*2.2. Bioconjugate Preparation*

Bioconjugates of haptens **AL***a* and **AL***b* were prepared by the active ester method. The activated haptens were dissolved in dimethyl sulfoxide (DMSO) instead of DMF to improve the solubility. Moreover, the number of hapten equivalents required for efficiently labelling the studied proteins was higher than usual. Commonly, 20-fold hapten-to-protein molar excess for bovine serum albumin (BSA), and 10-fold excess for ovalbumin (OVA) and horseradish peroxidase (HRP) are usually employed in our laboratory. For these haptens, 40-fold and 15-fold excess was used for BSA and HRP conjugates, respectively. Moreover, extremely slow addition of the hapten over the protein solution was required. These concentrations and procedures were necessary, probably due to low hapten solubility in buffer and potential intermolecular polymerization reactions that inactivate the hapten. The obtained bioconjugates were purified by size-exclusion chromatography and characterized

by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis. The two BSA conjugates had similar hapten densities, with haptento-protein molar ratios of 15.2 and 18.6 for BSA-**AL***a* and BSA-**AL***b*, respectively, which is considered optimal for immunogens—excessive molar ratios could lead to low protein solubility, and higher or lower hapten densities could be counter-productive for highaffinity antibody generation. Regarding ovalbumin (OVA) conjugates, molar ratios were lower than those of BSA conjugates—around 3 for both haptens –, as it is desirable for coating conjugates to enhance the competitive reaction with the target analyte. Finally, the hapten densities of the enzyme tracers were estimated to be 2.0 and 2.2 for haptens **AL***a* and **AL***b*, respectively, which is within the expected range for HRP conjugates. The MALDI spectra of the prepared bioconjugates can be seen in Figure 2.

**Figure 2.** MALDI-TOF mass spectra (singly charged ions) of proteins (blue) and bioconjugates with hapten **AL***a* (green) and hapten **AL***b* (brick-red). (**a**) Normalized spectra of BSA and BSA conjugates, (**b**) Spectra of OVA and OVA conjugates, and (**c**) Spectra of HRP and HRP conjugates.

#### *2.3. Assessment of the Immune Response*

Four polyclonal antibodies were generated in this study, two from each BSA-hapten conjugate. To evaluate the immune response to the prepared synthetic haptens, binding of the antibodies to the homologous conjugate—the conjugate with the same hapten that was used to generate the corresponding antibody—was studied by checkerboard competitive ELISA, using the direct and the indirect assay formats.

Concerning direct assays, the IC50 values for AOH of the obtained antibodies were in the low nanomolar range (Table 1). AL*a*-type antibodies showed equal or similar IC50 values for AOH and AME. In particular, antibody AL*a*#1 showed very high affinity— IC50 values were 2.2 nM—for both mycotoxins, and the cross-reactivity (CR) values of antibodies AL*a*#1 and AL*a*#2 for AME were 100% and 199%, respectively. These are the first reported polyclonal antibodies with equivalent recognition to both *Alternaria* toxins. To date, only one monoclonal antibody with such specificity has been published [15]. In contrast, AL*b*-type antibodies bound AOH with high affinity, but their recognition for AME was negligible—CR values were below 1% (Table 1). The IC50 values to AOH of these specific antibodies were 1.2 nM, an affinity comparable to that of previously published polyclonal antibodies [11,13,16]. The position of the spacer arm in hapten **AL***a* provided a closer mimic of the alkylated hydroxyl group of AME (C-9 position), whereas in hapten **AL***b* the hydroxyl groups at C-7 and C-9 were unsubstituted, as in the molecule of AOH (Figure 1). Therefore, display of the hydroxyl group at C-9 was maximized in hapten **AL***b*, which explains the much lower affinity of AL*b*-type antibodies for AME compared to AOH.

**Table 1.** Antibody characterization by checkerboard direct and indirect competitive ELISA using the corresponding homologous conjugate (n = 3) a.


<sup>a</sup> The Amax values were higher than 1.0. <sup>b</sup> Dilution factor × <sup>10</sup>−3. <sup>c</sup> Bioconjugate concentration in ng/mL. <sup>d</sup> Values are in nM. <sup>e</sup> Crossreactivity values with AOH as reference.

> Regarding the indirect assay format, the four antibodies bound the corresponding homologous coating conjugate. As observed with the direct format, the AL*a*-derived antibodies recognized AOH and AME, whereas the AL*b*-derived antibodies were more specific to AOH (Table 1). The IC50 values were consistent with previously published results for indirect competitive ELISA with polyclonal antibodies [10,11,16]. Our strategy to prepare immunizing haptens with opposite linker tethering sites clearly demonstrated that the linker position strongly determines the specificity of antibodies to these *Alternaria* mycotoxins.

### *2.4. Assessment of Heterologous Conjugates*

Heterologous conjugates constitute a well-known strategy for improving the sensitivity of immunoassays. To further characterize the generated antibodies, competitive assays were carried out using the heterologous conjugate, i.e., assay conjugates of haptens **AL***a* and **AL***b* for AL*b*- and AL*a*-type antibodies, respectively. In the direct assay format, low binding to the heterologous tracer—with the linker on the opposite side of the AOH molecule compared to the immunizing conjugate—was observed (Amax values were below 0.6). In contrast, the change in the linker attachment site was not detrimental to hapten recognition in the indirect format, as the four antibodies bound the corresponding heterologous coating conjugate (Table 2). Reasonably, higher antibody and/or conjugate concentrations were required with the heterologous conjugates to reach sufficient signal. The obtained IC50 values using the heterologous coating conjugate were mostly lower than those obtained with the homologous assays. Anyway, CR values did not significantly change with heterologous conjugates.


