Hapten Design and Antibody Generation for Immunoanalysis of Spirotetramat and Spirotetramat-enol

Abstract

Spirotetramat—a tetramic acid insecticide—is rapidly metabolized or degraded to give spirotetramat-enol; so, common residue definitions include the sum of both compounds. In the present study, two spirotetramat-functionalized derivatives (haptens) have been designed to generate immunoreagents to these molecules for rapid immunochemical analysis. Haptens have been synthesized with alternative linker tethering sites and, for the first time, high-affinity antibodies have been generated with different specificities to these active principles. Two sensitive assays have been developed using the same antibody in different formats, and by using linker-site heterologous haptens, the selectivity of the final immunoassay could be improved. A generic immunoassay with sensitivity similar to spirotetramat and spirotetramat-enol and a specific assay of spirotetramat-enol have been developed. The described antibody and bioconjugates showed great potential for sensitive immunosensor development and analysis of this complex analyte.

1. Introduction

Spirotetramat (SP) is the ISO common name for cis-4-(ethoxycarbonyloxy)-8-methoxy-3-(2,5-xylyl)-1-azaspiro[4.5]dec-3-en-2-one (IUPAC). This is a new-generation biocide that was discovered as a derivative of the natural antibiotic thiolactomycin.¹ In 2013, SP was included in Annex I to Regulation (EC) number 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on the market.² It belongs to the tetramic acid family, and it is structurally characterized by a spirocyclic group containing a dimethylphenyl group and an ethyl carbonate group as substituents (Figure 1). SP shows a new mode of action that consists of inhibiting the acetyl-CoA carboxylase activity, thus blocking the biosynthesis of lipids in a variety of target insect pests.³ Upon absorption by the organism, SP is transformed into a more polar enol derivative (SP-enol) by the hydrolytic cleavage of the ethyl carbonate group (Figure 1), thus facilitating its translocation.⁴ Moreover, SP is a labile compound that degrades at ambient temperature and particularly under alkaline conditions—the half-life at pH 9 and 25 °C is 7.6 h—SP-enol being the major degradation product.⁵ Further metabolism or degradation of SP-enol can give rise to several supplementary and frequently minor derivatives, such as spirotetramat-enol glucoside (SP-glc), spirotetramat-ketohydroxy (SP-keto), spirotetramat-monohydroxy (SP-mono), and spirotetramat-desmethyl-enol. SP shows moderate-to-low acute toxicity in mammals—LC₅₀ > 4.183 mg/L by inhalation and LD₅₀ > 2000 mg/kg via oral and dermal routes in rats, but it has been demonstrated to be rather toxic to aquatic organisms.⁵

Figure 1.

Synthesis of haptens SPc (A) and SPo (B) and the corresponding N-hydroxysuccinimidyl esters. Inset: molecular structure of SP and spirotetramat-enol.

Analysis of SP is commonly carried out by high-performance liquid chromatography with photometric^6,7 or mass spectrometry detection, frequently after quick, easy, cheap, effective, rugged, and safe extraction of samples.^8,9 However, the determination of SP residues in environmental and food samples can be quite complex because of the diversity of SP metabolites or degradates that can be present in the sample.¹⁰ For risk assessment studies, both the parent compound and the metabolites are taken into consideration, whereas a common definition of SP residues for enforcement or compliance with the maximum residue limits in plant commodities is the sum of SP and SP-enol, expressed as SP concentration, because both compounds mostly constitute the bulk of the residues.^11,12

Antibody-based analytical techniques are nowadays employed as complementary strategies for small chemical contaminant and residue monitoring. The competitive enzyme-linked immunosorbent assay (cELISA) is probably the most extended immunochemical method because of its large sample throughput and its ability to provide quantitative results.¹³ However, high-quality immunoreagents are required for attaining these goals. Covalent bioconjugates of the target compound should be primarily obtained and employed as immunogens to raise antibodies. Moreover, the bioconjugates are necessary to develop competitive immunoassays. To our knowledge, the preparation of bioconjugates and the generation of antibodies to SP have not been reported previously, and consequently no bioanalytical methods have been described so far for this complex analyte. The aim of our study was to raise valuable immunoreagents and to develop sensitive and rapid immunochemical techniques for the analysis of SP and SP-enol.

2. Results and Discussion

2.1. Hapten Design and Synthesis

Two functionalized derivatives of SP (haptens) with alternative linker tethering sites were designed and synthesized, namely SPc and SPo (Figure 1), to generate antibodies with different specificities. These haptens incorporated, through two of the oxygenated functions thereof, a carboxylated hydrocarbon chain that served as a spacer arm for its conjugation to the carrier proteins. In both cases, the SP framework was fully maintained, and the introduction of the spacer arm through a C–O bond ensured minimal modifications of the SP electronic properties, so that they could be deemed as adequate mimics of the analyte’s parent structure.

