, 1.7 Hz, 1H), 8.20 (d, J=8.4 Hz, 1H), 7.46 (t, J=1.5 Hz, 1H), 7.11 (d, J=1.5 Hz, 1H), 5.84 (s, 2H), 3.99 (s, 3H).
[0106] .sup.13C NMR (101 MHz, CDCl.sub.3): δ 164.7, 150.3, 148.3, 137.3, 135.1, 131.2, 131.1, 131.0, 130.3, 125.7, 117.3, 65.9, 53.2.
[0107] HRMS (C.sub.13H.sub.11N.sub.3O.sub.6): m/z (positive mode)=306.0718 (found [M+H].sup.+), 306.0721 (calc.).
Methyl 3-(methylcarbamoyloxy)methyl-4-nitrobenzoate (2)
[0108] ##STR00026##
[0109] Compound 2. To a solution of 1 (610 mg, 2 mmol) in dry CH.sub.2Cl.sub.2 (7 mL), cooled in ice-water bath, methylamine (2 M in THF; 3 mL, 6 mmol, 3 eq) was added dropwise. The reaction mixture was stirred at 0-5° C. for 1 h and evaporated on Celite. The product was isolated by flash chromatography on Biotage Isolera system (40 g Teledyne ISCO RediSep Rf cartridge, gradient 20% to 100% EtOAc/hexane) to yield 512 mg (96%) of 2 as white solid.
[0110] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.26 (s, 1H), 8.10 (s, 2H), 5.51 (s, 2H), 4.91 (br.s, 1H), 3.97 (s, 3H), 2.84 (d, J=4.9 Hz, 3H).
[0111] .sup.13C NMR (101 MHz, CDCl.sub.3): δ 165.2, 156.3, 150.0, 134.6, 133.6, 130.3, 129.8, 125.1, 62.9, 53.0, 27.8.
[0112] HRMS (C.sub.11H.sub.12N.sub.2O.sub.6): m/z (positive mode)=269.0767 (found [M+H].sup.+), 269.0768 (calc.).
Methyl 3-(carbamoyloxy)methyl-4-nitrobenzoate (3)
[0113] ##STR00027##
[0114] Compound 3. To a solution of 1 (196 mg, 0.64 mmol) in THF (2.5 mL), cooled in ice-water bath, aq. ammonia (25%, ˜13 M in water; 0.25 mL, 3.21 mmol, 5 eq) was added dropwise. The reaction mixture was stirred at 0-5° C. for 30 min, diluted with methanol and evaporated to dryness on Celite. The product was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 μm cartridge, gradient 20% to 100% EtOAc/hexane) to yield 103 mg (63%) of 3 as white solid.
[0115] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.21 (d, J=8.5 Hz, 1H), 8.18 (d, J=2.0 Hz, 1H), 8.10 (dd, J=8.5, 2.0 Hz, 1H), 6.98 (br.s, 1H), 6.69 (br.s, 1H), 5.35 (s, 2H), 3.92 (s, 3H).
[0116] .sup.13C NMR (101 MHz, DMSO-d.sub.6): δ 164.7, 156.0, 149.7, 133.8, 133.7, 129.5, 129.3, 125.4, 61.3, 52.9.
[0117] HRMS (C.sub.10H.sub.10N.sub.2O.sub.6): m/z (positive mode)=255.0611 (found [M+H].sup.+), 255.0612 (calc.).
tert-Butyl (3-((6-(azidomethyl)pyridin-3-yl)amino)-3-oxopropyl)carbamate (4)
[0118] ##STR00028##
[0119] Compound 4. To a solution of 6-(azidomethyl)pyridin-3-amine [Jiang et al. Bioconjugate Chem. 2014, 25, 698-706] (132 mg, 0.88 mmol), N,N-ethyldiisopropylamine (DIEA; 0.62 mL, 3.52 mmol, 4 eq) and Boc-β-Ala-OH (333 mg, 1.76 mmol, 2 eq) in dry DMF (1.5 mL), cooled in ice-water bath, solid HATU (502 mg, 1.32 mmol, 1.5 eq) was added in portions over 5 min. The resulting yellow-green solution was warmed up to rt and stirred for 1.5 h. The reaction mixture was poured into sat. aq. NaHCO.sub.3 (50 mL) and extracted with EtOAc (3×25 mL). The combined extracts were washed with water, brine, dried over Na.sub.2SO.sub.4, filtered and evaporated on Celite. The product 4 was isolated by flash chromatography on Biotage Isolera system (12 g Interchim SiHP 30 μm cartridge, gradient 50% to 100% EtOAc/hexane) and freeze-dried from dioxane to give 264 mg (94%) of pinkish solid.
[0120] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.72 (br.s, 1H), 8.62 (d, J=2.5 Hz, 1H), 8.20 (dd, J=8.5, 2.5 Hz, 1H), 7.29 (d, J=8.5 Hz, 1H), 5.22 (br.t, J=7.7 Hz, 1H), 4.43 (s, 2H), 3.50 (q, J=6.3 Hz, 2H), 2.64 (t, J=5.9 Hz, 2H), 1.42 (s, 9H).
[0121] .sup.13C NMR (101 MHz, CDCl.sub.3): δ 170.4, 156.8, 150.9, 141.1, 134.6, 127.9, 122.4, 80.2, 55.4, 38.0, 36.5, 28.5.
[0122] HRMS (C.sub.14H.sub.20N.sub.6O.sub.3): m/z (positive mode)=321.1663 (found [M+H].sup.+), 321.1670 (calc.).
3-Amino-N-(6-(azidomethyl)pyridin-3-yl)propanamide, bis(trifluoroacetate) salt (5)
[0123] ##STR00029##
[0124] Compound 5. Trifluoroacetic acid (0.5 mL) was added to a solution of 4 (64 mg, 0.2 mmol) in CH.sub.2Cl.sub.2 (2 mL), and the resulting mixture was stirred at rt for 2 h. It was then diluted with toluene (5 mL), evaporated, chased with toluene-CH.sub.2Cl.sub.2 (5 mL) and dioxane (2×5 mL) and dried in vacuo to give the product 5 as viscous light brown oil (90 mg, ˜100%).
[0125] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 10.49 (s, 1H), 8.77 (d, J=2.6 Hz, 1H), 8.07 (dd, J=8.4, 2.6 Hz, 1H), 7.81 (br.s, 4H, NH.sub.3.sup.++NH.sup.+), 7.43 (d, J=8.4 Hz, 1H), 4.46 (s, 2H), 3.11 (q, J=6.1 Hz, 2H), 2.74 (t, J=6.8 Hz, 2H).
[0126] .sup.13C NMR (101 MHz, DMSO-d.sub.6): δ 169.0, 158.4 (q, .sup.2J.sub.C-F=36.1 Hz, CF.sub.3CO.sub.2), 150.0, 140.3, 135.1, 127.2, 122.8, 115.77 (q, .sup.1J.sub.C-F=291.9 Hz, CF.sub.3CO.sub.2), 54.0, 34.8, 33.2.
