4.2. Synthesis of the N-hydroxysuccinimidyl Ester of Hapten ALa
4.2.1. Preparation of methyl 5-((5-hydroxy-2,2-dimethyl-4-oxo-4H-benzo[d][1,3]dioxin-7-yl)oxy)pentanoate (3)
Methyl 5-bromovalerate (186 µL, 266 mg, 1.36 mmol, 1.1 equiv) was added to a solution of 1,3-benzodioxinone 1 (260 mg, 1.237 mmol), KI (83 mg, 0.500 mmol, 0.4 equiv), Bu4NBr (0.5 mg, 1.6 µmol) y K2CO3 (188 mg, 1.36 mmol, 1 equiv) in dry acetone (9 mL) under nitrogen. After heating the mixture at reflux for 16 h, the acetone was eliminated at reduced pressure and the resulting brownish residue was diluted with water and extracted with Et2O. The combined organic layers were washed with water and brine, dried over anhydrous MgSO4 and concentrated under vacuum. The obtained crude product was purified by chromatography on silica gel, using hexane-EtOAc mixtures from 9:1 to 7:3 as eluent, to afford, in order of elution, dialkylated derivative 2 (67.7 mg, 12.5%) and monoalkylated compound 3 (300 mg, 75%) as a white solid. Mp 97.3–98.2 °C (crystallized from hexane-EtOAc) IR (ATR) νmax (cm−1) 3017 (w), 1740 (s), 1672 (s), 1251 (s), 1159 (s), 840 (s), 794 (s); 1H NMR (300 MHz, CDCl3) δ 10.42 (s, 1H, OH), 6.11 (d, J = 2.2 Hz, 1H, H-6), 5.97 (d, J = 2.2 Hz, 1H, H-8), 3.98 (t, J = 5.8 Hz, 2H, H2-5), 3.68 (s, 3H, OCH3), 2.39 (t, J = 7.0 Hz, 2H, H2-2), 1.81 (m, 4H, H2-3 and H2-4), 1.72 (s, 6H, 2×CH3); 13C NMR (75 MHz, CDCl3) δ 173.8 (CO2CH3), 167.2 (CO), 165.3 (C-7), 163.2 (C-8a), 156.9 (C-5), 107.0 (C-2), 96.3 (CH-6), 95.1 (CH-8), 93.1 (C-4a), 68.1 (CH2-5), 51.7 (OCH3), 33.7 (CH2-2), 28.4 (CH2-4), 25.8 (2×CH3), 21.6 (CH2-3); HRMS (TOF MS ES+) m/z calculated for C16H20O7 [M + H]+ 325.1282, found 325.1283.
4.2.2. Preparation of methyl 5-((2,2-dimethyl-4-oxo-5-(((trifluoromethyl)sulfonyl)oxy)-4H-benzo[d][1,3]dioxin-7-yl)oxy)pentanoate (4)
Triflic anhydride (230 µL, 1.369 mmol, 1.5 equiv) was added to a solution of phenol 3 (296 mg, 0.913 mmol) in anhydrous pyridine (4.5 mL) at 0 °C under nitrogen. The reaction mixture was allowed to warm to rt and stirred for 20 h, then cooled down to 0 °C and treated with a saturated aqueous solution of NaHCO3, stirred for a few minutes at rt and then extracted with Et2O. The organic layers were washed with water, a 1% (w/v) aqueous solution of CuSO4 and brine, dried over anhydrous MgSO4 and concentrated at reduced pressure. The obtained residue was chromatographed on silica gel, using hexane-EtOAc mixtures from 9:1 to 8:2 as eluent, to give aryl triflate 4 (378.8 mg, 91%) as a white semisolid. IR (ATR) νmax (cm−1) 3114 (w), 1746 (s), 1733 (s), 1381 (s), 1228 (s), 1167 (s), 869 (s); 1H NMR (300 MHz, CDCl3) δ 6.51 (d, J = 2.3 Hz, 1H, H-6), 6.45 (d, J = 2.3 Hz, 1H, H-8), 4.02 (t, J = 5.8 Hz, 2H, H2-5), 3.68 (s, 3H, CO2CH3), 2.40 (t, J = 6.9 Hz, 2H, H2-2), 1.85 (m, 4H, H2-3 and H2-4), 1.73 (s, 6H, 2xCH3); 13C NMR (75 MHz, CDCl3) δ 173.7 (CO2CH3), 165.0 (CO), 158.9 (C-7), 157.2 (C-8a), 150.1 (C-5), 106.7 (C-2), 105.7 (CH-6), 101.6 (CH-8), 101.0 (C-4a), 68.9 (CH2-5), 51.8 (OCH3), 33.6 (CH2-2), 28.3 (CH2-4), 25.7 (2×CH3), 21.5 (CH2-3); 19F NMR (282 MHz, CDCl3) δ 73.1 (s, CF3); HRMS (TOF, ES+) m/z calculated for C17H23F3NO9S [M + NH4]+ 474.1040; found 474.1027.