The synthesis of hapten SPc (Figure 1) started from the metabolite SP-enol (1), which is commercially available and also readily obtainable from SP by the basic hydrolysis of the enol-carbonate moiety at the C-4 position.¹ The incorporation of the spacer arm at the enolic oxygen atom was carried out in a three-step sequence starting with the formation of allyl carbonate 2, via the reaction of SP-enol with allyl chloroformate, followed by a microwave-assisted cross-metathesis reaction with 4-pentenoic acid to form a ca. 9:1 trans/cis mixture of the corresponding olefin 3. Finally, double-bond hydrogenation under homogeneous conditions using Wilkinson’s catalyst completed the synthesis of hapten SPc. Thus, the synthesis of hapten SPc was accomplished from 1 via three steps in a 49% overall yield. The structure of the hapten and those of the intermediates were confirmed by infrared (IR) spectroscopy, proton nuclear magnetic resonance (¹H NMR), carbon (¹³C) NMR, and high-resolution mass spectrometry (HRMS) (data can be found in section 4.2).

The preparation of hapten SPo (Figure 1) was readily undertaken from demethylated SP (5). This compound can be directly obtained from SP itself,¹⁴ although for this work we prepared it from ketone 4, which was in turn obtained through a synthetic route similar to the one previously described.¹⁵ Thus, the ketone carbonyl group of 4 was stereoselectively reduced under the Luche conditions to the equatorial hydroxyl group from which the spacer chain was introduced through an acylation reaction with glutaric anhydride and catalytic zinc perchlorate hexahydrate under microwave irradiation.¹⁶ Hapten SPo was thus obtained with an overall yield for the two steps of about 44%. The chemical structures of the hapten and the intermediates were verified by IR spectroscopy, ¹H NMR, ¹³C NMR, and HRMS (extended data are reported in section 4.3).

2.2. Preparation of Bioconjugates

Prior to coupling with proteins, haptens SPc and SPo were activated through the formation of active esters. This activation procedure and the subsequent chromatographic purification led to the corresponding N-hydroxysuccinimidyl esters—SPc-NHS and SPo-NHS esters (Figure 1)—in practically pure form, thus allowing a precise control of the coupling conditions and avoiding undesired secondary reactions.

Hapten SPo was coupled to bovine serum albumin (BSA) at basic pH to better promote the formation of amide bonds between the carboxyl group of the hapten and the amine groups of the protein. However, under these conditions, we cannot rule out the fact that partial hydrolysis of the ethyl carbonate group of the analyte might take place; if so, the immunogen would be a mixture between SP and SP-enol, which, far from being a drawback, may be convenient because both substances are included in the SP residue definition. A hapten-to-protein molar ratio (MR) of 13 was obtained, which is an optimum value for an immunizing conjugate (Figure 2A). On the other hand, hapten SPc was coupled to BSA at neutral pH to avoid the hydrolysis of the carbonate group which, in this case, would have caused the loss of the SP backbone, thus forming a bioconjugate with probably only the spacer arm. A rather low hapten-to-protein MR of 8 was attained in this conjugate (Figure 2A). The lower hapten-to-protein MR of the SPc conjugate, as compared with that of hapten SPo, was most probably due to the greater coupling efficiency of the active ester method at basic pH, as already mentioned. Assay conjugates based on ovalbumin (OVA) and horseradish peroxidase (HRP) were also prepared for both haptens at neutral pH and, in terms of applicability, good MRs (3–4 for OVA and 1 for HRP) were obtained (Figure 2).

Figure 2.

MALDI-TOF-MS spectra of bioconjugates. (A) BSA (blue) and conjugates BSA–SPc (green) and BSA–SPo (orange). (B) HRP (blue) and conjugates HRP–SPc (green) and HRP–SPo (orange). (C) OVA (blue) and OVA–SPc conjugate (green). (D) OVA (blue) and conjugate OVA–SPo (green).

2.3. Antibody Generation and Characterization

Two antisera were obtained from each BSA conjugate, which were named after the immunizing hapten, and were consecutively numbered (SPc#1, SPc#2, SPo#1, and SPo#2). The affinity of the four antibodies to SP and SP-enol was estimated by cELISA in three different formats, indirect, direct, and capture antibody direct, using the corresponding homologous assay conjugate (the conjugate that holds the same hapten as the immunizing conjugate).

