Concise and modular synthesis of regioisomeric haptens for the production of high-affinity and stereoselective antibodies to the strobilurin azoxystrobin

Article abstract of DOI:10.1016/j.tet.2010.11.054

The immune response to regioisomeric haptens of azoxystrobin with varied derivatization sites was studied. Based on the Sonogashira and Suzuki-Miyaura couplings and following a straightforward modular design, we have synthesized four haptens with the same linker anchored through C-C bonds and located at different sites of the molecule. The most stereoselective antibodies were produced from immunogens with the spacer arm at a distal position from the β-methoxyacrylate moiety characteristic of strobilurins. Moreover, we observed that assay cross-reactivity was reliant on the functionalization site of the competitor derivative. Finally, the antibody binding site was explored using synthetic chemical analogues.

Full text of DOI:10.1016/j.tet.2010.11.054

Tetrahedron 67 (2011) 624e635
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Concise and modular synthesis of regioisomeric haptens for the production of
high-affinity and stereoselective antibodies to the strobilurin azoxystrobin
a
a
a,
ꢀ b
*
,
Javier Parra , Josep V. Mercader , Consuelo Agullo , Antonio Abad-Fuentes
Antonio Abad-Somovilla b
Department of Biotechnology, IATAeCSIC, Agustí Escardino 7, 46980 Paterna, Valencia, Spain
,
*
a
ꢁ
b
ꢁ
ꢁ
Department of Organic Chemistry, Universitat de Valencia, Doctor Moliner 50, 46100 Burjassot, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 October 2010
Received in revised form 8 November 2010
Accepted 15 November 2010
Available online 24 November 2010
The immune response to regioisomeric haptens of azoxystrobin with varied derivatization sites was
studied. Based on the Sonogashira and SuzukieMiyaura couplings and following a straightforward
modular design, we have synthesized four haptens with the same linker anchored through CeC bonds
and located at different sites of the molecule. The most stereoselective antibodies were produced from
immunogens with the spacer arm at a distal position from the b-methoxyacrylate moiety characteristic
of strobilurins. Moreover, we observed that assay cross-reactivity was reliant on the functionalization site
of the competitor derivative. Finally, the antibody binding site was explored using synthetic chemical
analogues.
Keywords:
Hapten design
CeC coupling
Linker attachment site
Landsteiner
Ó 2010 Published by Elsevier Ltd.
Molecular recognition
Immunochemistry
1. Introduction
functionalized derivatives of the parent compound is one of the
most important steps in the process to produce high-affinity anti-
The generation of antibodies against small organic molecules
has a broad applicability in the medical, environmental and ana-
lytical sciences. During the past decades, antibodies have been
raised and immunoassays have been developed for the detection
and quantification of chemical substances of clinical interest, such
as hormones1 and drugs of abuse,2 as well as antibiotic3 and pes-
ticide4 residues in environmental and food samples.
In order to produce antibodies against a small organic molecule
and for the further development of a sensitive immunoassay, it is
necessary to covalently link the molecule (or a synthetic analogous
hapten) to a carrier protein. This bioconjugate is capable of stimu-
lating the immune system and producing antibodies against the
target molecule. The usual way of coupling a hapten to a carrier
antigen is to synthesize, first, a derivative that incorporates a spacer
arm, generally consisting in a linear hydrocarbon chain of a certain
length, most frequently between three and six carbon atoms, which
is ended in a functional group that can be used for the subsequent
conjugationtothecarrier molecule. Thedesign andsynthesis ofsuch
bodies.5 Hapten design must be guided by the principle of maxi-
mizing steric, electronic and geometric similarity with the analyte.
An ideal or optimum immunizing hapten should maintain the in-
tegrity of the framework and functional groups of the parent mol-
ecule, incorporating the linker chain at the site that induces the
lowest possible changes in the electronic and conformational
properties of the target compound.
As originally stated by Karl Landsteiner,6 the position of the
spacer arm that connects the hapten to the carrier protein is critical
in determining the specificity of the generated antibody, which is
usually maximal for those sites distal to the point of conjugation. In
accordance with this so-called Landsteiner’s principle, antibody
specificity is directed primarily to the portion of the hapten located
furthest from or opposite to the functional group linking it to the
carrier protein. To our knowledge, however, very few studies have
been carried out that allow an in-depth evaluation of the signi-
ficance of the derivatization site over the affinity and specificity of
the raised antibodies, and to what extent it is possible to modulate
antibody specificity by using bioconjugates with different linker
positions during immunization.7
Based on these principles, we designed four regioisomeric hap-
tens for azoxystrobin as a model molecule (Fig. 1). Azoxystrobin was
the first discovered and patented antifungal of a new class of
* Corresponding authors. Tel.: þ34 963636301; fax: þ34 963900022 (A.A.-F.);
tel.: þ34 963544509; fax: þ34 963544328 (A.A.-S.); e-mail addresses: aabad@
iata.csic.es (A. Abad-Fuentes), antonio.abad@uv.es (A. Abad-Somovilla).
0040-4020/$ e see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.tet.2010.11.054

J. Parra et al. / Tetrahedron 67 (2011) 624e635
625
agrochemicals, the strobilurins,8 and it is currently the world’s best
selling proprietary fungicide, with global annual sales over $1 billion
in 2008.9 Each of the four haptens incorporated the same hydro-
carbon spacerarm, but at different positions, which encompasses the
entire azoxystrobin skeleton. All derivatives maintained the whole
structure and characteristic groups of the target molecule, while the
spacer arm was connected via a carbonecarbon single bond at a site
where a carbonehydrogen bond previouslyexisted, thus introducing
the least possible changes on the electronic properties of the tricyclic
azoxystrobin framework.
Fig. 1. Structure of azoxystrobin and regioisomeric haptenic derivatives.
This comprehensive approach, which has rarely been used
before in the design of haptens for the production of antibodies
against structurally complex non-rigid small organic molecules,
allowed a broad diversity of orientations in which the antigen was
presented to the immune system, thereby increasing the chance of
producing antibodies with enhanced binding properties. Moreover,
the regioisomeric nature of the synthesized immunizing haptens
ensured that the differences in the binding affinity and/or speci-
ficity of the derived antibodies could be only attributed to the
variation in the linker attachment site. Antisera, representing the
unbiased whole-response of the animal immune system, were
chosen as the source of antibodies, and competitive enzyme-linked
immunosorbent assay (cELISA) was employed to evaluate the
affinity and selectivity of the produced antibodies.
Scheme 1. Modular synthetic approach (a/ab/abc) for the preparation of azox-
ystrobin haptens.