4.2.3. Preparation of methyl 5-((5-(2,4-bis(methoxymethoxy)-6-methylphenyl)-2,2-dimethyl-4-oxo-4H-benzo[d][1,3]dioxin-7-yl)oxy)pentanoate (7)
(i) Preparation of boronic acid
6. A solution of
n-BuLi in hexane (1.3 M, 336 µL, 0.436 mmol, 1.05 equiv) was dropwise added to a solution of aryl bromide
5 (122.3 mg, 0.420 mmol) in anhydrous THF (2.5 mL) at −78 °C under nitrogen. The reaction mixture was stirred at this temperature for 40 min, B(O
iPr)
3 (322 µL, 1.386 mmol, 3.3 equiv) was then added and the mixture stirred for 1.5 h. After this time, the dry ice bath was replaced by an ice bath and the mixture treated with an aqueous saturated solution of NH
4Cl (0.7 mL), then diluted with water and extracted with Et
2O. The organic layers were washed with brine, dried over anhydrous Na
2SO
4 and concentrated under reduced pressure to give boronic acid
6 (100.0 mg, 93%) as a thick oil that was immediately used in the next reaction without further purification since it is relatively prone to protodeboronation [
26].
1H NMR (300 MHz, DMSO-
d6) δ 7.91 (s, 1H, BOH), 6.49 (d,
J = 2.1 Hz, 1H, H-4), 6.47 (d,
J = 2.10 Hz, 1H, H-6), 5.12 and 5.09 (each s, 2H each, 2×OCH
2O), 3.37 and 3.34 (each s, 3H each, 2×OCH
3), 2.20 (s, 3H, CH
3).
(ii) Coupling reaction between aryl triflate 4 and boronic acid 6. A mixture of the above obtained boronic acid 6 (47.4 mg, 0.185 mmol), aryl triflate 4 (41.6 mg, 0.091 mmol), powdered K2CO3 (43.2 mg, 0.312 mmol) and Pd(PPh3)4 (11.4 mg, 9.9 µmol) under nitrogen was dissolved in anhydrous DMF (1.2 mL), previously degassed by three freeze-vacuum-thaw cycles. The mixture was heated at 93 °C and stirred at this temperature for 24 h. The mixture was cooled to rt, quenched with water and extracted with EtOAc. The combined organic layers were successively washed with water, a 1.5% (w/v) aqueous solution of LiCl and brine, and dried over anhydrous MgSO4. The obtained residue after evaporation of the solvent was chromatographed on silica gel, using hexane-EtOAc 8:2 as eluent, to afford biaryl compound 7 (35.2 mg, 75%) as a yellowish oil. 1H NMR (300 MHz, CDCl3) δ 6.71 (d, J = 2.3 Hz, 1H, H-6), 6.64 (d, J = 2.3 Hz, 1H, H-8), 6.42 (d, J = 2.5 Hz, 1H, H-5), 6.40 (d, J = 2.5 Hz, 1H, H-3), 5.18 (AB system, J = 6.7 Hz, 2H, OCH2O), 4.98 (AB system, J = 6.6 Hz 2H, OCH2O), 3.99 (t, J = 5.6 Hz, 2H, H2-5), 3.67 (s, 3H, CO2CH3), 3.50 and 3.29 (each s, 3H each, 2×OCH3), 2.39 (t, J = 6.8 Hz, 2H, H2-2), 2.04 (s, 3H, CH3 Ph), 1.81 (m, 4H, H2-3 and H2-4), 1.71 (s, 6H, 2×CH3); 13C NMR (75 MHz, CDCl3) δ 173.9 (CO2CH3), 164.1 (CO), 159.2 (C-7), 158. 5 (C-8a), 157.4 (C-4), 154.8 (OC-2), 142.9 (C-6), 136.9 (C-5), 123.9 (C-1), 113.8 (CH-3), 110.6 (CH-8), 106.8 (C-2), 105.1 (CH-5), 101.2 (CH-6), 94.9 and 94.7 (2×OCH2O), 68.1 (CH2-5), 56.3 and 55.9 (2×OCH3), 51.8 (CO2CH3), 33.7 (CH2-2), 28.6 (CH2-4), 26.3 and 25.2 (2×CH3), 21.7 (CH2-3), 20.7 (CH3 Ph). HRMS (TOF, ES+) m/z calculated for C27H34O10 [M + H]+ 519.2225; found 519.2212.
4.2.4. Preparation of methyl 5-((3,7-dihydroxy-1-methyl-6-oxo-6H-benzo[c]chromen-9-yl)oxy)pentanoate (8)
A 3 M solution of HCl in MeOH (150 µL, 0.450 mmol) was added to a solution of biaryl compound 7 (26.1 mg, 0.050 mmol) in anhydrous MeOH (1.5 mL) and the reaction mixture was stirred at rt for 22 h. After concentration under vacuum, the residue was dissolved in anhydrous CH2Cl2 (4 mL) and treated with trifluoroacetic acid (430 µL). Following stirring for 20 h at rt, thin layer chromatography showed the formation of a single compound and all the volatiles were removed under vacuum, using CHCl3 to co-evaporate the last traces of TFA. The obtained residue was purified by chromatography, using CHCl3 as eluent, to give benzochromenone derivative 8 (18.1 mg, 97%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 11.79 and 10.33 (each s, 1H each, 2×OH), 7.13 (d, J = 2.2 Hz, 1H, H-10), 6.69 (d, J = 2.6 Hz, 1H, H-2), 6.61 (d, J = 2.6 Hz, 1H, H-4), 6.55 (d, J = 2.2 Hz, 1H, H-8), 4.11 (t, J = 5.9 Hz, 2H, H2-5), 3.59 (s, 3H, OCH3), 2.68 (s, 3H, CH3), 2.41 (t, J = 7.0 Hz, 2H, H2-2), 1.85–1.56 (m, 4H, H2-3 and H2-4); 13C NMR (75 MHz, DMSO-d6) δ 173.2 (CO2CH3), 165.5 (CO), 164.6 (C-9), 164.1 (C-7), 158.5 (C-3), 152.6 (C-4a), 138.4 (C-1), 137.7 (C-10a), 117.5 (CH2-2), 108.8 (C-10b), 103.6 (CH-10), 101.6 (CH-4), 99.5 (CH-8), 98.3 (C-6a), 67.8 (CH2-5), 51.2 (CO2CH3), 32.8 (CH2-2), 27.8 (CH2-4), 25.0 (CH3), 21.1 (C-3); HRMS (TOF, ES+) m/z calculated for C20H21O7 [M + H]+ 373.1282; found 373.1278.