[0127] HRMS (C.sub.9H.sub.12N.sub.6O): m/z (positive mode)=221.1140 (found [M+H].sup.+), 221.1145 (calc.).
##STR00030##
[0128] Compound 6. In a flame-dried 10 mL tube, loaded with anhydrous K.sub.3PO.sub.4 (119 mg, 0.56 mmol, 4 eq) and 3 Å molecular sieves (56 mg), compound A1 [Grimm et al. Nat. Methods 2015, 12, 244-250] (98 mg, 0.14 mmol), compound 2 (90 mg, 0.34 mmol, 2.4 eq), JackiePhos Pd G3 precatalyst (24.5 mg, 0.021 mmol, 15 mol %) and JackiePhos ligand (16.7 mg, 0.021 mmol, 15 mol %) were loaded. The tube was sealed, anhydrous toluene (1.4 mL) was injected, the mixture was degassed on a Schlenk line and stirred at 110° C. for 6 h. It was then diluted with CH.sub.2Cl.sub.2, filtered through a plug of Celite (washing with CH.sub.2Cl.sub.2 and EtOAc—CH.sub.2Cl.sub.2), the filtrate was evaporated on Celite and the product was isolated by flash chromatography on Biotage Isolera system (25 g Interchim SiHP 30 μm cartridge, gradient 20% to 80% EtOAc/hexane) and freeze-dried from dioxane to yield 92 mg (70%) of 6 as white solid.
[0129] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.25 (dd, J=8.0, 1.3 Hz, 1H), 8.17 (br.s, 2H), 8.12-8.05 (m, 5H), 7.84 (s, 1H), 7.27 (d, J=2.2 Hz, 2H), 7.05 (dd, J=8.5, 2.2 Hz, 2H), 6.82 (d, J=8.5 Hz, 2H), 5.68-5.52 (m, 4H), 3.90 (s, 6H), 3.39 (s, 6H), 1.54 (s, 9H).
[0130] .sup.13C NMR (101 MHz, CDCl.sub.3): δ 168.5, 165.0, 164.1, 154.4, 153.0, 151.5, 150.0, 145.1, 138.7, 134.6, 132.8, 131.3, 130.5, 130.0, 129.4, 128.8, 125.4, 125.23, 125.19, 121.5, 116.3, 114.1, 82.8, 82.2, 64.2, 53.0, 37.8, 28.1.
[0131] HRMS (C.sub.47H.sub.40N.sub.4O.sub.17): m/z (positive mode)=933.2462 (found [M+H].sup.+), 933.2461 (calc.).
##STR00031##
[0132] Compound 7. To a solution of compound 6 (40 mg, 42.9 μmol) in THF (400 μL) and methanol (200 μL), a solution of LiOH—H.sub.2O (5.4 mg, 129 μmol, 3 eq) in water (85 μL) was added, and the resulting mixture was stirred vigorously at rt for 1 h. Acetic acid (200 μL) was then added, the reaction mixture was evaporated to dryness and dried in vacuo. The product was isolated by preparative HPLC (column: Thermo Scientific 250×21.2 mm 5 am Hypersil Gold C18; gradient 40/60.fwdarw.90/10 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 7 as yellowish solid (33 mg, 85%).
[0133] .sup.1H NMR (400 MHz, acetone-d.sub.6): δ 8.31 (dd, J=8.0, 1.3 Hz, 1H), 8.27 (br.s, 2H), 8.20 (d, J=8.3 Hz, 2H), 8.16 (dd, J=8.3, 1.6 Hz, 2H), 8.13 (dd, J=8.0, 0.8 Hz, 1H), 7.96 (dd, J=1.3, 0.8 Hz, 1H), 7.48 (d, J=2.2 Hz, 2H), 7.23 (dd, J=8.6, 2.2 Hz, 2H), 6.95 (d, J=8.6 Hz, 2H), 5.67-5.53 (m, 4H), 3.42 (s, 6H), 1.51 (s, 9H).
[0134] .sup.13C NMR (101 MHz, acetone-d.sub.6): δ 168.5, 165.8, 164.8, 154.9, 153.7, 152.2, 151.0, 146.4, 139.4, 135.8, 133.7, 132.0, 131.1, 130.8, 130.5, 129.4, 126.00, 125.98, 125.8, 122.2, 117.0, 114.6, 83.0, 82.7, 64.6, 37.7, 28.1.
[0135] HRMS (C.sub.45H.sub.36N.sub.4O.sub.17): m/z (positive mode)=905.2150 (found [M+H].sup.+), 905.2148 (calc.).
##STR00032##
[0136] Compound 8. To a solution of compound 7 (28 mg, 30.9 μmol) in DMF (500 μL) and water (100 μL), taurine (27 mg, 216 μmol, 7 eq) and N,N-ethyldiisopropylamine (DIEA; 300 μL) were added followed by HATU (59 mg, 155 μmol, 5 eq; dissolved in 200 μL DMF). The resulting mixture was stirred vigorously at rt overnight (16 h). It was then evaporated to dryness and dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 am Uptisphere Strategy PhC4; gradient 20/80.fwdarw.70/30 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 8 as light orange solid (29 mg), which was used directly in the next step.
[0137] HRMS (C.sub.49H.sub.46N.sub.6O.sub.21S.sub.2): m/z (positive mode)=1119.2229 (found [M+H].sup.+), 1119.2230 (calc.).
##STR00033##
[0138] Compound 9. A solution of compound 8 (28 mg, 25.0 μmol) in CH.sub.2Cl.sub.2 (600 μL) and TFA (300 μL) was stirred at rt for 1.5 h. The resulting mixture was diluted with CH.sub.2Cl.sub.2 and toluene, evaporated to dryness and dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 μm Uptisphere Strategy PhC4; gradient 20/80.fwdarw.60/40 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 9 as light orange solid (15 mg, 46% over 2 steps).
[0139] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.81 (t, J=5.3 Hz, 2H), 8.27 (dd, J=7.9, 1.3 Hz, 1H), 8.21-8.14 (m, 3H), 8.03 (br.s, 2H), 7.92 (dd, J=8.5, 1.9 Hz, 2H), 7.87 (t, J=1.0 Hz, 1H), 7.48 (d, J=2.2 Hz, 2H), 7.19 (dd, J=8.6, 2.2 Hz, 2H), 6.88 (d, J=8.6 Hz, 2H), 5.54-5.44 (m, 4H), 3.51 (q, J=6.8 Hz, 4H), 3.34 (s, 6H), 2.78-2.67 (t, J=6.8 Hz, 4H).
[0140] .sup.13C NMR (101 MHz, DMSO-d.sub.6): δ 167.7, 166.0, 163.8, 153.9, 152.4, 150.7, 148.6, 145.0, 138.9, 137.6, 132.1, 131.3, 129.0, 128.4, 128.2, 127.4, 125.6, 125.2, 124.8, 121.5, 115.4, 113.3, 81.7, 63.8, 50.0, 37.1, 36.3.