2.1.1. Synthesis of hapten AZa6 (2). The synthesis of hapten AZa6
started with the preparation of the phenolic a-ring synthon (i.e., 13)
from 2,4-dibromophenol (Scheme 2). First, the phenolic hydroxyl
group was protected with a conventional benzyl group using
standard conditions. Regioselective lithiumebromide exchange by
treatment of the obtained dibromobenzylether with BuLi at low
temperature,12 followed by quenching with triisopropyl borate
afforded the corresponding diisopropyl arylboronate, which was
then hydrolyzed in situ to the arylboronic acid 8 by treatment with
hydrochloric acid at rt. The SuzukieMiyaura cross-coupling
reaction between the arylboronic acid 8 and the iodoacrylate 913
2. Results and discussion
2.1. Hapten synthesis
The preparation of each of the four regioisomeric haptens (AZa6,
AZb6, AZc6 and AZo6) was undertaken via a concise, modular and
convergent synthesis based on two consecutive nucleophilic aro-
matic substitutions of the chlorine atoms of a 4,6-dichloro-
pyrimidine (ring b) with two conveniently functionalized phenols
(rings a and c) (Scheme 1).10 The saturated carboxylated alkyl chain
that constitutes the spacer arm in each hapten is introduced at the
appropriate position of the aromatic ring subunit by means of
a Sonogashira coupling reaction between the suitable aryl halide
and tert-butyl hex-5-ynoate, followed by hydrogenation of the
acetylenic triple bond. The incorporation of the carboxylated spacer
arm as its tert-butyl ester is made on the basis of its easy acid hy-
drolysis to the corresponding carboxylic acid in presence of the
that completed the incorporation of the
b-methoxyacrylate
moiety, was undertaken using similar reaction conditions to the
optimized once developed by Heo’s group for related couplings, i.e.,
Pd(PPh)4 as the palladium catalyst and K3PO4 as the base in a THF/
H2O system at 85 ꢀC.14 Under these conditions, the coupling of 8
and 9 took place very efficiently to give the
a-aryl-b-methox-
yacrylate 10 in 81% overall yield from starting dibromophenol (6).
The incorporation of the hydrocarbon spacer arm was based on
the Sonogashira cross-coupling reaction between the aryl bromide
10 and the terminal alkyne-ester 11, which had been obtained from
commercially available hex-5-ynoic acid.15 The cross-coupling
reaction was carried out efficiently using Pd(OAc)2 as the source of
the palladium catalyst and 1,4-diazabicyclo[2.2.2]octane (DABCO)
as the base in acetonitrile at rt.16 The next and last step that com-
pleted the preparation of the required arylic a-ring synthon in-
volved the hydrogenation of the acetylenic compound 12, using 10%
palladium-on-carbon as the catalyst. Under these conditions, both
complete hydrogenation of the triple bond and hydrogenolysis of
base sensitive
b-methoxyacrylate moiety. On the other hand, the
incorporation of the
b-methoxyacrylate moiety to the phenolic
a-ring synthon is carried out by means of a SuzukieMiyaura cross-
coupling reaction between the appropriate arylboronic acid and
methyl (Z)-2-iodo-3-alkoxy-acrylate, a kind of reaction that has
been successfully used for the elaboration of the
b-methoxyacrylate
moiety in related systems.11

626
J. Parra et al. / Tetrahedron 67 (2011) 624e635
Scheme 2. Synthesis of hapten AZa6 (2).
the benzyl protecting group took place to afford the desired phenol
13 in almost quantitative yield.
reagent,17 or 2-methyl-4,6-dichloropyrimidine, via alkylation re-
action of the benzylic-type carbanion derived from abstraction of
a proton from the methyl group,18 were unsuccessful. Therefore, we
used the alternative route based on a Sonogashira cross-coupling
reaction starting from the commercially available 2-amino-
pyrimidine 18. The 2-iodination of 18 was performed in good yield
by the method of Nair and Fasbender,19 using tert-butyl nitrite to
generate the corresponding pyrimidin-2-yl radical and diiodo-
methane as the source of iodine. Subsequent Sonogashira cross-
coupling reactions with the terminal alkyne 11 took place under
very smooth conditions affording the 2-alkenyl pyrimidine de-
rivative 20. It should be mentioned that, in spite of the recognized
much better ability of iodine, as compared with chlorine, as leaving
group in this type of coupling, the reaction also led to variable
amounts of the coupling products formed through the chlorinated
positions. This circumstance limits the yield obtained for 20 and can
be attributed to the intrinsically higher reactivity of the 4/6-py-
rimidine positions towards the oxidative addition step.20 Comple-
tion of the introduction of the hydrocarbon chain at the C-2 position
of the pyrimidine nucleus was undertaken by hydrogenation of the
triple bond of compound 20 under low hydrogen pressure and
careful control of the progress of the reaction by TLC analysis to
avoid excessive hydrogenolysis of the CeCl bonds. The desired 2-
alkyl pyrimidine 21 was thus obtained in about 68% yield from 2-
iodopyrimidine 19.
With compound 13 at hand, the synthesis of the hapten AZa6
was readily completed following the designed modular strategy.
The phenol 13 was reacted with 4,6-dichloropyrimidine (14) using
caesium carbonate as base in DMF at 0 ꢀC. Nucleophilic displace-
ment of the chlorine atom by the phenoxide moiety took place
smoothly under these conditions to afford the corresponding
substituted pyrimidine in high yield. It must be mentioned that
both, the low temperature and the use of caesium carbonate as the
base was essential to achieve a good yield for this reaction. Thus,
higher temperatures or the use of other bases habitually used in
this type of transformation, such as other alkaline carbonates, gave
less clean reactions resulting in lower yields of compound 15. In
particular, substantial amounts of benzofurano-2(3H)-one and
benzofurano-type derivatives, originated by intramolecular cycli-
zation of the phenolic hydroxyl group of 13 with the b-methox-
yacrylate moiety, were formed in the above conditions at rt or
when K2CO3 or NaH were used as bases.
The tricyclic carbon skeletal framework of the target hapten was
completed via another nucleophilic substitution reaction of the
chlorine atom of chloropyrimidine 15 with 2-cyanophenol (16). In
this case, this chloride substitution reaction was much more diffi-
cult than the former and required heating of the reaction mixture at
85 ꢀC. Anyway, the reaction was also very efficient affording the
tert-butyl ester of hapten AZa6 (17) in an excellent 93% yield.
Finally, chemoselective trifluoroacetic acid-promoted hydrolysis of
the tert-butyl ester moiety of 17 afforded the hapten AZa6 (2) in 93%
yield after its chromatographic purification.
With 21 available, further elaboration of the oxygen-bridged
tricyclic framework of hapten AZb6 was achieved, as before,
through two consecutive nucleophilic aromatic substitution
reactions. First, the dichloropyrimidine 21 was reacted under very
smooth basic conditions with methyl (E)-2-(2-hydroxyphenyl)-3-
methoxyacrylate (22) to afford the pyrimidinyl-aryl ether 25 in
2.1.2. Synthesis of hapten AZb6 (3). Following the designed con-
vergent synthetic route outlined in Scheme 1 for the preparation of
the haptens, the synthesis of hapten AZb6 was accomplished with
the previous preparation of the synthon corresponding to the pyr-
imidinyl b-ring, the 2-alkyl-4,6-dichloropyrimidine 21 (Scheme 3).