For the conjugate-coated indirect cELISA format, microplate coating was carried out either at pH 7.4 or 9.6, so as to evaluate the potential instability of the conjugates at high pH. With SPc-type antibodies, and to achieve A_max values around 1.0, a much higher coating conjugate concentration was required in the plates coated at basic pH than at neutral pH (Table 1). This result may be indicative of a partial hydrolysis of the assay hapten when the coating was carried out at basic pH. In addition, no inhibition was observed, either with SP or SP-enol, using SPc-type antibodies. We hypothesized that the hydrolysis of the carbonate group of hapten SPc might have taken place in the animal body during the immunization process; hence, the hapten molecule would be released from the conjugate, and the antibodies recognizing SP or SP-enol would not have been generated. Concerning SPo-type antibodies, high A_max values were obtained at very low homologous coating conjugate concentrations, regardless of the coating pH. More significantly, both SP and SP-enol inhibited the immunochemical reaction between the antibodies SPo#1 and SPo#2 and the coating conjugate, even though the IC₅₀ values were lower for SP-enol than for SP. This result could be explained by a partial loss of the ethyl carbonate group in the SPo hapten during conjugation and immunization; so, in practice, animals would have been immunized with a mixture of a conjugate of SP and an in vivo-formed conjugate of SP-enol.

Table 1. Checkerboard Titration of Antibodies and Bioconjugates by Indirect cELISA Using the Homologous Coating Conjugate (n = 3).

					curve parameter
coating conjugate	coating pH	[C]a	pAb	antibody titer	Amax	slope	IC50 (nM) SP	IC50 (nM) SP-enol
OVA–SPc	9.6	3000	SPc#1	3000	1.06	b
		3000	SPc#2	3000	1.67
	7.4	100	SPc#1	3000	1.56
		100	SPc#2	10 000	1.03
OVA–SPo	9.6	10	SPo#1	100 000	0.92	0.52	561	36.6
		10	SPo#2	10 000	1.32	0.54	963	56.2
	7.4	10	SPo#1	30 000	1.42	0.56	1295	97.7
		10	SPo#2	10 000	1.08	0.71	805	70.3

^aConjugate concentrations are in ng/mL.

^bNo inhibition was observed.

The obtained antibodies were also evaluated by the antibody-coated direct and the capture antibody direct cELISA formats, and similar results were obtained among each other. Not surprisingly, binding between SPc-type antibodies and HRP–SPc tracer was not observed (Table 2). On the contrary, binding to the homologous enzyme tracer (HRP–SPo) was found with both SPo-type antibodies in the two formats, and, more importantly, inhibition was observed with SP and SP-enol. Interestingly, both antibodies showed a much higher affinity to SP-enol than to SP, probably because of the partial hydrolysis of hapten SPo in the immunizing conjugate, as already mentioned. The lowest IC₅₀ value for SP-enol (1.5 nM) was found with the antibody SPo#1 in the capture format.

Table 2. Checkerboard Titration of Antibodies and Bioconjugates by Direct and Capture Antibody Direct cELISA (n = 3).

					curve parameter
assay format	enzyme tracer	[T]a	pAb	antibody titer	Amax	slope	IC50 (nM) SP	IC50 (nM) SP-enol
direct	HRP–SPc	300	SPc#1	3000	n.s.b
		300	SPc#2	3000	n.s.
	HRP–SPo	30	SPo#1	10 000	0.69	0.74	165	10.8
		300	SPo#2	3000	1.02	0.78	233	19.6
capture	HRP–SPc	300	SPc#1	3000	n.s.
		300	SPc#2	3000	n.s.
	HRP–SPo	100	SPo#1	30 000	1.07	0.71	113	1.5
		300	SPo#2	3000	0.56	0.65	381	22.7

^aTracer concentrations are in ng/mL.

^bNo signal was observed.

2.4. Immunoassay Development

The performance of the four antibodies in combination with the heterologous conjugate—in which the hapten is different to that of the immunizing conjugate—was assessed in the three studied cELISA formats. Unfortunately, none of the antibodies recognized the heterologous enzyme tracer; so, no signal was observed with the direct and the capture assay formats. The situation was quite similar for the indirect format, in which the antibodies poorly recognized the heterologous coating conjugates (Table 3); so, no feasible assays could be developed. Higher MR values of the OVA–SPo conjugate (MR = 15) did not improve the A_max values. The only exception to this general finding was antibody SPo#1; in plates coated at neutral pH with the OVA–SPc heterologous conjugate, an inhibition curve with an A_max value around 1.0 could be obtained. More notably, the observed IC₅₀ values with the immunoreagent combination were 100-fold (9.47 nM) and 30-fold (3.60 nM) lower for SP and SP-enol, respectively, than the values obtained with the homologous assay. Accordingly, further work was carried out only with antibody SPo#1, either combined with the heterologous conjugate (OVA–SPc) in the indirect format or together with the homologous tracer (HRP–SPo) in the capture antibody direct format.