4.2.5. Preparation of 5-((3,7-dihydroxy-1-methyl-6-oxo-6H-benzo[c]chromen-9-yl)oxy)pentanoic acid (Hapten ALa)
Lipase from Candida antarctica immobilized on acrylic resin (23 mg) was added to a solution of methyl ester 8 (16.6 mg, 0.0446 mmol) in a 4:1 mixture of 100 mM sodium phosphate buffer (pH 7.4) and THF (1.5 mL) at 30 °C. The resulting heterogeneous mixture was smoothly stirred for 24 h at rt and then filtered to separate the enzyme. The filtrated and washing THF phases were combined, diluted with EtOAc, washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo to afford hapten ALa (14.9 mg, 93%) as a white amorphous solid. 1H NMR (300 MHz, THF-d8) δ 11.99 and 9.19 (each s, 1H each, 2×OH), 7.27 (d, J = 2.2 Hz, 1H, H-10), 6.67 (d, J = 2.7 Hz, 1H, H-2), 6.61 (d, J = 2.6 Hz, 1H, H-4), 6.56 (d, J = 2.2 Hz, 1H, H-8), 4.13 (t, J = 6.1 Hz, 2H, H2-5), 2.78 (s, 3H, CH3), 2.33 (t, J = 7.1 Hz, 2H, H2-2), 1.91–1.77 (m, 4H, H2-3 and H2-4); 13C NMR (126 MHz, THF-d8) δ 174.5 (CO2H), 167.1 (CO), 166.4 (C-9), 166.2 (C-7), 159.9 (C-3), 154.5 (C-4a), 139.5 (C-1), 139.2 (C-10a), 118.5 (CH-2), 110.7 (C-10b), 105.1 (CH-10), 102.8 (CH-4), 100.3 (CH-8), 100.0 (C-6a), 69.2 (CH2-5), 34.0 (CH2-2), 29.6 (CH2-4), 25.0 (CH3, overlapped with solvent signal), 22.6 (CH2-3); HRMS (TOF, ES+) m/z calculated for C19H18O7 [M + H]+ 359.1125; found 359.1122.
4.2.6. Preparation of 2,5-dioxopyrrolidin-1-yl 5-((3,7-dihydroxy-1-methyl-6-oxo-6H-benzo[c]chromen-9-yl)oxy)pentanoate (ALa-NHS Ester)
A solution of hapten ALa (11.0 mg, 30.7 µmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC∙HCl) (7.0 mg, 36.8 µmol, 1.2 equiv) and N-hydroxisuccinimide (NHS) (5.0 mg, 43.4 µmol, 1.4 equiv) in anhydrous DMF (0.6 mL) was stirred at rt under nitrogen overnight. The reaction mixture was diluted with CH2Cl2, washed with water, a 1.5% (w/v) aqueous solution of LiCl and brine, dried over anhydrous MgSO4 and concentrated under reduced pressure to give the N-hydroxysuccinimidyl ester of hapten ALa, ALa-NHS ester, (13.8 mg, ca. 99% of crude product) as a slightly yellowish oil which was used immediately for the preparation of the corresponding protein bioconjugates. 1H NMR (500 MHz, THF-d8) δ 11.99 and 9.06 (each s, 1H each, 2×OH), 7.28 (d, J = 2.2 Hz, 1H, H-10), 6.66 (d, J = 2.6 Hz, 1H, H-2), 6.60 (d, J = 2.7 Hz, 1H, H-4), 6.57 (d, J = 2.2 Hz, 1H, H-8), 4.17 (t, J = 5.8 Hz, 2H, H2-5), 2.78 (s, 3H, CH3), 2.75 (br s, 4H, COCH2CH2CO), 2.72 (t, J = 7.0 Hz, 2H, H2-2), 1.95 (m, 4H, H2-3 and H2-4).