A key step in the preparation of this synthon was the incorporation
of the hydrocarbon spacer arm at the C-2 position of the pyrimidine
ring. Several attempts to prepare 20 starting from 4,6-dichloropyr-
imidine, via hydride substitution at C-2 with a lithium acetylide
60% yield. Although the preparation of a-aryl-b-methoxyacrylate
22 has already been described,21 it was more easily obtained in this
case from commercial boronic acid 23, in only two steps and 85%
overall yield, via SuzukieMiyaura cross-coupling reaction with
iodoacrylate 9 to give 24 and hydrogenolysis of the protective
benzyl group. Completion of the tricyclic aromatic ring system was
achieved through a second nucleophilic aromatic substitution
reaction between chloropyrimidine derivative 25 and 2-

J. Parra et al. / Tetrahedron 67 (2011) 624e635
627
Scheme 3. Synthesis of hapten AZb6 (3).
cyanophenol (16) catalyzed by Cs2CO3 in DMF at 85 ꢀC. Finally, the
synthesis of the required hapten AZb6 (3) was completed very
efficiently by trifluoroacetic acid-promoted hydrolysis of the tert-
butyl ester moiety of 26.
hydrocarbon spacer arm at this aromatic system via a palladium-
catalyzed cross-coupling process. This was achieved using the io-
dine-silver sulfate system in dichloromethane, which afforded
regioselectively the iodo-phenol 27 in an excellent 84% yield. Several
attempts to directly incorporate the hydrocarbon chain from the
iodo-phenol 27, via a Sonogashira reaction with tert-butyl hex-5-
ynoate (11), failed, even under the conditions described previously
for related transformations.22 Therefore, the phenolic hydroxyl
group of 27 was transformed into the corresponding benzyl ether by
alkylation with benzyl bromide in basic medium. The resulting
benzyl ether 28 underwent the Sonogashira palladium-catalyzed
2.1.3. Synthesis of hapten AZc6 (4). The synthesis of hapten AZc6,
which incorporated the hydrocarbon spacer arm at the cyanophe-
noxy moiety, required the previous preparation of the synthon cor-
responding to the arylic c-ring, the phenol 30 (Scheme 4). The first
step for the preparation of this intermediate was the iodination of
2-cyanophenol (16), necessary for the further incorporation of the
Scheme 4. Synthesis of hapten AZc6 (4).

628
J. Parra et al. / Tetrahedron 67 (2011) 624e635
cross-coupling reaction with the alkyne-ester 11, under similar
conditions as those previously used in the preparation of hapten
AZa6, to afford the aryl-alkyne 29 in almost quantitative yield. Fur-
ther catalytic hydrogenation of the triple bond, with concomitant
O-benzyl group hydrogenolysis, completed the preparation of the
key phenol intermediate 30.
smoothly to afford the corresponding a-aryl-b-alkoxyacrylate 36,
which was transformed under hydrogenolysis conditions into the
required phenol 37 in 68% overall yield. It must be noted that the
hydrogenolysis reaction of the O-benzyl group of 36 resulted more
problematic that it might be expected on the basis of the results
previously obtained for the related reaction of benzyl ether 24. In
With phenol intermediate 30 available, further elaboration of
the whole framework of hapten AZc6 was straightforward follow-
ing essentially the same strategy as that described above for the
preparation of the other haptens. Thus, reaction of 30 with
4-phenyloxy-6-chloropyrimidine 31, in the same conditions de-
scribed above for the preparation of regioisomeric compounds 17
and 26, afforded the tert-butyl ester of hapten AZc6 (32) in 91%
yield after silica-gel column chromatographic purification. The
4-aryloxy-6-chloropyrimidine 31 was prepared in an improved
way (84% yield) by modification of a previously described pro-
cedure,23 involving the reaction between 4,6-dichloropyrimidine
(14) and 2-(2-hydroxyphenyl)-3-methoxy acrylate (22) catalyzed
by Cs2CO3 in DMF at 0 ꢀC.
this case, the b-alkoxyacrylate moiety was partially hydrogenated
(up to 50%) to the corresponding saturated system under the
hydrogenation conditions previously used for 24. This side reac-
tion could be minimized using a low pressure of hydrogen and
limiting the reaction time to about 1 h.
The final steps to complete the synthesis of hapten AZo6 were,
as designed, the same as those previously used for the preparation
of hapten AZa6. Thus, reaction of phenol 37 with 4,6-dichloropyr-
imidine (14) followed by the reaction with 2-cyanophenol (16) and
the final acid-catalyzed hydrolysis of the tert-butyl ester moiety
completed the synthesis of hapten AZo6 (5), with an overall yield of
about 52% for the three steps.
Finally, acid hydrolysis of the tert-butyl ester group of 32 fur-
nishes the corresponding carboxylic acid, thus completing the
synthesis of hapten AZc6 (4).
2.2. Immunological response
For rabbit immunization, the synthesized haptens were con-
verted to NHS-active esters and covalently linked to bovine serum
albumin (BSA) to afford bioconjugates with similar hapten-to-pro-
tein molar ratios (15e20). After the fourth boost, all of the immu-
nized animals gave rise to high titres (over 105) against the
homologous ovalbumin (OVA) conjugate. The obtained antisera also
bound tightly to free azoxystrobin, showing affinity values in the
low nanomolar range (Table 1). Interestingly, rabbits that were
immunized with BSAeAZb6 (the hapten that was functionalized at
the central pyrimidine ring) produced the antisera with the lowest
affinity to azoxystrobin in homologous assays, whereas the antisera
derived from the three other immunogens showed a very similar
binding to the free compound (Fig. 2). The IC50 values in competitive
assays using AZb6-type sera could be lowered if heterologous con-
jugates were employed, but still these antisera provided the least
sensitive assays, with IC50 values over 3 nM. The highest affinity
was observed with AZo6-derived antisera in combination with the
heterologous conjugate OVAeAZa6 (IC50¼0.3 nM), in which
the derivatization site was at the aromatic ring located proximal to
the methoxyacrylate moiety where the linker in AZo6 was located.