[0141] HRMS (C.sub.45H.sub.38N.sub.6O.sub.21S.sub.2): m/z (positive mode)=1063.1604 (found [M+H].sup.+), 1063.1604 (calc.).
##STR00034##
[0142] Compound 10. In a flame-dried 10 mL tube, loaded with anhydrous K.sub.3PO.sub.4 (119 mg, 0.56 mmol, 4 eq) and 3 Å molecular sieves (56 mg), compound A2 [Butkevich et al. Angew. Chem. Int. Ed. 2016, 55, 3290-3294] (108 mg, 0.14 mmol), compound 2 (90 mg, 0.34 mmol, 2.4 eq), JackiePhos Pd G3 precatalyst (24.5 mg, 0.021 mmol, 15 mol %) and JackiePhos ligand (16.7 mg, 0.021 mmol, 15 mol %) were loaded. The tube was sealed, anhydrous toluene (1.4 mL) was injected, the mixture was degassed on a Schlenk line and stirred at 110° C. for 6 h. It was then diluted with CH.sub.2Cl.sub.2, filtered through a plug of Celite (washing with CH.sub.2Cl.sub.2 and EtOAc—CH.sub.2Cl.sub.2), the filtrate was evaporated on Celite and the product was isolated by flash chromatography on Biotage Isolera system (25 g Interchim SiHP 30 μm cartridge, gradient 20% to 80% EtOAc/hexane) and freeze-dried from dioxane to yield 89 mg (66%) of 10 as white solid.
[0143] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.20 (dd, J=8.0, 1.3 Hz, 1H), 8.17 (br.s, 2H), 8.10-8.05 (m, 5H), 7.68 (br.s, 1H), 7.60 (br.s, 2H), 7.09 (dd, J=8.5, 2.2 Hz, 2H), 6.78 (d, J=8.5 Hz, 2H), 5.62-5.50 (m, 4H), 3.92 (s, 6H), 3.38 (s, 6H), 1.83 (s, 3H), 1.73 (s, 3H), 1.53 (s, 9H).
[0144] .sup.13C NMR (101 MHz, CDCl.sub.3): δ 169.6, 165.0, 164.3, 154.9, 154.7, 150.1, 146.0, 143.9, 138.3, 134.6, 133.0, 130.7, 130.5, 129.9, 129.2, 128.8, 125.3, 125.2, 125.1, 124.2, 85.7, 82.7, 64.1, 53.0, 38.5, 38.0, 34.8, 33.3, 28.1.
[0145] HRMS (C.sub.50H.sub.46N.sub.4O.sub.16): m/z (positive mode)=959.2976 (found [M+H].sup.+), 959.2982 (calc.).
##STR00035##
[0146] Compound 11. To a solution of compound 10 (40 mg, 41.7 μmol) in THF (400 μL) and methanol (200 μL), a solution of LiOH—H.sub.2O (5.3 mg, 125 μmol, 3 eq) in water (85 μL) was added, and the resulting mixture was stirred vigorously at rt for 1 h. Acetic acid (200 μL) was then added, the reaction mixture was evaporated to dryness and dried in vacuo. The product was isolated by preparative HPLC (column: Thermo Scientific 250×21.2 mm 5 am Hypersil Gold C18; gradient 40/60.fwdarw.90/10 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 11 as light pink solid (33 mg, 85%).
[0147] .sup.1H NMR (400 MHz, acetone-d.sub.6): δ 8.25 (dd, J=8.0, 1.3 Hz, 1H), 8.25 (br.s, 2H), 8.21-8.15 (m, 4H), 8.12 (dd, J=8.0, 0.8 Hz, 1H), 7.87 (d, J=2.2 Hz, 2H), 7.67 (dd, J=1.3, 0.8 Hz, 1H), 7.26 (dd, J=8.6, 2.2 Hz, 2H), 6.87 (d, J=8.6 Hz, 2H), 5.56 (s, 4H), 3.39 (s, 6H), 1.87 (s, 3H), 1.76 (s, 3H), 1.50 (s, 9H).
[0148] .sup.13C NMR (101 MHz, acetone-d.sub.6): δ 169.6, 165.9, 164.7, 156.1, 155.1, 151.2, 146.6, 145.1, 139.1, 135.8, 133.8, 131.4, 131.2, 130.8, 130.0, 129.3, 129.1, 126.2, 126.0, 125.3, 125.2, 125.1, 85.9, 83.0, 64.4, 39.1, 37.9, 34.6, 33.8, 28.1.
[0149] HRMS (C.sub.48H.sub.42N.sub.4O.sub.16): m/z (positive mode)=931.2661 (found [M+H].sup.+), 931.2669 (calc.).
##STR00036##
[0150] Compound 12. To a solution of compound 11 (30 mg, 32.2 μmol) in DMF (500 μL) and water (100 μL), taurine (40 mg, 322 μmol, 10 eq) and N,N-ethyldiisopropylamine (DIEA; 350 μL) were added followed by HATU (98 mg, 258 μmol, 8 eq; dissolved in 300 μL DMF). The resulting mixture was stirred vigorously at rt overnight (16 h). It was then evaporated to dryness and dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 am Uptisphere Strategy PhC4; gradient 20/80.fwdarw.70/30 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 8 as light pink solid (24 mg, 65%).
[0151] .sup.1H NMR (400 MHz, CD.sub.3OD): δ 8.23 (dd, J=8.0, 1.3 Hz, 1H), 8.14-8.07 (m, 3H), 8.04 (br.s, 2H), 7.92 (br.d, J=8.5 Hz, 2H), 7.76 (br.s, 2H), 7.60 (s, 1H), 7.18 (dd, J=8.6, 2.1 Hz, 2H), 6.81 (d, J=8.6 Hz, 2H), 5.52 (s, 4H), 3.79 (t, J=6.7 Hz, 4H), 3.70 (hept, J=6.6 Hz, DIEA-H.sup.+), 3.37 (s, 6H), 3.20 (q, J=7.4 Hz, DIEA-H.sup.+), 3.11 (t, J=6.7 Hz, 4H), 1.80 (s, 3H), 1.70 (s, 3H), 1.49 (s, 9H), 1.37-1.32 (m, DIEA-H.sup.+).
[0152] .sup.13C NMR (101 MHz, CD.sub.3OD): δ 171.1, 167.4, 165.3, 156.6, 156.4, 150.6, 147.2, 145.3, 140.2, 139.6, 133.9, 131.8, 130.1, 129.9, 129.7, 129.4, 128.7, 126.4, 126.2, 125.8, 125.5, 86.9, 83.6, 65.4, 55.8, 51.1, 43.8, 39.5, 38.2, 37.4, 34.8, 33.8, 28.2, 18.7, 17.3, 13.2 (including the signals of DIEA-H.sup.+ counterion).
[0153] HRMS (C.sub.52H.sub.52N.sub.6O.sub.20S.sub.2): m/z (positive mode)=1145.2753 (found [M+H].sup.+), 1145.2751 (calc.).