4.3. Synthesis of the N-hydroxysuccinimidyl Ester of Hapten ALb
4.3.1. Preparation of 3,5-bis(benzyloxy)-2′,4′-bis(methoxymethoxy)-6′-methyl-[1,1′-biphenyl]-2-carbaldehyde (10)
An ampoule containing a mixture of freshly prepared aryl boronic acid 6 (104.5 mg, 0.408 mmol, 2 equiv), 2,4-bis(benzyloxy)-6-bromobenzaldehyde 9 (80.9 mg, 0.204 mmol), K2CO3 (63.6 mg, 0.460 mmol, 2.2 equiv) and Pd(PPh3)4 (26.6 mg, 0.023 mmol, 0.1 equiv) in anhydrous DMF (2 mL) was exhaustively degassed by freeze-thaw cycles. The ampoule was closed under vacuum and heated at 95 °C for 19 h. After cooling, the ampoule was opened and the reaction mixture was poured onto water and extracted with EtOAc. The combined organic extracts were washed with water, a 1.5% (w/v) aqueous solution of LiCl and brine, dried under anhydrous MgSO4 and concentrated under vacuum. The resulting crude reaction mixture was chromatographed on silica gel to give biaryl-2-carbaldehyde 10 (93.6 mg, 77%) as a viscous yellowish oil. 1H NMR (500 MHz, CDCl3) δ 10.02 (s, 1H, CHO), 7.54–7.48 (m, 2H, 2×CH Ph), 7.44–7.36 (m, 6H, 6xCH Ph), 7.36–7.29 (m, 2H, 2×CH Ph), 6.72 (d, J = 2.4 Hz, 1H, H-6), 6.65 (d, J = 2.3 Hz, 1H, H-4), 6.64 (d, J = 2.3 Hz, 1H, H-5′), 6.37 (d, J = 2.3 Hz, 1H, H-3′), 5.21–5.16 (m, two overlapped AB systems, 4H, OCH2O and OCH2Ph), 5.09 and 5.06 (AB system, J = 11.7 Hz, 1H each, OCH2Ph), 5.08 and 4.97 (AB system, J = 6.7 Hz, 1H each, OCH2O), 3.51 and 3.27 (each s, 3H each, 2×OCH3), 1.96 (s, 3H, CH3 Ph); 13C NMR (126 MHz, CDCl3) δ 189.6 (CHO), 163.5 (C-3), 162.1 (C-5), 157.6 (C-4′), 155.1 (C-2′), 144.8 (C-6′), 138.1 (C-1), 136.4 and 136.1 (2×C Ph), 128.9 (2×CH Ph), 128.8 (2×CH Ph), 128.4 (CH Ph), 128.1 CH Ph), 127.7 (2×CH Ph), 127.2 (2×CH Ph), 123.1 (C-1′), 118.6 (C-2), 110.7 (CH-5′), 109.6 (CH-3′), 101.2 (CH-6), 100.1 (CH-4), 94.7 and 94.6 (2×OCH2O), 70.7 and 70.4 (2×OCH2Ph), 56.3 and 56.1 (2×OCH3), 20.6 (CH3 Ph); HRMS (TOF, ES+) m/z calculated for C32H33O7 [M + H]+ 529.2221, found 529.2205.
4.3.2. Preparation of 3,5-bis(benzyloxy)-2′,4′-bis(methoxymethoxy)-6′-methyl-[1,1′-biphenyl]-2-carboxylic Acid (11)
NaH2PO4·H2O (58.6 mg, 0.425 mmol, 2.8 equiv), 2-methylbut-2-ene (322.1 µL, 3.04 mmol, 20 equiv) and NaClO2 (45.3 mg, 0.501 mmol, 3.3 equiv) were successively added to a solution of biaryl-2-carbaldehyde 10 (80.4 mg, 0.152 mmol) in tBuOH (3.2 mL) and milli-Q water (0.4 mL) at 0 °C. The mixture was allowed to warm at rt and stirred for 5 h, then diluted with an aqueous saturated solution of NH4Cl and extracted with EtOAc. The combined organic layers were washed with brine and dried over anhydrous MgSO4. Chromatography on silica gel of the residue left after evaporation of the solvent at reduced pressure, using 8:2 hexane-EtOAc as eluent, gave the biaryl-2-carboxylic acid 11 (79.5 mg, 96%) as a semi solid. 