2.1.4. Synthesis of hapten AZo6 (5). The synthesis of the hapten
AZo6 began with the previous elaboration of the aryl a-ring syn-
thon that introduced the hydrocarbon spacer arm at the b-alkoxyl
group of the acrylate moiety (Scheme 5). The preparation of this
intermediate, the phenol 37, was accomplished following a similar
strategy to that previously used for the preparation of the related
phenolic compound 22 (see Scheme 3). The synthesis of 37 began
with the achievement of
b-alkoxyacrylate 34, which was un-
dertaken in very high yield by the stereoselective reaction of tert-
butyl 6-hydroxyhexanoate (33) with methyl propiolate in the
presence of a catalytic amount of tri-n-butyl phosphine.24 Sub-
sequent acetoxy-iodination of the vinyl ether moiety of 34 with
N-iodosuccinimide (NIS) and AcOH, followed by elimination of
AcOH from the resulting mixture of diastereoisomeric iodoace-
tates using Et3N,25 afforded exclusively the methyl (Z)-
a-iodo-b-
alkoxyacrylate isomer 35 in good overall yield. The subsequent
SuzukieMiyaura cross-coupling reaction between the iodoacryla-
tye 35 and the commercial arylboronic acid 23 proceeded
Scheme 5. Synthesis of hapten AZo6 (5).

J. Parra et al. / Tetrahedron 67 (2011) 624e635
629
Table 1
reactivity values for this isomer that were achieved by cELISA with
homologous and heterologous coating conjugates. A dissimilar re-
sponse was clearly found for the different antisera. Following
Landsteiner’s principle, the highest stereospecificity was obtained
with AZc6-derived antisera, independently of the hapten conjugate
that was employed in the assay. Interestingly, the Landsteiner’s
principle was also applicable to the coating conjugate because, for
all sorts of antisera, the cross-reactivity values could be lowered
below 10% if OVAeAZc6 was used for coating, independently of the
employed antiserum. Therefore, not only AZc6-type antisera but
also AZc6 conjugates, in which the linker was placed at the aromatic
Antibody affinity for azoxystrobin by cELISAa
Antiserumc
Coating conjugateb
OVAeAZa6
OVAeAZb6
OVAeAZc6
OVAeAZo6
AZa6
AZb6
AZc6
AZo6
1.64 ꢁ 0.26
3.05 ꢁ 0.29
1.01 ꢁ 0.17
0.30 ꢁ 0.04
1.62 ꢁ 0.02
19.04 ꢁ 1.20
0.79 ꢁ 0.09
1.40 ꢁ 0.13
0.94 ꢁ 0.19
3.47 ꢁ 0.23
1.85 ꢁ 0.05
2.89 ꢁ 0.37
1.39 ꢁ 0.16
3.32 ꢁ 0.21
1.68 ꢁ 0.38
0.93 ꢁ 0.12
a
Values are the mean of three independent experiments. Immunoreagents were
used at limiting concentrations. Amax values were between 0.7 and 1.5. Affinity
values are expressed in nM.
b
Coating conjugate concentration was 0.1 mg/mL.
Results are for one antiserum from each immunogen.
ring located distal to the b-methoxyacrylate group, clearly afforded
c
the best stereospecificity. On the contrary, if plates were coated with
OVAeAZb6, AZa6-derived antisera bound similarly both stereoiso-
mers (cross-reactivity close to 90%).
AZa6-type Ab
AZb6-type Ab
AZc6-type Ab
AZo6-type Ab
100
80
60
40
20
0
Finally, in order to further enquire into the specificity properties
of the antibody binding site, the capacity of the produced antisera
to distinguish between structurally quite similar molecules was
investigated. With this aim, two structural analogues of azox-
ystrobin (compounds 40 and 41, Fig. 3) were prepared in our lab-
oratory following a similar strategy to that used for the preparation
of the synthetic haptens (see the Supplementary data). Each of
them contained just one modified moiety located at opposite sites,
either at the cianophenyl ring or at the acrylate group. These
minimal changes in the azoxystrobin structure drastically reduced
the affinity of the antisera for such analogues (cross-reactivity
values were lower than 1%), thus confirming the high selectivity of
the produced antibodies. However, differential binding behaviours
to compounds 40 and 41 could be observed for the four types of
antisera. As shown in Fig. 3, AZo6- and AZa6-derived antisera dis-
played a lower affinity towards analogue 41 (the compound with-
out the nitrile group) than to analogue 40 (the compound without
the methoxymethylene group). On the contrary, AZc6-type anti-
bodies bound worst to compound 40 than to compound 41. In
both cases, the affinity was lower towards the compound in
which the modification was distal from the derivatization site of the
corresponding immunizing hapten, demonstrating again that the
specificity of the immune response was mainly directed towards
the chemical moieties that were located opposite to the linker.
0
10-2 10-1 100 101 102 103 104
[AZ] (nM)
Fig. 2. Inhibition curves for azoxystrobin in homologous competitive assays using
antibodies derived from AZa6 (circles), AZb6 (up triangles), AZc6 (squares) and AZo6
(down triangles).
Concerning selectivity, all of the generated antisera were highly
specific to their target and none of them recognized any of the other
strobilurin fungicides. In fact, this family of agrochemicals only
shares the small b-methoxyacrylate group or a modification of this
toxophore moiety. Instead, the bulk of the molecule is particular and
distinct for each compound. Azoxystrobin exists in two stereoiso-
meric forms, although the fungicide activity strongly relies on the
E-isomer, which accounts for up to 98% of the technical commercial
product. On the other hand, the Z-isomer is the main degradation
product, and it appears primarily by photochemical reaction.26
During hapten synthesis, especial precaution was taken to pre-
serve the E-conformation of the toxophore in all derivatives. Anti-
bodies are biomolecules most often displaying exquisite specificity
to their target, being able to discriminate even between chiral
molecules and geometric isomers.27 Accordingly, we thought it was
worthwhile to challenge the generated antisera with the Z-isomer
to further explore the fine specificity of their binding sites and to
study the relationship between their stereoselectivity and the linker
position in the parental hapten. The Z-isomer of azoxystrobin was
prepared as described by Clough et al.,28 and competitive assays
with both isomers were run in parallel. Table 2 lists the cross-
N
N
N
N
O
O
O
O
O
O
O
CN
41
OCH3
40
OCH3
20
15
10
5
Table 2
Cross-reactivities for the azoxystrobin Z-isomer (%)a
Antiserumc
Coating conjugateb
AZa6
AZb6
AZc6
AZo6
AZa6
AZb6
AZc6
AZo6
16.0 ꢁ 2.9
51.9 ꢁ 6.8
8.0 ꢁ 0.6
88.1 ꢁ 4.2
15.7 ꢁ 0.9
2.0 ꢁ 0.1
1.7 ꢁ 0.2
7.3 ꢁ 0.8
2.4 ꢁ 0.3
3.0 ꢁ 1.0
16.4 ꢁ 1.3
45.5 ꢁ 7.7
2.7 ꢁ 0.3
0
AZo6
AZa6
AZb6
AZc6
21.8 ꢁ 5.1
20.8 ꢁ 3.6
11.9 ꢁ 3.1
Polyclonal Antiserum
a
Values are the mean of four independent experiments.
b
c
Coating conjugate concentration was 0.1
mg/mL.