##STR00037##
[0154] Compound 13. A solution of compound 12 (24 mg, 21.0 μmol) in CH.sub.2Cl.sub.2 (600 μL) and TFA (300 μL) was stirred at rt for 1.5 h. The resulting mixture was diluted with CH.sub.2Cl.sub.2 and toluene, evaporated to dryness, the residue was redissolved in aq. dioxane and freeze-dried to give 13 as light pink solid (20 mg, 88%).
[0155] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.84 (t, J=5.4 Hz, 2H), 8.24-8.13 (m, 4H), 8.06 (s, 2H), 7.94 (d, J=8.5 Hz, 2H), 7.81 (d, J=2.2 Hz, 2H), 7.54 (s, 1H), 7.22 (dd, J=8.6, 2.1 Hz, 2H), 6.74 (d, J=8.6 Hz, 2H), 5.47 (s, 4H), 3.66-3.57 (m, DIEA-H.sup.+), 3.54 (t, J=7.1 Hz, 4H), 3.33 (s, 6H), 3.18-3.09 (m, DIEA-H.sup.+), 2.76 (t, J=7.1 Hz, 4H), 1.74 (s, 3H), 1.65 (s, 3H), 1.28-1.22 (m, DIEA-H.sup.+).
[0156] .sup.13C NMR (101 MHz, DMSO-d.sub.6): δ 168.7, 166.0, 163.8, 154.7, 154.0, 148.8, 145.4, 143.7, 138.9, 137.5, 132.1, 130.8, 128.5, 128.4, 127.9, 127.5, 127.4, 125.8, 125.1, 124.4, 123.9, 84.9, 63.7, 53.6, 50.0, 41.9, 37.9, 37.4, 36.4, 33.7, 33.2, 18.1, 16.7, 12.5 (including the signals of DIEA-H.sup.+ counterion).
[0157] HRMS (C.sub.48H.sub.44N.sub.6O.sub.2S.sub.2): m/z (positive mode)=1089.2129 (found [M+H].sup.+), 1089.2125 (calc.).
##STR00038##
[0158] Compound 14. In a flame-dried 10 mL tube, loaded with anhydrous K.sub.3PO.sub.4 (115 mg, 0.54 mmol, 4 eq) and 3 Å molecular sieves (55 mg), compound A3 [Butkevich et al. Chem. Eur. J. 2017, 23, 12114-12119] (100 mg, 0.135 mmol), compound 3 (83 mg, 0.325 mmol, 2.4 eq), JackiePhos Pd G3 precatalyst (24 mg, 0.02 mmol, 15 mol %) and JackiePhos ligand (16 mg, 0.02 mmol, 15 mol %) were loaded. The tube was sealed, anhydrous toluene (1.4 mL) was injected, the mixture was degassed on a Schlenk line and stirred at 110° C. for 6 h. It was then diluted with CH.sub.2Cl.sub.2, filtered through a plug of Celite (washing with CH.sub.2Cl.sub.2 and EtOAc—CH.sub.2Cl.sub.2), the filtrate was evaporated on Celite and the product was isolated by flash chromatography on Biotage Isolera system (25 g Interchim SiHP 30 μm cartridge, gradient 10% to 80% EtOAc/hexane) and freeze-dried from dioxane to yield 85 mg (66%) of 14 as white solid.
[0159] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.30 (t, J=1.1 Hz, 2H), 8.15-8.09 (m, 5H), 7.99 (dd, J=8.0, 0.7 Hz, 1H), 7.80 (br.s, 2H), 7.76 (dd, J=1.3, 0.7 Hz, 1H), 7.30 (dd, J=8.8, 2.5 Hz, 2H), 7.14 (br.s, 2H), 7.07 (d, J=8.8 Hz, 2H), 5.60 (s, 4H), 3.95 (s, 6H), 1.53 (s, 9H), 0.70 (s, 3H), 0.58 (s, 3H).
[0160] .sup.13C NMR (101 MHz, CDCl.sub.3): δ 170.2, 165.1, 164.0, 155.2, 152.6, 150.0, 139.1, 137.8, 137.4, 135.9, 134.7, 132.7, 130.4, 130.1, 127.7, 127.4, 126.1, 125.3, 124.5, 123.7, 120.5, 89.8, 82.7, 63.4, 53.1, 28.2, −0.26, −0.34.
[0161] HRMS (C.sub.47H.sub.42N.sub.4O.sub.16S.sub.1): m/z (positive mode)=947.2438 (found [M+H].sup.+), 947.2438 (calc.).
##STR00039##
[0162] Compound 15. To a solution of compound 14 (40 mg, 42.2 μmol) in THF (400 μL) and methanol (200 μL), a solution of LiOH—H.sub.2O (5.3 mg, 127 μmol, 3 eq) in water (85 μL) was added, and the resulting mixture was stirred vigorously at rt for 1 h. Acetic acid (200 μL) was then added, the reaction mixture was evaporated to dryness and dried in vacuo. The product was isolated by preparative HPLC (column: Thermo Scientific 250×21.2 mm 5 μm Hypersil Gold C18; gradient 40/60.fwdarw.90/10 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 15 as white solid (30 mg, 77%).
[0163] .sup.1H NMR (400 MHz, acetone-d.sub.6): δ 9.29 (br.s, 2H), 8.39 (dd, J=1.8, 0.8 Hz, 2H), 8.25 (d, J=8.3 Hz, 2H), 8.21 (dd, J=8.3, 2.0 Hz, 2H), 8.17 (dd, J=8.0, 1.3 Hz, 1H), 8.09-8.02 (m, 3H), 7.81 (d, J=0.6 Hz, 1H), 7.57 (dd, J=8.8, 2.5 Hz, 2H), 7.12 (d, J=8.8 Hz, 2H), 5.63 (s, 4H), 1.53 (s, 9H), 0.75 (s, 3H), 0.61 (s, 3H).
[0164] .sup.13C NMR (101 MHz, acetone-d.sub.6): δ 170.1, 165.9, 164.5, 156.4, 153.8, 151.0, 139.7, 139.3, 138.5, 136.01, 135.95, 133.8, 131.03, 131.01, 130.9, 128.5, 128.0, 126.8, 126.1, 124.9, 124.2, 121.1, 90.1, 82.9, 63.5, 28.1, −0.2, −0.3.
[0165] HRMS (C.sub.45H.sub.38N.sub.4O.sub.16S.sub.1): m/z (positive mode)=919.2124 (found [M+H].sup.+), 919.2125 (calc.).
##STR00040##
[0166] Compound 16. To a solution of compound 15 (28 mg, 30.5 μmol) in DMF (500 μL) and water (150 μL), taurine (38 mg, 306 μmol, 10 eq) and N,N-ethyldiisopropylamine (DIEA; 350 μL) were added followed by HATU (92 mg, 244 μmol, 8 eq; dissolved in 300 μL DMF). The resulting mixture was stirred vigorously at rt overnight (16 h). It was then evaporated to dryness and dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 am Uptisphere Strategy PhC4; gradient 20/80.fwdarw.70/30 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 16 as pale violet solid (15.5 mg, 45%).