1H NMR (500 MHz, CDCl3) δ 9.32 (s, 1H, CO2H), 7.61–7.32 (m, 10H, 10xCH Ph), 6.68 (d, J = 2.4 Hz, 2H, H-6 and H-4), 6.65 (d, J = 2.4 Hz, 1H, H-5′), 6.44 (d, J = 2.3 Hz, 1H, H-3′), 5.21–5.14 (m, two overlapped AB systems, 4H, OCH2O and OCH2Ph), 5.07 and 5.04 (AB system, J = 11.7 Hz, 1H each, OCH2Ph), 4.99 (br s, 2H, OCH2O), 3.50 and 3.16 (each s, 3H each, 2×OCH3), 2.01 (s, 3H, CH3 Ph); 13C NMR (126 MHz, CDCl3) δ 165.7 (CO2H), 161.1 (C-3), 157.8 (C-5), 157.6 (C-4′), 155.1 (C-2′), 141.3 (C-6′), 138.4 and 135.7 (2×C Ph), 128.9 (2×CH Ph), 128.8 (2×CH Ph), 128.6 (CH Ph), 128.4 (CH Ph), 127.7 (2×CH Ph), 127.6 (2×CH Ph), 125.1 (C-2), 115.9 (C-1′), 111.6 (CH-5′), 109.9 (CH-3′), 102.6 (CH-6), 100.3 (CH-4), 96.1 and 94.7 (2×OCH2O), 71.5 and 70.4 (2×OCH2Ph), 56.3 and 56.1 (2×OCH3), 20.5 (CH3 Ph); HRMS (TOF, ES+) m/z calculated for C32H33O8 [M + H]+ 545.2170, found 545.2156.
4.3.3. Preparation of 7,9-bis(benzyloxy)-3-hydroxy-1-methyl-6H-benzo[c]chromen-6-one (12)
A 50:1 (v/v) mixture of iPrOH and concentrated HCl (1.7 mL) was added to a solution of biaryl-2-carboxylic acid 11 (69.4 mg, 0.127 mmol) in THF (5.1 mL) at rt under nitrogen. The mixture was thermostated at 55 °C in an oil bath and stirred at this temperature for 24 h. After this time, the mixture was cooled to rt, diluted with a concentrated aqueous solution of NaHCO3 and extracted with Et2O. The organic phase was washed with brine, dried over anhydrous MgSO4 and concentrated under vacuum to give 7,9-bis(benzyloxy)alternariol 12 (54.7 mg, 98%) as an amorphous whitish solid. The crude reaction product thus obtained was sufficiently pure, as judged by its NMR spectroscopic data, to be used in the next step without further purification. 1H NMR (500 MHz, DMSO-d6) δ 7.58 (d, J = 7.4 Hz, 2H, 2×CH Ph), 7.48 (d, J = 7.1 Hz, 2H, 2×CH Ph), 7.44–7.31 (m, 6H, 6xCH Ph), 7.28 (d, J = 2.2 Hz, 1H, H-10), 6.90 (d, J = 2.2 Hz, 1H, H-8), 6.63 (d, J = 2.7 Hz, 1H, H-2), 6.53 (d, J = 2.7 Hz, 1H, H-4), 5.31 and 5.29 (each s, 2H each, 2×OCH2Ph), 2.63 (s, 3H, CH3 Ph); 13C NMR (126 MHz, DMSO-d6) δ 163.6 (CO), 162.4 (C-9), 158.4 (C-7), 156.5 (C-3), 153.7 (C-4a), 140.0 (C-1), 138.0 (C-10a), 136.8 and 136.3 (2×CH Ph), 128.7 (2×CH Ph), 128.5 (2×CH Ph), 128.3 (CH Ph), 127.9 (2×CH Ph), 127.7 (CH Ph), 127.0 (2×CH Ph), 116.7 (CH-2), 109.1 (C-10b), 103.2 (C-6a), 102.8 (CH-10), 100.9 (CH-4), 99.8 (CH-8), 70.1 and 69.9 (2×OCH2Ph), 25.0 (CH3 Ph); HRMS (TOF, ES+) m/z calculated for C28H23O5 [M + H]·+ 439.1540, found 439.1530.