Fig. 3. Affinity of the different types of antisera towards two analogues of azox-
ystrobin. Black bars: compound 40; grey bars: compound 41.
Results are for one antiserum from each immunogen.

630
J. Parra et al. / Tetrahedron 67 (2011) 624e635
3. Conclusion
4.1.1.3. (E)-Methyl 2-(2-(benzyloxy)-5-bromophenyl)-3-methox-
yacrylate (10). A mixture of iodoacrylate 9 (214 mg, 0.886 mmol),
arylboronic acid 8 (272 mg, 0.886 mmol), K3PO4 (564.2 mg,
2.658 mmol) and Pd(PPh3)4 (51 mg, 0.044 mmol) in a mixture of
A concise modular approach for the synthesis of regioisomeric
haptens for azoxystrobin has been designed. Distinct functional-
ized haptens with the same linker located at varying positions
of the azoxystrobin framework were prepared. Following this
approach, we could generate antibodies showing superior speci-
ficity and higher affinity than the previously reported polyclonal
antibodies for this compound.29 It could also be concluded that the
hapten in which the linker was placed at the central ring of azox-
ystrobin afforded antisera with lower affinity for the free molecule,
probably because of a less efficient display of the chemical to the
immune system. The prepared haptens provided expanded possi-
bilities for tuning the assay sensitivity and specificity through the
selection of optimum haptens for the preparation of solid surface-
coating bioconjugates. Our study showed that the observed cross-
reactivity for an antiserum can greatly depend on the employed
coating conjugate. This observation is probably exclusive of poly-
clonal antibodies and it might be a result of the heterogeneity of
this sort of immunoreagent. For comparison, further studies are
being conducted to generate a collection of monoclonal antibodies
using the described synthetic haptens. Finally, the produced anti-
bodies and conjugates will be highly valuable reagents for the
development of sensitive immunoassays applicable to monitoring
programmes for the analysis of azoxystrobin in, for example, food
and environmental samples.
dioxane (1.15 mL) and water (233
mL), previously degasified by
bubbling nitrogen under ultrasonic irradiation for a period of 10 min,
was stirred under nitrogen at 85 ꢀC for 4.45 h. After this time, the
reaction mixture was cooled down, poured into water and worked
up using EtOAc as extraction solvent. Column chromatography,
eluting with hexane/EtOAc (from 9:1 to 8:2) as eluent, gave methyl
acrylate derivative 10 (312 mg, 93%) as a solid. Mp 121e122 ꢀC (from
hexane/EtOAc). 1H NMR (300 MHz, CHCl3):
d
¼7.50 (s, 1H), 7.342 (m,
5H), 7.341 (dd, J¼8.8, 2.5 Hz, 1H), 7.32 (d, J¼2.5 Hz, 1H), 6.81 (d,
J¼8.8 Hz,1H), 5.04 (s, 2H), 3.81 (s, 3H), 3.66 (s, 3H) ppm. HRMS (ES):
calcd for C18H1779BrNaO4 [MþþNa]þ 399.0208, found 399.0207.
4.1.1.4. tert-Butyl 6-(3-((E)-1-(methoxycarbonyl)-2-methoxyvinyl)-
4-(benzyloxy)phenyl)hex-5-ynoate (12). A solution of tert-butyl hex-
5-ynoate (11, 194 mg, 1.154 mmol) in anhydrous degassed DMF
(1.03 mL) was added to a mixture of aryl bromide 10 (152.3 mg,
0.40 mmol), CuI (12 mg, 0.063 mmol) and (Ph3P)2PdCl2 (38 mg,
0.054 mmol) followed anhydrous Et3N (1.03 mL) under nitrogen
atmosphere. The reaction mixture, initially yellow that turned
deep-orange after a few minutes, was stirred in an oil bath at 70 ꢀC
for 22 h. It was then cooled down to rt, poured into water and
worked up in the usual manner. Chromatography of the crude
product with hexane/EtOAc (from 9:1 to 7:3) as eluent afforded
alkyne 12 (162 mg, 88%) as a solid. Mp 94e96 ꢀC (from hexane). 1H
4. Experimental
NMR (300 MHz, CHCl3):
d
¼7.50 (s, 1H), 7.34 (m, 5H), 7.31 (dd, J¼8.5,
4.1. Hapten synthesis
2.0 Hz, 1H), 7.25 (d, J¼2.0 Hz, 1H), 6.84 (d, J¼8.5 Hz, 1H), 5.06 (s,
OCH2, 2H), 3.80 (s, 3H), 3.64 (s, 3H), 2.43 (t, J¼7.2 Hz, 2H), 2.39 (t,
J¼7.2 Hz, 2H), 1.86 (quint, J¼7.2 Hz, 2H), 1.45 (s, 9H) ppm. HRMS
(EI): calcd for C28H32O6 464.2199, found 464.2196.
General experimental details and full characterisation data for
all of the described compounds are given as Supplementary data.
4.1.1. Synthesis of hapten AZa6 (2).
4.1.1.5. tert-Butyl 6-(3-((E)-1-(methoxycarbonyl)-2-methoxyvinyl)-
4-(benzyloxy)phenyl)hexanoate(13). A mixture ofalkyne 12 (90.3 mg,
0.195 mmol) and 10% Pd/C in EtOAc (2.4 mL) was stirred under an
atmosphere of hydrogen at 4 atm overnight. The reaction mixture
was filtered through a short silica-gel column, using EtOAc as the
eluent. The filtrate was concentrated to dryness to give pure the title
compound 13 (73.5 mg, 100%) as a colourless oil. 1H NMR (300 MHz,
4.1.1.1. 1-(Benzyloxy)-2,4-dibromobenzene (7). Benzyl bromide
(592
mL, 4.983 mmol) was added to a solution of 2,4-dibromo-
benzene (1.142 g, 4.53 mmol) and anhydrous K2CO3 (688.7 mg,
4.982 mmol) in dry DMF (9.5 mL) under nitrogen. The mixture was
stirred at rt for 1.30 h, poured into water and worked up as usual,
using EtOAc to extract. Chromatography of the residue left after
evaporation of the solvent, using hexane/EtOAc (from 9:1 to 8:2) as
eluent, afforded the benzyl ether 7 (1.475 g, 96%) as a white solid.
CHCl3):
d
¼7.61 (s, 1H), 7.02 (dd, J¼8.2, 2.1 Hz, 1H), 6.94 (d, J¼2.1 Hz,
1H), 6.88(d, J¼8.2 Hz,1H), 6.15 (br s,1H), 3.88 (s, 3H), 3.76 (s, 3H), 2.53
(t, J¼7.6 Hz, 2H), 2.20 (t, J¼7.6 Hz, 2H), 1.60 (quint, J¼7.6 Hz, 2H),1.59
(quint, J¼7.6 Hz, 2H,), 1.43 (s, 9H), 1.34 (quint, J¼7.6 Hz, 2H) ppm.