Table 3. Checkerboard Titration of Antibodies and Bioconjugates by Indirect cELISA Using the Heterologous Coating Conjugate (n = 3).

					curve parameter
coating conjugate	coating pH	[C]a	pAb	antibody titer	Amax	slope	IC50 (nM) SP	IC50 (nM) SP-enol
OVA–SPo	9.6	3000	SPc#1	3000	0.33	0.77	4.10	b
		3000	SPc#2	3000	0.49	0.75	78.9
	7.4	3000	SPc#1	3000	0.24	0.89	5.32
		3000	SPc#2	3000	0.31	0.79	96.6
OVA–SPc	9.6	3000	SPo#1	3000	0.32	0.66	3.27	3.58
		3000	SPo#2	3000	0.10	0.52	12.0	1.71
	7.4	3000	SPo#1	3000	1.02	0.94	9.47	3.60
		3000	SPo#2	3000	0.28	0.63	9.96	20.1

^aConjugate concentrations are in ng/mL.

^bNo inhibition.

The selectivity of both immunoassays to the four main metabolites of SP, that is, SP-enol, SP-glc, SP-keto, and SP-mono, was assessed. The homologous capture assay was pretty specific of SP-enol, as previously described. The cross-reactivity (CR) values, referred to SP-enol, were 1.3% for SP, 2.2% for SP-glc, and almost negligible for SP-keto and SP-mono. However, the heterologous indirect assay exhibited a different recognition pattern, with SP and SP-enol being recognized to a closer extent, whereas the other compounds were again much less recognized. The CR values were 38 and 100% for SP and SP-enol, respectively, 2.3% for SP-glc, and lower than 0.5% for the rest of the metabolites (Table 4).

Table 4. Immunoassay Cross-Reactivity (%, n = 3).

analyte	indirect	capture
SP	38	1.3
SP-enol	100	100
SP-glc	2.3	2.2
SP-keto	0.2	<0.1
SP-mono	<0.1	0.1

Interestingly, the two selected immunoassays employed the same antibody but different assay conjugates. The heterology introduced by hapten SPc in the indirect assay made the IC₅₀ value for SP closer to that of SP-enol, thus resulting in a more generic assay for both compounds. The presence of the carbonate group in SPc probably selects the fraction of antibody molecules that bind the nonhydrolyzed hapten, thus increasing the apparent affinity to SP. The standard curves for SP and SP-enol in the two optimized immunoassays are shown in Figure 3. The logarithm of the odds (LOD) values, calculated as IC₁₀ of the standard curve, were 0.5 and 0.6 nM for SP and SP-enol, respectively, in the indirect assay. On the other hand, the LOD values for the capture assay were almost 100 times higher for SP than for SP-enol (5 and 0.06 nM, respectively). These values are in the same range of those of the reference chromatographic methods,^10,17,18 and they suggest the great potential of the developed immunoassays for SP and SP-enol residue determination.

Figure 3.

Standard curves of the selected immunoassays. The A_max value was around 1.0, and the background signal was always near zero. The values are the average of three independent experiments.

3. Conclusions

Two functionalized derivatives of SP have been synthesized with equivalent linkers located at different sites of the molecule. The lability of the carbonate group of the molecule was the determinant for the generation of antibodies to SP and SP-enol. A cELISA specific of SP-enol was established with the capture antibody format using the antibody SPo#1 and the homologous enzyme tracer. Moreover, a generic cELISA for SP and SP-enol was characterized in the conjugate-coated indirect format using the same antibody but combined with a heterologous conjugate. Highly sensitive immunoassays, with the LOD values in the low nanomolar scale, have been presented as a proof of principle for the immunoanalysis of this complex chemical residue.

4. Materials and Methods

4.1. Reagents and Instruments

SP, spirotetramat-enol, and the rest of the metabolites were of Pestanal grade and were purchased from Merck (Madrid, Spain). Further information about the reagents and equipment is provided in the Supporting Information.

4.2. Synthesis of Hapten SPc

This hapten was prepared in three synthetic steps as schematized in Figure 1. The ¹H NMR spectrum of hapten SPc is provided in the Supporting Information.