[0167] .sup.1H NMR (400 MHz, CD.sub.3OD): δ 8.26 (br.s, 2H), 8.19 (d, J=8.5 Hz, 2H), 8.15 (dd, J=8.1, 1.3 Hz, 1H), 8.02 (dd, J=8.1, 0.7 Hz, 1H), 7.95 (dd, J=8.4, 1.8 Hz, 2H), 7.92 (d, J=2.3 Hz, 2H), 7.74 (app.t, J=1.0 Hz, 1H), 7.49 (dd, J=8.8, 2.4 Hz, 2H), 7.00 (d, J=8.8 Hz, 2H), 5.58 (s, 4H), 3.82 (br.t, J=6.4 Hz, 4H), 3.70 (hept, J=6.6 Hz, DIEA-H.sup.+), 3.18 (q, J=7.4 Hz, DIEA-H.sup.+), 3.13 (br.t, J=6.4 Hz, 4H), 1.53 (s, 9H), 1.36-1.30 (m, DIEA-H.sup.+), 0.73 (s, 3H), 0.62 (s, 3H).
[0168] .sup.13C NMR (101 MHz, CD.sub.3OD): δ 171.61, 167.58, 165.2, 157.0, 154.9, 150.2, 140.5, 140.0, 139.4, 139.0, 136.7, 134.4, 131.3, 129.1, 128.9, 128.6, 128.0, 127.0, 126.3, 125.3, 124.7, 121.4, 91.3, 83.7, 64.1, 55.8, 51.1, 43.8, 37.4, 28.3, 18.7, 17.3, 13.2, −0.25, −0.27 (including the signals of DIEA-H.sup.+ counterion).
[0169] HRMS (C.sub.49H.sub.48N.sub.6O.sub.20S.sub.2Si): m/z (positive mode)=1133.2208 (found [M+H].sup.+), 1133.2207 (calc.).
##STR00041##
[0170] Compound 17. A solution of compound 16 (15 mg, 13.3 μmol) in CH.sub.2Cl.sub.2 (600 μL) and TFA (300 μL) was stirred at rt for 1 h. The resulting mixture was diluted with CH.sub.2Cl.sub.2 and toluene, evaporated to dryness and dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 μm Uptisphere Strategy PhC4; gradient 20/80.fwdarw.60/40 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 17 as light violet solid (9.3 mg, 65%).
[0171] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 10.08 (s, 2H), 8.84 (t, J=5.3 Hz, 2H), 8.28-8.20 (m, 4H), 8.14 (dd, J=8.0, 1.3 Hz, 1H), 8.09 (d, J=7.7 Hz, 1H), 8.01-7.92 (m, 4H), 7.72 (s, 1H), 7.52 (dd, J=8.8, 2.4 Hz, 2H), 6.99 (d, J=8.8 Hz, 2H), 5.53 (s, 4H), 3.57 (q, J=6.9 Hz, 4H), 2.76 (t, J=6.9 Hz, 4H), 0.66 (s, 3H), 0.56 (s, 3H).
[0172] .sup.13C NMR (101 MHz, DMSO-d.sub.6): δ 169.1, 165.9, 164.0, 154.7, 152.8, 148.4, 139.2, 138.7, 137.4, 136.8, 134.9, 132.5, 130.4, 128.3, 127.4, 127.2, 126.9, 126.4, 125.3, 124.0, 123.3, 120.1, 89.2, 62.6, 50.0, 36.4, −0.5, −0.9.
[0173] HRMS (C.sub.45H.sub.40N.sub.6O.sub.20S.sub.2Si): m/z (positive mode)=1077.1583 (found [M+H].sup.+), 1077.1581 (calc.).
##STR00042##
[0174] Compound 18. In a flame-dried 10 mL tube, loaded with anhydrous K.sub.3PO.sub.4 (115 mg, 0.54 mmol, 4 eq) and 3 Å molecular sieves (55 mg), compound A3 [Butkevich et al. Chem. Eur. J. 2017, 23, 12114-12119] (100 mg, 0.135 mmol), compound 2 (87 mg, 0.325 mmol, 2.4 eq), JackiePhos Pd G3 precatalyst (24 mg, 0.02 mmol, 15 mol %) and JackiePhos ligand (16 mg, 0.02 mmol, 15 mol %) were loaded. The tube was sealed, anhydrous toluene (1.4 mL) was injected, the mixture was degassed on a Schlenk line and stirred at 110° C. for 6 h. It was then diluted with CH.sub.2Cl.sub.2, filtered through a plug of Celite (washing with CH.sub.2Cl.sub.2 and EtOAc—CH.sub.2Cl.sub.2), the filtrate was evaporated on Celite and the product was isolated by flash chromatography on Biotage Isolera system (25 g Interchim SiHP 30 μm cartridge, gradient 20% to 80% EtOAc/hexane) and freeze-dried from dioxane to yield 68 mg (52%) of 18 as white solid.
[0175] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.20-8.05 (m, 7H), 8.01 (dd, J=8.1, 0.7 Hz, 1H), 7.91 (br.s, 1H), 7.62 (d, J=2.4 Hz, 2H), 7.23 (dd, J=8.6, 2.4 Hz, 2H), 7.16 (d, J=8.6 Hz, 2H), 5.54 (s, 4H), 3.91 (s, 6H), 3.36 (s, 6H), 1.55 (s, 9H), 0.72 (s, 3H), 0.59 (s, 3H).
[0176] .sup.13C NMR (101 MHz, CDCl.sub.3): δ 169.8, 165.0, 164.1, 154.6, 154.2, 150.1, 142.7, 141.8, 137.7, 136.2, 134.6, 132.9, 131.0, 130.6, 129.9, 128.1, 127.4, 127.2, 126.2, 125.2, 125.1, 89.7, 82.7, 64.1, 53.0, 37.9, 28.2, −0.1, −0.6.
[0177] HRMS (C.sub.49H.sub.46N.sub.4O.sub.16Si): m/z (positive mode)=975.2741 (found [M+H].sup.+), 975.2751 (calc.).
##STR00043##
[0178] Compound 19. To a solution of compound 18 (20 mg, 20.5 μmol) in THF (200 μL) and methanol (100 μL), a solution of LiOH—H.sub.2O (2.6 mg, 61.5 μmol, 3 eq) in water (40 μL) was added, and the resulting mixture was stirred vigorously at rt for 1 h. Acetic acid (100 μL) was then added, the reaction mixture was evaporated to dryness and dried in vacuo. The product was isolated by preparative HPLC (column: Thermo Scientific 250×21.2 mm 5 am Hypersil Gold C18; gradient 40/60.fwdarw.90/10 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 19 as white solid (14.5 mg, 75%).
[0179] .sup.1H NMR (400 MHz, acetone-d.sub.6): δ 8.25 (br.s, 2H), 8.21-8.15 (m, 5H), 8.07 (dd, J=8.0, 0.7 Hz, 1H), 7.90 (dd, J=1.3, 0.7 Hz, 2H), 7.88 (d, J=2.4 Hz, 2H), 7.41 (dd, J=8.7, 2.4 Hz, 2H), 7.21 (d, J=8.7 Hz, 2H), 5.54 (s, 4H), 3.36 (s, 6H), 1.52 (s, 9H), 0.74 (s, 3H), 0.60 (s, 3H).