HRMS (EI): calcd for C21H30O6 378.2042, found 378.2031.
Mp 65e67 ꢀC (from hexane). 1H NMR (300 MHz, CDCl3):
J¼2.2 Hz, 1H), 7.40 (m, 5H), 7.33 (dd, J¼8.8, 2.2 Hz, 1H), 6.80 (d,
J¼8.8 Hz, 1H), 5.14 (s, 2H). HRMS (EI): calcd for C13H1079Br2O
339.9098, found 339.9097.
d¼7.69 (d,
4.1.1.6. tert-Butyl 6-(4-(6-chloropyrimidin-4-yloxy)-3-((E)-1-(meth-
oxycarbonyl)-2-methoxyvinyl) phenyl)hexanoate (15). A mixture of
phenol 13 (60.8 mg, 0.16 mmol), Cs2CO3 (104.5 mg, 0.32 mmol) and
4,6-dichloropyrimidine (49.3 mg, 0.32 mmol) was dissolved in dry
DMF (1.2 mL) at 0 ꢀC under nitrogen. The mixture was stirred at this
temperature for 2½ h, poured into water and worked up as usual
using EtOAc as the extraction solvent. Chromatographic purifica-
tion, eluting with hexane/EtOAc (9:1), afforded the chloropyr-
imidine 15 (68.4 mg, 87%) as a slightly coloured oil. 1H NMR
4.1.1.2. 2-(Benzyloxy)-5-bromophenylboronic acid (8). A 1.43 M
solution of BuLi in hexane (1.4 mL, 2 mmol) was added drop wise
into a white slurry of benzyl ether 7 (685 mg, 2 mmol) in anhydrous
Et2O (7.5 mL) at ꢂ60 ꢀC. The reaction solution soon turned into
a clear yellowish solution that was stirred at the same temperature
for 30 min and then treated with B(OiPr)3 (462
mL, 2 mmol). The
white slurry formed was stirred for 30 min at ꢂ60 ꢀC and then for
1 h at rt. The reaction mixture was quenched with 1 M aqueous HCl
solution (5 mL), then stirred for 45 min and poured into water.
Work up as usual, using EtOAc to extract, yielded a solid residue,
which was purified by chromatography with hexane/EtOAc (from
9:1 to 8:2) as eluent, to yield the arylboronic acid 8 (557 mg, 91%) as
a white solid. Mp 99e100 ꢀC (from hexane/MeOH). 1H NMR
(300 MHz, CHCl3):
d
¼8.56 (s,1H), 7.42 (s,1H), 7.18 (dd, J¼8.3, 2.0 Hz,
1H), 7.11 (d, J¼2.0 Hz, 1H), 7.05 (d, J¼8.3 Hz, 1H), 6.75 (s, 1H), 3.72 (s,
3H), 3.58 (s, 3H), 2.63 (t, J¼7.6 Hz, 2H), 2.21 (t, J¼7.6 Hz, 2H), 1.66
(quint, J¼7.6 Hz, 2H), 1.62 (quint, J¼7.6 Hz, 2H), 1.43 (s, 9H), 1.25
(quint, J¼7.6 Hz, 2H) ppm. HRMS (EI): calcd for C25H3135ClN2O6
490.1871, found 490.1860.
(300 MHz, CHCl3):
d
¼7.97 (d, J¼2.6 Hz, 1H), 7.51 (dd, J¼8.8, 2.6 Hz,
1H), 7.41 (m, 5H), 6.86 (d, J¼8.8 Hz, 1H), 6.05 (s, 2H), 5.12 (s, 2H).
HRMS (ES): calcd for C13H12B79BrNaO3 [MþþNa]þ 328.9961, found
328.9974.
4.1.1.7. tert-Butyl 6-(4-(6-(2-cyanophenoxy)pyrimidin-4-yloxy)-3-
((E)-1-(methoxycarbonyl)-2-methoxyvinyl)phenyl)hexanoate (17). A
mixture of chloropyrimidine 15 (61.8 mg, 0.126 mmol), Cs2CO3

J. Parra et al. / Tetrahedron 67 (2011) 624e635
631
(82 mg, 0.252 mmol) and 2-cyanophenol (16, 60 mg, 0.504 mmol) in
dry DMF (0.87 mL) was stirred at 85 ꢀC under nitrogen for 2½ h. The
resulting orange suspension was cooled to rt, diluted with water and
worked up as usual using EtOAc as the extraction solvent. Chro-
matographic purification, eluting with hexane/EtOAc (from 8:2 to
7:3), yielded the tert-butyl ester17 (67.2 mg, 93%) as an oil. 1H NMR
73%) as a yellowish oil. 1H NMR (300 MHz, CHCl3):
d
¼7.31 (s, 1H),
2.53 (t, J¼7.2 Hz, 2H), 2.38 (t, J¼7.2 Hz, 2H), 1.91 (quint, J¼7.2 Hz,
2H), 1.43 (s, 9H) ppm. HRMS (ES): calcd for C14H1635Cl2N2NaO2
[MþþNa]þ 337.0487, found 337.0490.
4.1.2.3. tert-Butyl 6-(4,6-dichloropyrimidin-2-yl)hexanoate (21). To
a solution of alkynylpyrimidine 20 (101.6 mg, 0.31 mmol) in EtOAc
(4 mL) was added 10% palladium-on-carbon (39.2 mg) and the
stirred mixture was hydrogenated at rt under balloon pressure of
hydrogen. After 18 h, the mixture was filtered through a pad of
silica gel, washing with EtOAc. The filtrate was concentrated under
reduced pressure and the residue was chromatographed on silica
gel, using hexane/EtOAc (9:1) as eluent, to yield alkylpyrimidine 21
(300 MHz, CHCl3):
d¼8.40 (d, J¼0.8 Hz, 1H), 7.71 (dd, J¼7.7, 1.7 Hz,
1H), 7.66 (ddd, J¼7.7, 8.3, 1.7 Hz, 1H), 7.47 (s, 1H), 7.36 (td, J¼7.7,
1.0 Hz, 1H), 7.29 (dd, J¼8.3, 1.0 Hz, 1H), 7.20 (dd, J¼8.2, 2.3 Hz, 1H),
7.13 (d, J¼2.3 Hz, 1H), 7.11 (d, J¼8.2 Hz, 1H), 6.40 (d, J¼0.8 Hz, 1H),
3.74 (s, 3H), 3.63 (s, 3H), 2.64 (t, J¼7.7 Hz, 2H), 2.22 (t, J¼7.4 Hz, 2H),
1.67 (quint, J¼7.7 Hz, 2H), 1.64 (quint, J¼7.4 Hz, 2H), 1.44 (s, 9H), 1.40
(m, 2H) ppm. HRMS (EI): calcd for C32H35N3O7 573.2475, found
573.2481.