4.2.1. Preparation of Allyl(3-(2,5-dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro[4.5]dec-3-en-4-yl) Carbonate (2)

Ethyl chloroformate (73 μL, 0.69 mmol) was added to a solution of 1 (97.5 mg, 0.323 mmol) and Et₃N (101 μL, 0.72 mmol) in dry CH₂Cl₂ (4 mL) at 0 °C under nitrogen. The mixture was stirred at room temperature for 2 h, then diluted with CH₂Cl₂, washed with brine, and dried over MgSO₄. Chromatographic purification of the residue obtained after the evaporation of the solvent at reduced pressure, using CHCl₃/MeOH 99:1 as the eluent, afforded allyl carbonate 2 (86.5 mg, 85%). IR ν_max (cm^–1): 3197, 3017, 2944, 1778, 1693, 1498, 1499, 1210, 1105, 931, 735; ¹H NMR (CDCl₃, 300 MHz): δ 7.09 (1H, d, J = 7.9 Hz, H-3 Ph), 7.03 (1H, dd, J = 7.9, 1.5 Hz, H-4 Ph), 6.97 (1H, d, J = 1.5 Hz, H-6 Ph), 6.94 (1H, br s, NH), 5.65 (1H, ddt, J = 16.0, 10.0, 6.0 Hz, H-2′), 5.18 (1H, dq, J = 10.0, 1.5 Hz, H-3′), 5.17 (1H, dq, J = 16.0, 1.2 Hz, H′-3′), 4.41 (2H, dt, J = 6.0, 1.3 Hz, H₂-1′), 3.37 (3H, s, OMe), 3.23 (1H, tt, J = 10.7, 4.1 Hz, H-8), 2.28 and 2.21 (3H each, each s, 2 × Me-Ph), 2.19–2.12 (2H, m, H₂-Cy), 1.91 (2H, td, J = 13.6, 3.9 Hz, H₂-Cy), 1.83–1.70 (2H, m, H₂-Cy), 1.55–1.34 (2H, m, H₂-Cy); ¹³C NMR (CDCl₃, 75 MHz): δ 169.9 (C-2), 164.6 (C-4), 149.7 (OCO₂), 134.9 (C-5 Ph), 134.0 (C-1 Ph), 130.1 (C-2′, C-3 Ph), 129.9 (C-4 Ph), 129.4 (C-6 Ph), 127.7 (C-2 Ph), 121.7 (C-3), 119.7 (C-3′), 77.2 (C-8), 69.8 (C-1′), 60.2 (C-5), 55.7 (OMe), 31.6 (C-7/C-9), 28.3 (C-6/C-10), 20.8 and 19.1 (2 × Me-Ph); HRMS: calcd for C₂₂H₂₈NO₅ [M + H]^+•, 386.1962; found, 386.1969.

4.2.2. Preparation of 6-((((3-(2,5-Dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro[4.5]dec-3-en-4-yl)oxy)carbonyl)oxy)hex-4-enoic Acid (3)

A solution of allyl carbonate 2 (84.5 mg, 0.217 mmol), pent-4-enoic acid (68 μL, 0.666 mmol), second-generation Grubbs catalyst (11.3 mg, 0.013 mmol, 6%), and CuI (1.7 mg, 0.009 mmol, 4%) in dry Et₂O (2.5 mL) was stirred and refluxed under nitrogen for 5 h. The solvents were then removed under reduced pressure, and the crude mixture was purified by flash chromatography, eluting first with Et₂O and then with CHCl₃/MeOH mixtures from 100:0 to 90:10, to afford acid 3 (85 mg, 85%) as an approximately 8:2 mixture of trans/cis double-bond isomers. IR ν_max (cm^–1): 3273, 2935, 1776, 1693, 1443, 1204, 1099, 968, 728; ¹H NMR (CD₃OD, 300 MHz), only the signals of the trans isomer are given: δ 7.90 (1H, s, NH), 7.12 (1H, d, J = 7.8 Hz, H-3 Ph), 7.07 (1H, dd, J = 7.8, 1.5 Hz, H-4 Ph), 6.91 (1H, d, J = 1.5 Hz, H-6 Ph), 5.78–5.61 (1H, m, H-4), 5.34 (1H, dtt, J = 15.6, 6.4, 1.4 Hz, H-5), 4.38 (2H, br d, J = 6.4, 1.1 Hz, H₂-6), 3.39 (3H, s, OMe), 3.30 (1H, m overlapped with the solvent signal, H-8′), 2.35–2.25 (4H, m, H₂-2, H₂-3), 2.29 and 2.18 (3H each; each s, 2 × Me-Ph), 2.18–2.10 (2H, m, 2H, m, H₂-Cy), 1.95 (2H, td, J = 13.6, 3.8 Hz, H₂-Cy), 1.68 (2H, m, H₂-Cy), 1.64–1.48 (2H, m, H₂-Cy); ¹³C NMR (CDCl₃, 75 MHz): δ 177.1 (CO₂H), 171.4 (C-2′), 165.2 (C-4′), 149.6 (OCO₂), 135.9 (C-4), 135.0 (C-5 Ph), 134.0 (C-1 Ph), 130.2 (C-3 Ph), 129.9 (C-6 Ph), 129.6 (C-2 Ph), 127.5 (C-2 Ph), 123.1 (C-5), 121.3 (C-3′), 77.3 (C-8′), 69.8 (C-6), 60.9 (C-5′), 55.8 (OMe), 33.1 (C-2), 31.5 (C-6′/C-10′), 28.3 (C-7′/C-9′), 27.2 (C-3), 20.8 and 19.1 (2 × Me-Ph); HRMS: calcd for C₂₅H₃₂NO₇ [M + H]^+•, 458.2173; found, 458.2161.