4.3.4. Preparation of methyl 5-((7,9-bis(benzyloxy)-1-methyl-6-oxo-6H-benzo[c]chromen-3-yl)oxy)pentanoate (13)
Methyl bromovalerate (29.5 mg, ca. 22 µL, 0.151 mmol, 1.1 equiv) was added via syringe to a stirred suspension of Cs2CO3 (57.8 mg, 0.177 mmol, 1.3 equiv) and phenol 12 (60.1 mg, 0.137 mmol) in anhydrous DMF (2 mL) at rt under nitrogen and the mixture was stirred for 19 h. The resulting pale yellowish reaction mixture was diluted with water and extracted with EtOAc. The combined organic extracts were washed successively with water, a 1.5% (w/v) aqueous solution of LiCl and brine, dried over anhydrous MgSO4 and concentrated under reduced pressure. The crude reaction product was purified by chromatography on silica gel, using CHCl3 as eluent, to afford the O-alkylated product 13 (71.4 mg, 94%) as a pale yellowish semi-solid. 1H NMR (500 MHz, CDCl3) δ 7.59 (m, 2H, CH Ph), 7.43–7.34 (m, 8H, 8×CH Ph), 7.30 (d, J = 2.3 Hz, 1H, H-2), 6.67 (d, J = 2.7 Hz, 1H, H-10), 6.64 (d, J = 4.6 Hz, 2H, H-4), 6.64 (s, 1H, H-8), 5.28 and 5.16 (each s, 2H each, 2×OCH2Ph), 4.00 (t, J = 5.5 Hz, 2H, H2-5), 3.68 (s, 3H, CO2CH3), 2.67 (s, 3H, CH3), 2.41 (t, J = 6.9 Hz, 2H, H2-2), 1.84 (m, 4H, H2-3 and H2-4); 13C NMR (126 MHz, CDCl3) δ 173.9 (CO2CH3), 163.7 (CO), 162.9 (C-9), 159.5 (C-7), 157.8 (C-3), 154.3 (C-4a), 140.7 (C-1), 137.4 (C-10a), 136.5 and 135.9 (2×C Ph), 129.0 (2×CH Ph), 128.8 (2×CH Ph), 128.6 (CH Ph), 127.9 (CH Ph), 127.5 (2×CH Ph), 126.8 (2×CH Ph), 116.7 (CH-2), 111.0 (C-10b), 104.5 (C-6a), 103.9 (CH-10), 100.0 (CH-4), 99.9 (CH-8), 71.0 and 70.5 (each OCH2Ph), 67.7 (CH2-5), 51.7 (OCH3), 33.8 (CH2-2), 28.6 (CH2-4), 25.6 (CH3), 21.7 (CH2-3); HRMS (TOF, ES+) m/z calculated for C34H33O7 [M + H]+ 553.2221, found 553.2112.
4.3.5. Preparation of 5-((7,9-bis(benzyloxy)-1-methyl-6-oxo-6H-benzo[c]chromen-3-yl)oxy)pentanoic Acid (14)
The hydrolysis of the methyl ester moiety of 13 was performed following the same procedure reported for the hydrolysis of ester 8 to obtain hapten ALa. The methyl ester 13 (17.5 mg, 0.032 mmol), lipase from Candida antarctica immobilized on acrylic resin (16 mg) and a 4:1 mixture of 100 mM sodium phosphate buffer (pH 7.4) and THF (1.1 mL). Workup as described for the hydrolysis of 8 yielded acid 14 (17.0 mg, 99%) as a whitish semi-solid. 1H NMR (500 MHz, THF-d8) δ 7.68 (m, 2H, 2×CH Ph), 7.46 (m, 2H, 2×CH Ph), 7.40–7.30 (m, 6H, 4×CH Ph and H-2), 7.25 (br t, J = 7.5 Hz, 1H, CH Ph), 6.85 (d, J = 2.2 Hz, 1H, H-10), 6.71 (d, J = 2.8 Hz, 1H, H-4), 6.69 (d, J = 2.8 Hz, 1H, H-8), 5.26 (s, 4H, 2×OCH2Ph), 4.05 (t, J = 6.2 Hz, 2H, H2-5), 2.71 (s, 3H, Ar-CH3), 2.32 (t, J = 7.2 Hz, 2H, H2-2), 1.78 (m, 4H, H2-3 and H2-4). 13C NMR (126 MHz, THF-d8) δ 174.5 (CO2H), 164.8 (CO), 163.9 (C-9), 160.9 (C-7), 156.7 (C-3), 155.7 (C-4a), 141.4 (C-1), 138.5 (C-10a), 138.3 and 137.9 (each C Ph), 129.5 (2×CH Ph), 129.2 (2×CH Ph), 129.0 (CH Ph), 128.5 (2×CH Ph), 128.2 (CH Ph), 127.6 (2×CH Ph), 117.2 (CH-2), 111.8 (C-10b), 105.4 (C-6a), 104.5 (CH-10), 100.7 (CH-4), 100.5 (CH-8), 71.4 and 71.1 (each OCH2Ph), 68.8 (CH2-5), 34.0 (CH2-2), 29.7 (CH2-4), 26.5 (CH3), 22.6 (CH2-3); HRMS (TOF, ES+) m/z calculated for C33H31O7 [M + H]+ 539.2064, found 539.2073.