(93.8 mg, 92%) as a colourless oil. 1H NMR (300 MHz, CHCl3):
d¼7.23
(s, 1H), 2.89 (t, J¼7.7 Hz, 2H), 2.20 (t, J¼7.7 Hz, 2H), 1.80 (quint,
J¼7.7 Hz, 2H), 1.61 (quint, J¼7.7 Hz, 2H), 1.41 (s, 9H), 1.38 (m, 2H)
ppm. HRMS (EI): calcd for C14H2035Cl2N2O2 318.0902, found
318.0909.
4.1.1.8. 6-(4-(6-(2-Cyanophenoxy)pyrimidin-4-yloxy)-3-((E)-1-
(methoxycarbonyl)-2-methoxyvinyl) phenyl)hexanoic acid (hapten
AZa6, 2). A solution of tert-butyl ester17 (57.7 mg, 0.10 mmol) in
dry CH2Cl2 (700 mL) was treated with CF3CO2H (700 mL) under ni-
trogen. The mixture was stirred at rt for 20 min, then diluted with
benzene and concentrated to dryness in vacuum; the residue was
purified by chromatography, using CHCl3/MeOH (95:5) as eluent, to
give hapten AZa6 (2, 48 mg, 93%) as a viscous oil. IR (NaCl):
4.1.2.4. Methyl(E)-2-(2-hydroxyphenyl)-3-methoxyacrylate(22). A
mixture of iodoacrylate 9 (634 mg, 2.62 mmol), arylboronic acid 23
(896.2 mg, 3.93 mmol), K3PO4 (1.668 g, 7.86 mmol) and Pd(PPh3)4
(121.1 mg, 0.105 mmol) in a previously degasified mixture of di-
oxane (13 mL) and water (2.62 mL) was stirred under nitrogen at
90 ꢀC for 10 h. After this time, the reaction mixture was cooled
down, diluted with ether and worked up. Column chromatography,
eluting first with CHCl3 to separate the excess of boronic acid and
then with hexane/EtOAc (from 9:1 to 8:2), gave (E)-methyl 2-(2-
(benzyloxy)phenyl)-3-methoxyacrylate (24, 687 mg, 88%) as an oil,
which had physical and spectroscopic properties identical with
those described in the literature.30
A mixture of 24 (690 mg, 0.065 mmol) and 10% Pd/C (120 mg) in
EtOAc (6.3 mL) was stirred at rt under a pressure of hydrogen of
4 atm for 6 h. The mixture was filtered through a short column of
silica gel, eluting with EtOAc, to afford pure compound 22 (467 mg,
97%) as an amorphous solid. The physical and spectroscopic prop-
erties of 22 were also identical to those described previously in the
literature for this compound.31
nmax¼3219 (m), 2230 (m), 1709 (s), 1636 (s), 1589 (s), 1571 (s) cmꢂ1
.
1H NMR (300 MHz, CHCl3):
d
¼8.41 (s, 1H, H-2 pyrim), 7.71 (dd,
J¼7.8, 1.6 Hz, 1H, H-3 C`NePh), 7.66 (td, J¼8.0, 1.6 Hz, 1H, H-5
CNePh), 7.48 (s, 1H, H-20), 7.36 (br t, J¼7.8 Hz, 1H, H-4 CNePh), 7.30
(br d, J¼8.0, Hz, 1H, H-6 CNePh), 7.20 (dd, J¼8.2, 2.1 Hz, 1H, H-6 Ph),
7.13 (d, J¼2.1 Hz, 1H, H-2Ph), 7.11 (d, J¼8.2 Hz, 1H, H-5 Ph), 6.41 (s,
1H, H-5 pyrim), 3.74 (s, 3H, OCH3), 3.64 (s, 3H, CO2CH3), 2.65 (t,
J¼7.5 Hz, 2H, H2-6), 2.36 (t, J¼7.5 Hz, 2H, H2-2),1.69 (quint, J¼7.5 Hz,
4H, H2-5 and H2-3), 1.43 (m, 2H, H2-4) ppm. 13C NMR (75 MHz,
CHCl3):
d
¼171.93 (C-6 pyrim), 171.89 (COOH), 170.02 (C-4 pyrim),
167.56 (CO2Me), 160.69 (C-20), 157.91 (C-2 pyrim), 154.10 (C-1
CNePh), 148.08 (C-4 Ph), 139.93 (C-1 Ph), 134.18 (C-5 CNePh),
133.56 (C-3 CNePh), 132.48 (C-2 Ph), 129.09 (C-6 Ph), 126.05 (C-4
CNePh), 125.46 (C-3 Ph), 123.03 (C-6 CNePh), 121.67 (C-5 Ph),
115.19 (CN), 107.25 (C-2 CNePh), 106.99 (C-10), 92.33 (C-5 pyrim),
61.97 (OCH3), 51.61 (CO2Me), 35.02 (C-6), 33.82 (C-2), 30.67 (C-5),
28.59 (C-4), 24.45 (C-3) ppm. MS (EI): m/z (%)¼517 (Mþ, 18), 502
(35), 486 (18), 474 (10), 467 (4), 458 (100), 398 (13), 305 (12), 229
(40), 189 (22), 119 (8), 75 (25). HRMS (EI): calcd for C28H27N3O7
517.1849, found 517.1857.
4.1.2.5. tert-Butyl 6-(4-chloro-6-(2-((E)-1-(methoxycarbonyl)-2-
methoxyvinyl)phenoxy)pyrimidin-2-yl)hexanoate (25). A mixture of
4,6-dichloro pyrimidine 21 (117 mg, 0.366 mmol), Cs2CO3 (118.9 mg,
0.366 mmol) and phenol 22 (38.2 mg, 0.184 mmol) was dissolved in
dry DMF (1.4 mL) at 0 ꢀC under nitrogen. The mixture was stirred at
this temperature for 2½ h. After an additional 1 h at rt, the mixture
was poured into water and worked up as usual using EtOAc as the
extraction solvent. Chromatographic purification, eluting with hex-
ane/EtOAc (from 95:5 to 8:2), afforded the chloropyrimidine 25
4.1.2. Synthesis of hapten AZb6 (3).
4.1.2.1. 4,6-Dichloro-2-iodopyrimidine (19). tBuONO (431
mmol) was added to a solution of aminopyrimidine 18 (129.3 mg,
0.788 mmol) and CH2I2 (3.27 mL) in anhydrous CH3CN (820 L)
mL, 3.625
m
(54.1 mg, 60%)as a yellowishoil.1H NMR (300 MHz, CHCl3):
d¼7.45 (s,
under nitrogen. The reaction mixture was heated in an oil bath at
80 ꢀC for 3½ h, then cooled to rt and concentrated under reduced
pressure. The residue was directly chromatographed on silica gel,
eluting with hexane, to give the iodopyrimidine 19 (160 mg, 74%) as
a white solid. Mp 97e98 ꢀC (from hexane). 1H NMR (300 MHz,
1H), 7.39 (ddd, J¼6.2, 8.0, 2.4 Hz,1H), 7.33 (dd, J¼6.2, 2.4 Hz,1H), 7.29
(td, J¼6.2, 1.2 Hz, 1H), 7.16 (dd, J¼8.0, 1.2 Hz, 1H), 6.48 (s, 1H), 3.73 (s,
3H),3.59(s, 3H),2.79(t,J¼7.7Hz, 2H), 2.19(t,J¼7.6Hz, 2H),1.76(quint,
J¼7.7 Hz, 2H),1.59 (quint, J¼7.6 Hz, 2H),1.43 (s, 9H),1.36 (m, 2H) ppm.