4.2.3. Preparation of 6-((((3-(2,5-Dimethylphenyl)-8-methoxy-2-oxo-1-azaspiro[4.5]dec-3-en-4-yl)oxy)carbonyl)oxy)hexanoic Acid (Hapten SPc)

A solution of acid 3 (59 mg, 0.129 mmol) and Wilkinson catalyst (10 mg, 0.011 mmol, 8%) in anhydrous tetrahydrofuran (2.5 mL) was evacuated and purged under an atmosphere of hydrogen gas. The hydrogen pressure was regulated to 55 psi and the reaction mixture was stirred at room temperature overnight. Then, the solvent was removed under vacuum, and the residue was purified by chromatography, using CHCl₃/MeOH 99:1 as the eluent, to give hapten SPc (40.5 mg, 68%). IR ν_max (cm^–1): 3268, 2935, 2864, 1777, 1695, 1209, 1094; ¹H NMR (CDCl₃, 300 MHz): δ 7.69 (1H, br s, NH), 7.10 (1H, d, J = 7.8 Hz, H-3 Ph), 7.04 (1H, dd, J = 7.8, 1.6 Hz, H-4 Ph), 6.97 (1H, d, J = 1.6 Hz, H-6 Ph), 3.95 (2H, t, J = 6.6 Hz, H₂-6), 3.38 (3H, s, OMe), 3.25 (1H, m, H-8′), 2.29 (2H, t, J = 7.4 Hz, H₂-2), 2.28, and 2.20 (3H each; each s, 2 × Me-Ph), 2.20 (2H, m, H₂-Cy), 1.92 (2H, td, J = 13.7, 3.9 Hz, H₂-Cy), 1.74 (2H, m, H₂-Cy), 1.58 (2H, quint, J = 7.5 Hz, H₂-3), 1.51–1.40 (4H, m, H₂-5 and H₂-Cy), 1.31–1.17 (2H, m, H₂-4); ¹³C NMR (CDCl₃, 75 MHz): δ 178.2 (CO₂H), 171.2 (C-2′), 165.2 (C-4′), 149.9 (OCO₂), 135.0 (C-5 Ph), 134.0 (C-1 Ph), 130.2 (C-3 Ph), 129.9 (C-4 Ph), 129.6 (C-6 Ph), 127.5 (C-2 Ph), 121.4 (C-3′), 77.3 (C-8′), 69.4 (C-6), 60.8 (C-5′), 55.8 (OMe), 31.6 (C-6′/C-10′), 31.5 (C-2), 28.3 (C-7′/C-9′), 27.9 (C-5), 24.9 (C-4), 24.2 (C-3), 20.9 and 19.1 (2 × Me-Ph); HRMS: calcd for C₂₅H₃₄NO₇ [M + H]^+•, 460.2330; found, 460.2321.

4.3. Synthesis of Hapten SPo

To prepare this hapten (Figure 1), ketone 4 was obtained as described by Zhao et al.¹⁵ The ¹H NMR spectrum of hapten SPo is provided in the Supporting Information.