[0180] .sup.13C NMR (101 MHz, acetone-d.sub.6): δ 169.9, 165.8, 164.6, 155.7, 155.1, 151.2, 143.9, 142.3, 138.6, 136.4, 135.8, 133.7, 131.8, 131.4, 131.3, 130.9, 128.5, 128.2, 127.8, 127.0, 126.0, 125.2, 89.9, 83.1, 64.4, 37.9, 28.1, −0.3, −0.4.
[0181] HRMS (C.sub.47H.sub.42N.sub.4O.sub.16Si): m/z (positive mode)=947.2429 (found [M+H].sup.+), 947.2438 (calc.).
##STR00044##
[0182] Compound 20. To a solution of compound 19 (18.5 mg, 19.5 μmol) in DMF (300 μL) and water (100 μL), taurine (25 mg, 200 μmol, 10 eq) and N,N-ethyldiisopropylamine (DIEA; 100 μL) were added followed by HATU (45 mg, 117 μmol, 6 eq; dissolved in 150 μL DMF). The resulting mixture was stirred vigorously at rt overnight (16 h). It was then evaporated to dryness and dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 am Uptisphere Strategy PhC4; gradient 20/80.fwdarw.70/30 A:B, A=acetonitrile, B=50 mM ammonium formate in water, pH=3.5-4.0; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 20 as pale violet solid (23.5 mg, remainder ammonium formate).
[0183] .sup.1H NMR (400 MHz, CD.sub.3OD): δ 8.43 (s, 1H), 8.14 (dd, J=8.0, 1.3 Hz, 1H), 8.12-7.99 (m, 5H), 7.92 (dd, J=8.4, 1.9 Hz, 2H), 7.77 (s, 3H), 7.32 (dd, J=8.7, 2.4 Hz, 2H), 7.17 (d, J=8.7 Hz, 2H), 5.49 (s, 4H), 3.79 (t, J=6.7 Hz, 4H), 3.34 (s, 6H), 3.10 (t, J=6.7 Hz, 4H), 1.50 (s, 9H), 0.70 (s, 3H), 0.56 (s, 3H).
[0184] .sup.13C NMR (101 MHz, CD.sub.3OD): δ 171.4, 167.3, 165.2, 156.7, 156.4, 150.6, 144.1, 142.9, 140.2, 139.1, 136.7, 133.8, 132.5, 131.5, 129.8, 128.8, 128.4, 128.1, 127.2, 126.2, 125.3, 90.6, 83.7, 65.4, 51.1, 38.2, 37.4, 28.3, −0.1, −0.4.
[0185] HRMS (C.sub.51H.sub.52N.sub.6O.sub.20S.sub.2Si): m/z (positive mode)=1161.2518 (found [M+H].sup.+), 1161.2520 (calc.).
##STR00045##
[0186] Compound 21. A solution of compound 20 (23.5 mg, 19.5 μmol) in CH.sub.2Cl.sub.2 (500 μL) and TFA (250 μL) was stirred at rt for 1.5 h. The resulting mixture was diluted with CH.sub.2Cl.sub.2 and toluene, evaporated to dryness; dissolved the residue in CH.sub.2Cl.sub.2 (3 mL) and N,N-ethyldiisopropylamine (DIEA; 200 μL), evaporated to dryness and dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 am Uptisphere Strategy PhC4; gradient 20/80.fwdarw.100/0 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 21 as bluish solid (17.5 mg, 81% over 2 steps).
[0187] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.84 (t, J=5.3 Hz, 2H), 8.20-8.11 (m, 4H), 8.05 (br.s, 2H), 7.95 (dd, J=8.4, 1.9 Hz, 2H), 7.86-7.79 (m, 3H), 7.37 (dd, J=8.6, 2.5 Hz, 2H), 7.06 (d, J=8.6 Hz, 2H), 5.46 (s, 4H), 3.62-3.52 (m, 6H), 2.82-2.71 (m, 4H), 0.64 (s, 3H), 0.52 (s, 3H).
[0188] .sup.13C NMR (101 MHz, DMSO-d.sub.6): δ 168.8, 165.9, 163.8, 154.0, 153.9, 148.7, 142.6, 140.6, 138.9, 136.9, 135.4, 132.0, 130.8, 130.7, 128.5, 127.5, 127.3, 127.2, 126.8, 126.6, 125.2, 124.3, 89.0, 63.7, 50.0, 37.3, 36.4, −0.6, −1.3.
[0189] HRMS (C.sub.47H.sub.44N.sub.6O.sub.20S.sub.2Si): m/z (positive mode)=1105.1897 (found [M+H].sup.+), 1105.1894 (calc.).
Example 2
Synthesis of Photoactivatable Labels
[0190] ##STR00046##
[0191] Compound 9-NHS. A solution of compound 9 (8 mg, 7.5 μmol) and N,N-ethyldiisopropylamine (DIEA; 60 μL) in DMF (150 μL) was treated with N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (TSTU; 6.8 mg, 22.6 μmol in 50 μL DMF). The reaction mixture was stirred at rt for 1.5 h, the solvents were evaporated to dryness and the residue was dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 μm Uptisphere Strategy PhC4; gradient 20/80.fwdarw.60/40 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 9-NHS as light orange solid (7 mg, 81%).
[0192] HRMS (C.sub.49H.sub.41N.sub.7O.sub.23S.sub.2): m/z (positive mode)=1160.1767 (found [M+H].sup.+), 1160.1768 (calc.).
##STR00047##
[0193] Compound 13-NHS. A solution of compound 13 (7 mg, 6.43 μmol) and N,N-ethyldiisopropylamine (DIEA; 50 μL) in DMF (150 μL) was treated with N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (TSTU; 5.8 mg, 19.4 μmol in 50 μL DMF). The reaction mixture was stirred at rt for 1 h, the solvents were evaporated to dryness and the residue was dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 μm Uptisphere Strategy PhC4; gradient 20/80.fwdarw.60/40 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 13-NHS as light pink solid (9.3 mg, ˜100%, bis-DIEA salt).
[0194] HRMS (C.sub.52H.sub.47N.sub.7O.sub.22S.sub.2): m/z (positive mode)=1186.2287 (found [M+H].sup.+), 1186.2288 (calc.).
##STR00048##
[0195] Compound 17-NHS. A solution of compound 17 (6.5 mg, 4.6 μmol) and N,N-ethyldiisopropylamine (DIEA; 50 μL) in DMF (150 μL) was treated with N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (TSTU; 4.2 mg, 14 μmol in 40 μL DMF). The reaction mixture was stirred at rt for 1 h, at which time the LC/MS analysis showed incomplete conversion, so another portion of TSTU (4.2 mg, 14 μmol in 40 μL DMF) was added and the mixture was stirred for another 1 h period. The solvents were evaporated to dryness and the residue was dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 μm Uptisphere Strategy PhC4; gradient 20/80.fwdarw.60/40 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 17-NHS as light violet solid (7.2 mg, ˜100%, bis-DIEA salt).