**Table 2.** Antibody characterization by checkerboard indirect competitive ELISA using the corresponding heterologous coating conjugate (n = 3) a.

<sup>a</sup> The Amax values were higher than 1.0. <sup>b</sup> Dilution factor × <sup>10</sup><sup>−</sup>3. <sup>c</sup> Bioconjugate concentration in ng/mL. <sup>d</sup> Values are in nM.

#### **3. Conclusions**

In this study, two de novo synthesized and purified AOH haptens were comprehensively characterized by spectrometric methods, and bioconjugates with unique structure and composition were prepared for the first time. In this perspective, it is worth noting the challenges of obtaining stable enzyme tracers with high activity. This matter was most likely caused by the chemical characteristics of *Alternaria* toxins and their haptens, which could explain why no direct competitive immunoassays for these mycotoxins have been reported up to now. Once this issue was overcome, the resultant immunoreagents were thoroughly investigated utilizing both direct and indirect competitive ELISA, as well as homologous and heterologous conjugates. Remarkably, antibodies capable of binding AOH and AME with affinities in the low nanomolar range were eventually generated from both haptens. Given that the levels of these mycotoxins are not yet regulated, both specific and generic antibodies are relevant. Our findings showed that hapten **AL***a*, with the linker at the methylated hydroxyl group in AME (C-9 position), was particularly well-suited for producing antibodies that recognized similarly both toxins, whereas antibodies generated from hapten **AL***b*, with the spacer arm at the hydroxyl group in C-3 position, primarily bound AOH. In contrast to previous one-pot hapten synthesis and bioconjugation procedures, the strategy described here for producing AOH haptens with alternative linker tethering sites not only enabled high-affinity antibodies with different specificities, but it may also help to improve the sensitivity of immunoassays to *Alternaria* mycotoxins by using site heterologous haptens.

#### **4. Materials and Methods**

#### *4.1. Reagents and Instruments*

Standard AOH [3,7,9-trihydroxy-1-methyl-benzo[c]chromen-6-one, CAS registry number 641-38-3, Mw 258.23] and AME [3,7-dihydroxy-9-methoxy-1-methyl-benzo[c]chromen-6-one, CAS registry number 23452-05-3, Mw 272.25] from *Alternaria* sp. were purchased from Merck (Darmstadt, Germany). Mycotoxins were dissolved in anhydrous *N*,*N*dimethylformamide (DMF), and the stock solutions were stored at −20 ◦C. Phosphate buffered saline (PBS) 10× solution (Fisher BioReagents BP399-20) was from Thermo Fisher Scientific (Waltham, MA, USA). Immunizing bioconjugates were prepared with BSA, fraction V, obtained from Roche Applied Science (Mannheim, Germany). OVA, HRP, Freund's adjuvants, and adult bovine serum, were acquired from Merck (Darmstadt, Germany). Polyclonal goat anti-rabbit (GAR) immunoglobulins antibody and polyclonal goat antirabbit immunoglobulins antibody conjugated to peroxidase (GAR-HRP) were purchased from Rockland Immunochemicals Inc. (Pottstown, PA, USA) and BioRad (Madrid, Spain), respectively. 3,3 ,5,5 -Tetramethylbenzidine (TMB) liquid substrate for ELISA was obtained from Biopanda Reagents Ltd. (Belfast, UK). Other reagents, materials, and instruments employed for bioconjugate preparation and ELISA experiments are described in the Supplementary Materials file.

#### *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, *<sup>J</sup>* = 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]<sup>+</sup> 325.1282, found 325.1283.

4.2.2. Preparation of methyl 5-((2,2-dimethyl-4-oxo-5-(((trifluoromethyl)sulfonyl)oxy)-4Hbenzo[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, CF*3*); HRMS (TOF, ES+) *m/z* calculated for C17H23F3NO9S [M + NH4] <sup>+</sup> 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(Oi Pr)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 NH4Cl (0.7 mL), then diluted with water and extracted with Et2O. The organic layers were washed with brine, dried over anhydrous Na2SO4 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-*d*6) 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×OCH2O), 3.37 and 3.34 (each s, 3H each, 2×OCH3), 2.20 (s, 3H, CH3).

(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-vacuumthaw 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) <sup>δ</sup> 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-*d*6) 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-*d*6) 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 **AL***a*)

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 **AL***a* (14.9 mg, 93%) as a white amorphous solid. 1H NMR (300 MHz, THF-*d*8) 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-*d*8) 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]<sup>+</sup> 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 (**AL***a***-NHS** Ester)

A solution of hapten **AL***a* (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 **AL***a,* **AL***a***-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-*d*8) <sup>δ</sup> 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 *<sup>t</sup>* BuOH (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]<sup>+</sup> 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 <sup>i</sup> PrOH 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-*d*6) <sup>δ</sup> 7.58 (d, *<sup>J</sup>* = 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-*d*6) 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]·<sup>+</sup> 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]<sup>+</sup> 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 **AL***a*. 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-*d*8) <sup>δ</sup> 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-*d*8) 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]<sup>+</sup> 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 (**AL***b***-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 **AL***a* into **AL***a***-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) <sup>δ</sup> 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 **AL***b*, **AL***b***-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-*d*8/DMSO-d6) <sup>δ</sup> 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).