4.3.1. Preparation of 3-(2,5-Dimethylphenyl)-8-hydroxy-2-oxo-1-azaspiro[4.5]dec-3-en-4-yl Ethyl Carbonate (5)

CeCl₃·7H₂O (100 mg, 0.27 mmol) was added to a solution of ketone 4 (40 mg, 0.11 mmol) in a 1:1 mixture of MeOH and CH₂Cl₂ (3 mL). The resulting solution was cooled to −12 °C; then, NaBH₄ (10.2 mg, 0.27 mmol) was added and the mixture was stirred at the same temperature for 40 min. After this time, the mixture was treated with a few drops of acetone to destroy the excess of NaBH₄, stirred for 5 min, and poured into a saturated aqueous solution of sodium citrate and extracted with EtOAc. The combined organic extracts were washed with brine, dried over anhydrous MgSO₄, and concentrated at a reduced pressure to give alcohol 5 (36.6 mg, 88%) as a white solid. mp 244.6–246.0 °C (crystals obtained by the slow evaporation of a CHCl₃ solution). IR ν_max (cm^–1): 3361, 3247, 2939, 1780, 1683, 1214, 1065, 646; ¹H NMR (CD₃COCD₃, 300 MHz): δ 8.55 (1H, s, NH), 7.10 (1H, d, J = 7.7 Hz, H-3 Ph), 7.04 (1H, dd, J = 7.8, 1.8 Hz, H-4 Ph), 6.93 (1H, d, J = 1.8 Hz, H-6 Ph), 4.24 (1H, d, J = 3.8 Hz, OH), 4.02 (2H, q, J = 7.1 Hz, OCH₂Me), 3.65–3.48 (1H, m, H-8′), 2.26 and 2.19 (3H each, each s, 2 × Me-Ph), 1.99–1.82 (4H, m, 2 × H₂-Cy), 1.78–1.65 (2H, m, H₂-Cy), 1.65–1.52 (2H, m, H₂-Cy), 1.06 (3H, t, J = 7.1 Hz, OCH₂Me); ¹³C NMR (CD₃COCD₃, 75 MHz): δ 169.4 (C-2), 165.6 (C-4), 150.9 (OCO₂), 135.1 (C-5 Ph), 134.9 (C-1 Ph), 130.8 (C-6 Ph), 130.5 (C-3 Ph), 130.0 (C-2 Ph), 129.6 (C-4 Ph), 122.6 (C-3′), 68.9 (C-8′), 66.0 (OCH₂Me), 60.9 (C-5′), 32.9 (C-6′/C-10′), 32.2 (C-7′/C-9′), 20.8 and 19.4 (2 × Me-Ph), 13.9 (OCH₂Me); HRMS: calcd for C₂₀H₂₆NO₅ [M + H]^+•, 360.1805; found, 360.1816.

4.3.2. Preparation of 5-((3-(2,5-Dimethylphenyl)-4-((ethoxycarbonyl)oxy)-2-oxo-1-azaspiro[4.5]dec-3-en-8-yl)oxy)-5-oxopentanoic Acid (Hapten SPo)

Alcohol 5 (26.6 mg, 0.074 mmol), Zn(ClO₄)₂·6H₂O (0.5 mg, ca. 0.02 equiv), and glutaric anhydride (8.9 mg, 0.078 mmol) were suspended in dry benzene (0.7 mL) in a microwave vial. The vial was purged with nitrogen, then capped and heated under a microwave irradiation at 100 °C for 4 h in a CEM Discover apparatus. The mixture was cooled to room temperature, additional amounts of Zn(ClO₄)₂·6H₂O (0.5 mg, ca. 0.02 equiv) and glutaric anhydride (8.9 mg, 0.078 mmol) were added, and the mixture was heated again under the same conditions as above for additional 3 h. After being cooled, the mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine and dried over anhydrous MgSO₄. The chromatographic purification of the residue left after the evaporation of the solvent, using CHCl₃/MeOH mixtures from 100:0 to 97:3 as the eluent, afforded hapten SPo (17.7 g, 50.5%) as an amorphous solid. IR ν_max (cm^–1): 3263, 3013, 2927, 1776, 1725, 1701, 1638, 1198, 1172, 747; ¹H NMR (CDCl₃, 300 MHz): δ 9.00 (1H, s, NH), 7.10 (1H, d, J = 7.8 Hz, H-3 Ph), 7.03 (1H, dd, J = 7.8, 1.5 Hz, H-4 Ph), 6.96 (1H, d, J = 1.5 Hz, H-6 Ph), 4.87–4.70 (1H, m, H-8′), 4.02 (2H, q, J = 7.1 Hz, OCH₂Me), 2.48–2.37 (4H, m, H₂-2, H₂-4), 2.28 and 2.21 (3H each; each s, 2 × Me-Ph), 2.19–2.07 (2H, m, H₂-Cy), 2.03–1.90 (4H, m, H₂-3, H₂-Cy), 1.76–1.56 (4H, m, 2 × H₂-Cy), 1.10 (3H, t, J = 7.1 Hz, OCH₂Me); ¹³C NMR (CDCl₃, 75 MHz): δ 177.3 (C-1), 172.6 (C-5), 172.2 (C-2′), 165.3 (C-4′), 149.8 (OCO₂), 135.0 (C-5 Ph), 134.0 (C-1 Ph), 130.2 (C-3 Ph), 129.9 (C-6 Ph), 129.6 (C-4 Ph), 127.4 (C-2 Ph), 121.4 (C-3′), 71.1 (C-8′), 65.8 (OCH₂Me), 60.8 (C-5′), 33.7 (C-2), 33.1 (C-4), 31.6 (C-6′/C-10′), 28.1 (C-7′/C-9′), 20.0 (C-3), 20.8 and 19.2 (2 × Me-Ph), 13.7 (OCH₂Me); HRMS: calcd for C₂₅H₃₂NO₈ [M + H]^+•, 474.2122; found, 474.2107.