[0196] HRMS (C.sub.49H.sub.43N.sub.7O.sub.22S.sub.2Si): m/z (positive mode)=1174.1745 (found [M+H].sup.+), 1174.1745 (calc.).
##STR00049##
[0197] Compound 17-Halo. A solution of compound 17 (4 mg, 3.71 μmol) and N,N-ethyldiisopropylamine (DIEA; 30 μL) in DMF (100 μL) was treated with HaloTag(O2)-NH.sub.2 ligand (1.7 mg, 7.43 μmol in 25 μL DMF) and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP; 3.9 mg, 7.43 μmol in 30 μL DMF). The resulting light yellow clear solution was stirred at rt for 2 h, the solvents were then evaporated to dryness and the residue was dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 μm Uptisphere Strategy PhC4; gradient 20/80.fwdarw.70/30 A:B, A=acetonitrile, B=50 mM ammonium formate in water, pH=3.5-4.0; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 17-Halo as white solid (4 mg, 84%).
[0198] HRMS (C.sub.55H.sub.60ClN.sub.7O.sub.21S.sub.2Si): m/z (positive mode)=1282.2810 (found [M+H].sup.+), 1282.2814 (calc.).
##STR00050##
[0199] Compound 21-NHS. A solution of compound 21 (4.4 mg, 4 μmol) and N,N-ethyldiisopropylamine (DIEA; 40 μL) in DMF (150 μL) was treated with N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (TSTU; 3.7 mg, 12 μmol in 40 μL DMF). The reaction mixture was stirred at rt for 1 h, the solvents were evaporated to dryness and the residue was dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 am Uptisphere Strategy PhC4; gradient 20/80.fwdarw.60/40 A:B, A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 21-NHS as white solid (3.5 mg, 73%).
[0200] HRMS (C.sub.51H.sub.45N.sub.7O.sub.22S.sub.2Si): m/z (positive mode)=1202.2062 (found [M+H].sup.+), 1202.2058 (calc.).
##STR00051##
[0201] Compound 21-Maleimide. A solution of compound 21 (5 mg, 4.53 μmol) and N,N-ethyldiisopropylamine (DIEA; 45 μL) in DMF (100 μL) was treated with N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (TSTU; 4.1 mg, 13.6 μmol in 40 μL DMF). The reaction mixture was stirred at rt for 1 h, and DIEA (45 μL) was added followed by the solution of 1-(2-aminoethyl)maleimide hydrochloride (1.6 mg, 9.06 μmol in 30 μL DMF). The mixture was left stirring at rt overnight (18 h), the solvents were then evaporated to dryness and the residue was dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 μm Uptisphere Strategy PhC4; gradient 20/80.fwdarw.60/40 A:B, A=acetonitrile, B=50 mM triethylammonium bicarbonate in water, pH=7.0-7.5; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 21-Maleimide as yellowish solid (3.8 mg, 68%).
[0202] HRMS (C.sub.53H.sub.50N.sub.8O.sub.21S.sub.2Si): m/z (positive mode)=1227.2375 (found [M+H].sup.+), 1227.2374 (calc.).
##STR00052##
[0203] Compound 21-BG. A solution of compound 21 (5 mg, 4.53 μmol) and N,N-ethyldiisopropylamine (DIEA; 45 μL) in DMSO (100 μL) was treated with N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (TSTU; 4.1 mg, 13.6 μmol in 40 μL DMF). The reaction mixture was stirred at rt for 1 h, and DIEA (45 μL) was added followed by the solution of 6-((4-(aminomethyl)benzyloxy)-7H-purin-2-amine (BG-NH.sub.2; 2.4 mg, 9.06 μmol in 70 μL DMSO). The mixture was left stirring at rt overnight (18 h), the solvents were then evaporated to dryness and the residue was dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 μm Uptisphere Strategy PhC4; gradient 20/80.fwdarw.60/40 A:B, A=acetonitrile, B=50 mM triethylammonium bicarbonate in water, pH=7.0-7.5; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 21-BG as white solid (3.2 mg, 52%).
[0204] HRMS (C.sub.60H.sub.56N.sub.12O.sub.2S.sub.2Si): m/z (positive mode)=1357.3021 (found [M+H].sup.+), 1357.3017 (calc.).
##STR00053##
[0205] Compound 21-Halo. A solution of compound 21 (5 mg, 4.53 μmol) and N,N-ethyldiisopropylamine (DIEA; 30 μL) in DMF (100 μL) was treated with HaloTag(O2)-NH.sub.2 ligand (2 mg, 9.1 μmol in 25 μL DMF) and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP; 4.7 mg, 9.1 μmol in 30 μL DMF). The resulting light yellow clear solution was stirred at rt for 4 h, the solvents were then evaporated to dryness and the residue was dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 μm Uptisphere Strategy PhC4; gradient 20/80.fwdarw.70/30 A:B, A=acetonitrile, B=50 mM ammonium formate in water, pH=3.5-4.0; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 21-Halo as white solid (4.9 mg, 83%).
[0206] HRMS (C.sub.57H.sub.64ClN.sub.7O.sub.21S.sub.2Si): m/z (positive mode)=1310.3126 (found [M+H].sup.+), 1310.3127 (calc.).
##STR00054##
[0207] Compound 21-Picolyl azide. A solution of compound 21 (5 mg, 4.53 μmol) and N,N-ethyldiisopropylamine (DIEA; 45 μL) in DMF (100 μL) was treated with N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (TSTU; 4.1 mg, 13.6 μmol in 40 μL DMF). The reaction mixture was stirred at rt for 1 h, and DIEA (45 μL) was added followed by the solution of 5 (5 mg, 11.2 μmol in 30 μL DMF). The mixture was left stirring at rt overnight (18 h), the solvents were then evaporated to dryness and the residue was dried in vacuo. The product was isolated by preparative HPLC (column: Interchim 250×21.2 mm 5 μm Uptisphere Strategy PhC4; gradient 10/90.fwdarw.60/40 A:B A=0.1% v/v HCO.sub.2H in acetonitrile, B=0.1% v/v HCO.sub.2H in water; detection at 220 nm), fractions containing the product were evaporated (bath temperature 30° C.), and the residue was freeze-dried from aq. dioxane to give 21-Picolyl azide as off-white solid (4.9 mg, 83%).
[0208] HRMS (C.sub.56H.sub.54N.sub.12O.sub.20S.sub.2Si): m/z (positive mode)=1307.2860 (found [M+H].sup.+), 1307.2861 (calc.).
Example 3
Characterization of Exemplary Compounds of the Present Invention
[0209] General Materials and Methods
[0210] All chemical reagents (TCI, Sigma-Aldrich, Alfa Aesar) and dry solvents for synthesis (over molecular sieves, AcroSeal package, Acros Organics) were purchased from reputable suppliers and were used as received without further purification. The products were lyophilized from a suitable solvent system using Alpha 2-4 LDplus freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH).