4.3.6. Preparation of 2,5-dioxopyrrolidin-1-yl 5-((7,9-dihydroxy-1-methyl-6-oxo-6H-benzo[c]chromen-3-yl)oxy)pentanoate (ALb-NHS Ester)
The acid 14 obtained in the above step (15.1 mg, 28 µmol) was transformed into the corresponding N-hydroxysuccinimidyl ester 15 (17.2 mg) following the same procedure previously described for the transformation of hapten ALa into ALa-NHS ester, using EDC∙HCl (6.4 mg, 33.6 µmol, 1.2 equiv) and NHS (4.2 mg, 36.5 µmol, 1.3 equiv) in anhydrous DMF (1 mL). 1H NMR (300 MHz, CDCl3) δ 7.59 (m, 2H, 2×CH Ph), 7.45–7.32 (m, 8H, 8xCH Ph), 7.31 (d, J = 2.3 Hz, 1H, H-2), 6.69 (d, J = 2.7 Hz, 1H, H-10), 6.67 (d, J = 2.7 Hz, 1H, H-4), 6.65 (d, J = 2.2 Hz, 1H, H-8), 5.29 and 5.17 (each s, 2H each, 2×OCH2Ph), 4.04 (t, J = 5.6 Hz, 2H, H2-5), 2.85 (br s, 4H, COCH2CH2CO), 2.72 (t, J = 6.9 Hz, 1H, H2-2), 2.68 (s, 3H, CH3), 1.96 (m, 4H, H2-3 and H2-4).
Thereafter, a suspension of 5% Pd/C (8 mg) and 15 in acetone (3 mL) was degassed and purged with hydrogen by several cycles of freeze-pump-thaw using a water aspirator pump. The hydrogen pressure was adjusted to 1.5 atm and the mixture was stirred vigorously overnight at rt. The reaction mixture was filtered through a disposable Teflon membrane filter (0.45 µm), and the filtrate and washing THF phases were combined and concentrated at reduced pressure to give the N-hydroxysuccinimidyl ester of hapten ALb, ALb-NHS ester, (12.2 mg, 95% of crude product from 14) as a viscous colorless oil which was used immediately for the preparation of the corresponding protein bioconjugates. 1H NMR (500 MHz, THF-d8/DMSO-d6) δ 11.92 and 9.67 (each s, 1H each, 2×OH), 7.24 (d, J = 2.1 Hz, 1H, H-2), 6.83 (d, J = 2.7 Hz, 1H, H-4), 6.82 (d, J = 2.6 Hz, 1H, H-10), 6.36 (d, J = 2.1 Hz, 1H, H-8), 4.10 (t, J = 5.6 Hz, 1H, H2-5), 2.79 (s, 3H, CH3), 2.76 (br s, 4H, COCH2CH2CO), 2.71 (t, J = 6.9 Hz, 2H, H2-2), 1.93 (m, 4H, H2-3 and H2-4).