4.4. Preparation of Bioconjugates

Active ester formation was carried out with N,N-disuccinimidyl carbonate and Et₃N at 0 °C (Figure 1). The complete activation procedures as well as the ¹H NMR spectra of the corresponding NHS esters are provided in the Supporting Information. For conjugation, a 50 mM purified hapten solution in N,N-dimethylformamide was dropwise added to a 15 mg/mL BSA or OVA solution in 100 mM phosphate buffer, pH 7.4 (50 mM carbonate–bicarbonate buffer, pH 9.6, was used to couple hapten SPo to BSA) under gentle stirring. A 5 mM purified hapten solution was slowly mixed with a 2.5 mg/mL HRP solution in 100 mM phosphate buffer, pH 7.4, for tracer enzyme conjugate preparation. BSA immunizing conjugates were prepared with a 22-fold molar excess of purified activated hapten, whereas the OVA and HRP assay conjugates were prepared with an 8-fold molar excess of hapten. The mixtures were incubated for 2 h at room temperature, and the conjugates were purified by gel filtration chromatography using 100 mM phosphate buffer, pH 7.4, as the eluent. The BSA conjugate solutions were filter-sterilized with 0.45 μm pore filter units and stored frozen at −20 °C. The OVA conjugates were stored frozen, whereas the HRP conjugates were diluted 1:1 with phosphate-buffered saline (PBS) containing 1% (w/v) BSA and stored at 4 °C. The obtained hapten-to-protein MR was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The sample preparation and MS analysis of bioconjugates are described in the Supporting Information.

4.5. Antibody Generation

The experimental design was approved by the Bioethics Committee of the University of Valencia. The animals were manipulated according to Spanish laws (RD1201/2005 and law 32/2007) and in compliance with the EU Directive 2010/63EU about the protection of laboratory animals. Two groups of two female New Zealand white rabbits were subcutaneously immunized with 300 μg of either BSA–SPc or BSA–SPo. Four injections were applied at 21 day intervals. The immunogen consisted of a 1:1 emulsion between PBS and Freund’s adjuvant (complete for the first injection and incomplete for subsequent boosts). Ten days after the last injection, the animals were exsanguinated by intracardiac puncture, and blood was let to coagulate at 4 °C during 24 h. Then, the serum was separated by centrifugation at 3000g for 20 min. The immunoglobulins were partially purified from the animal sera by salting out twice with one volume of a cold saturated (3.90 M) ammonium sulfate solution. The antibodies were stored precipitated at 4 °C.

4.6. Competitive ELISA

Immunoassays were carried out in three different competitive ELISA formats. The indirect format was employed by immobilizing on the microplate wells the OVA–hapten conjugates and using an enzyme-labeled secondary antibody. The direct format was performed with antibody-coated plates and using the HRP–hapten enzyme tracers. Finally, the capture antibody direct format was evaluated with immobilized goat antirabbit immunoglobulin and the corresponding enzyme tracers in solution. Eight-point standard curves in PBS (10 mM phosphate buffer, pH 7.4, containing 140 mM NaCl) were prepared in borosilicate glass vials by serial dilution, including a blank without analyte. The ELISA absorbance values were fitted to a four-parameter logistic equation. Assay sensitivity was defined as the concentration of analyte at the inflexion point of the sigmoidal curve, typically corresponding to a 50% inhibition (IC₅₀) of the maximum signal (A_max). Antibody titer was defined as the antibody dilution affording an A_max value near to 1.0. The assay selectivity was estimated from the quotient between the IC₅₀ of the reference analyte and the IC₅₀ of the cross-reacting compound. For complete immunoassay procedures, see the Supporting Information.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01784.

• General procedures, materials, and instrumentation; preparation of N-hydroxysuccinimidyl esters of haptens; MALDI-MS analysis of bioconjugates; competitive ELISA procedures; and ¹H NMR spectra of the haptens SPc and SPo and their NHS esters (PDF)

Author Present Address

^§ Department of Chemical Processes, Technical University of Manabi (UTM), Avenue José María Urbina y Che Guevara, 130105 Portoviejo, Republic of Ecuador.

The authors declare no competing financial interest.