[0211] Thin Layer Chromatography
[0212] Normal phase TLC was performed on silica gel 60 F.sub.254 (Merck Millipore, Germany). For TLC on reversed phase silica gel 60 RP-18 F.sub.254s (Merck Millipore) was used. Compounds were detected by exposing TLC plates to UV-light (254 or 366 nm) or heating with vanillin stain (6 g vanillin and 1.5 mL conc. H.sub.2SO.sub.4 in 100 mL ethanol), unless indicated otherwise.
[0213] Flash Chromatography Preparative flash chromatography was performed with an automated Isolera One system with Spektra package (Biotage AG) using commercially available cartridges of suitable size as indicated (RediSep Rf series from Teledyne ISCO, Puriflash Silica HP 30 μm series from Interchim).
[0214] Nuclear Magnetic Resonance (NMR)
[0215] NMR spectra were recorded on a Bruker DPX 400 spectrometer. All spectra are referenced to tetramethylsilane as an internal standard (6=0.00 ppm). Multiplicities of the signals are described as follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet or overlap of non-equivalent resonances; br=broad signal. Coupling constants .sup.nJ.sub.X-Y are given in Hz, where n is the number of bonds between the coupled nuclei X and Y (J.sub.H-H are always listed as J without indices).
[0216] Mass-Spectrometry (MS)
[0217] Low resolution mass spectra (100-1500 m/z) with electro-spray ionization (ESI) were obtained on a Shimadzu LC-MS system described below. High resolution mass spectra (HRMS) were obtained on a maXis II ETD (Bruker) with electrospray ionization (ESI) at the Mass Spectrometry Core facility of the Max-Planck Institute for Medical Research (Heidelberg, Germany).
[0218] High-Performance Liquid Chromatography (HPLC)
[0219] Analytical liquid chromatography-mass spectrometry was performed on an LC-MS system (Shimadzu): 2×LC-20AD HPLC pumps with DGU-20A3R solvent degassing unit, SIL-20ACHT autosampler, CTO-20AC column oven, SPD-M30A diode array detector and CBM-20A communication bus module, integrated with CAMAG TLC-MS interface 2 and LCMS-2020 spectrometer with electrospray ionization (ESI, 100-1500 m/z). Analytical column: Hypersil GOLD 50×2.1 mm 1.9 μm, standard conditions: sample volume 1-2 μL, solvent flow rate 0.5 mL/min, column temperature 30° C. General method: isocratic 95:5 A:B over 2 min, then gradient 95:5.fwdarw.:100 A:B over 5 min, then isocratic 0:100 A:B over 2 min; solvent A=water+0.1% v/v HCO.sub.2H, solvent B=acetonitrile+0.1% v/v HCO.sub.2H.
[0220] Preparative high-performance liquid chromatography was performed on a Buchi Reveleris Prep system using the suitable preparative columns and conditions as indicated for individual preparations. Method scouting was performed on a HPLC system (Shimadzu): 2×LC-20AD HPLC pumps with DGU-20A3R solvent degassing unit, CTO-20AC column oven equipped with a manual injector with a 20 μL sample loop, SPD-M20A diode array detector, RF-20A fluorescence detector and CBM-20A communication bus module; or on a Dionex Ultimate 3000 UPLC system: LPG-3400SD pump, WPS-3000SL autosampler, TCC-3000SD column compartment with 2×7-port 6-position valves and DAD-3000RS diode array detector. The test runs were performed on analytical columns with matching phases (HPLC: Interchim 250×4.6 mm 10 μm C18HQ, Interchim 250×4.6 mm 5 μm PhC4, solvent flow rate 1.2 mL/min; UPLC: Interchim C18HQ or PhC4 75×2.1 mm 2.2 μm, ThermoFisher Hypersil GOLD 100×2.1 mm 1.9 μm, solvent flow rate 0.5 mL/min).
[0221] STED (Stimulated Emission Depletion) Microscopy
[0222] STED and confocal counterpart images were acquired using the commercially available STED 595/775 quad scanning microscope (Abberior Instruments, Göttingen, Germany) equipped with an Olympus IX83 microscope stand and an Olympus UplanSApo 100×/1.4 OIL objective. Dyes were excited respective their excitation (with 485 nm, 561 nm or 640 nm laser); the 595 nm and 775 nm STED lasers were pulsed at 40 MHz with a pulse length of ˜1 ns. Imaging and image processing was done with ImSpector software, and the images are displayed as raw data.
Example 4
STED Optical Microscopy of Cells Using Exemplary Novel Dyes of the Invention Coupled to Antibody
[0223] Amino-reactive NHS-esters of the present dyes were coupled to secondary antibody using a standard coupling protocol. In brief, the reactive dye (9-NHS, 13-NHS, 17-NHS or 21-NHS; 100 μg) was dissolved in anhydrous DMSO (10 μl), mixed with 1 mg antibody in PBS+0.1 M NaHCO.sub.3, stirred for 1 h and purified using a size exclusion column (PD 10, GE Healthcare). U2OS cells, grown on coverslips, were fixed using ice-cold methanol, permeabilzied with Triton X-100, blocked with 2% (w/v) BSA in PBS and treated with primary and secondary antibody in the same buffer with PBS washes in between. The samples were mounted on microscope slides with PBS and sealed with nail polish. In order to activate the dyes, the sample was illuminated with a 405 nm broadband LED for the duration of several seconds.
Example 5
STED Optical Microscopy of Cells Using Exemplary Novel Dyes of the Invention with Self-Labelling Enzymes
[0224] BG- or Halo-derivatives of the dyes of the present invention were dissolved in DMSO. Cells with stable expression of protein fusions with SNAP- or Halo-Tag [Ratz et al. Sci. Rep. 2015, 5, 9592; Butkevich et al. ACS Chem. Biol. 2018, 13(2), 475-480] or transfected cells were grown on coverslips and fixed with 2.4% (w/v) paraformaldehyde in PBS, permeabilized with Triton X-100, and treated with 21-BG or 21-Halo (200 nM in PBS) for one hour. After washing with PBS, the samples were mounted on microscope slides with PBS and sealed with nail polish. The caged dyes were activated by illumination with a 405 nm broadband LED for several seconds.
Example 6
STED Optical Microscopy of M13 Bacteriophages Labelled Using Click Chemistry
[0225] Purified M13 bacteriophages were modified with terminal alkyne groups using 3-(2-propyn-1-yloxy)propanoic acid N-hydroxysuccinimidyl ester (“Propargyl-N-hydroxysuccinimidyl ester”, CAS #1174157-65-3) using the same protocol as for the labelling of antibodies. The alkyne modified phages were deposited on poly-L-lysine coated coverslips and treated with the picolyl-azide conjugate of one of the dyes of the present invention (21-Picolyl azide) under standard copper-catalyzed click chemistry conditions [Jiang et al. Bioconjugate Chem. 2014, 25(4), 698-706]. The labelled samples were treated with primary and secondary antibodies before mounting (see