HRMS (EI): calcd for C25H3135ClN2O6 490.1871, found 490.1881.
CHCl3):
273.8561, found 273.8557.
d
¼7.40 (s, 1H) ppm. HRMS (EI): calcd for C4H35Cl2IN2
4.1.2.6. tert-Butyl 6-(4-(2-cyanophenoxy)-6-(2-((E)-1-(methoxycar-
bonyl)-2-methoxyvinyl)phenoxy) pyrimidin-2-yl)hexanoate (26). A
mixture of chloropyrimidine 25 (67.8 mg, 0.138 mmol), Cs2CO3
(89.9 mg, 0.276 mmol) and 2-cyanophenol (16, 65.7 mg,
0.552 mmol) in dry DMF (0.96 mL) was stirred at 85 ꢀC under ni-
trogen for 4 h. The reaction mixture was cooled to rt, diluted with
water and worked up as usual using EtOAc as the extraction solvent.
Chromatographic purification, eluting with hexane/EtOAc (from
9:1 to 7:3) as eluent, afforded the tert-butyl ester 26 (59.4 mg, 75%)
4.1.2.2. tert-Butyl 6-(4,6-dichloropyrimidin-2-yl)hex-5-ynoate (20).
A solution of alkyne 20 (86 mg, 0.512 mmol) in anhydrous DMF
(611 mL) and Et3N (611 mL) were added sequentially via syringe to
a mixture of iodopyrimidine 19 (127.7 mg, 0.464 mmol), CuI
(6.3 mg, 0.033 mmol) and (Ph3P)2PdCl2 (7.8 mg, 0.011 mmol) under
nitrogen. The deep-orange mixture formed was stirred at rt for
2½ h, then poured into water and worked up using EtOAc to extract.
Chromatographic purification, using hexane/EtOAc (from 9.5:0.5 to
8:2) as eluent, yielded the alkynylpyrimidine derivative 20 (106 mg,
as a yellowish oil. 1H NMR (300 MHz, CHCl3):
1.5 Hz, 1H), 7.63 (ddd, J¼7.7, 8.2, 1.5 Hz, 1H), 7.49 (s, 1H), 7.39 (ddd,
d¼7.68 (dd, J¼7.7,

632
J. Parra et al. / Tetrahedron 67 (2011) 624e635
J¼6.8, 8.0, 2.3 Hz, 1H), 7.33 (dd, J¼6.8, 2.3 Hz, 1H), 7.32 (td, J¼7.7,
1.4 Hz, 1H), 7.28 (td, J¼6.8, 1.2 Hz, 1H), 7.26 (br d, J¼8.2 Hz, 1H), 7.20
(dd, J¼8.0, 1.2 Hz, 1H), 6.08 (s, 1H), 3.75 (s, 3H), 3.62 (s, 3H), 2.65 (t,
J¼7.7 Hz, 2H), 2.14 (t, J¼7.7 Hz, 2H), 1.63 (quint, J¼7.7 Hz, 2H), 1.52
(quint, J¼7.7 Hz, 2H), 1.43 (s, 9H), 1.27 (m, 2H) ppm. HRMS (EI):
calcd for C32H35N3O7 573.2475, found 573.2471.
aryl iodide 28 (1.08 g, 3.2 mmol), CuI (80 mg, 0.42 mmol) and
(Ph3P)2PdCl2 (82 mg, 0.12 mmol) under nitrogen. The reaction
mixture, originally yellowish that turned deep-orange after a few
minutes, was stirred at rt for 5½ h, poured into water and worked up
with EtOAc. Chromatographic purification, using hexane/EtOAc
(9.5:0.5) as eluent, afforded the aryl-alkyne 29 (1.18 g, 98%) as awhite
solid. Mp 86e88 ꢀC (from benzene). 1H NMR (300 MHz, CHCl3):
4.1.2.7. 6-(4-(2-Cyanophenoxy)-6-(2-((E)-1-(methoxycarbonyl)-
2-methoxyvinyl)phenoxy)pyrimidin-2-yl)hexanoic acid (hapten AZb6,
d
¼7.60 (d, J¼2.1 Hz,1H), 7.49 (dd, J¼8.8, 2.1 Hz,1H), 7.38 (m, 5H), 6.91
(d, J¼8.8 Hz, 1H), 5.21 (s, 2H), 2.44 (t, J¼7.1 Hz, 2H), 2.38 (t, J¼7.1 Hz,
2H), 1.86 (quint, J¼7.1 Hz, 2H), 1.45 (s, 9H) ppm. HRMS (EI): calcd for
C24H25NO3 375.1834, found 375.1828.
3). Trifluoroacetic acid (522
m
L) was added to a solution of tert-butyl
ester 26 (43 mg, 0.075 mmol) in dry CH2Cl2 (522
m
L) at 0 ꢀC. The
mixture was stirred at rt for 20 min and then diluted with benzene.
The mixture was concentrated under vacuum without heating and
the residue obtained was purified by silica gel chromatography,
using CHCl3/MeOH (95:5) as eluent, to give hapten AZb6 (3, 37 mg,
95%) as a yellowish viscous oil. IR (NaCl): nmax¼3213 (m), 3017 (m),
4.1.3.4. tert-Butyl 6-(3-cyano-4-hydroxyphenyl)hexanoate (30). A
mixture of aryl-alkyne 29 (315.6 mg, 0.841 mmol) and 10% Pd/C
(33.4 mg) in EtOAc (4.3 mL) was purged with nitrogen, hydrogen
bubbled through the solution and the mixture kept under hydrogen
at atmospheric pressure (balloon) during 7 h at rt. The reaction
mixture was filtered through a short pad of Celite and the residue
left after evaporation of the solvent was chromatographed on silica
gel with hexane/Et2O (9:1) as eluent to give the phenol 30
2233 (m), 1710 (s), 1634 (m), 1588 (s), 1562 (s) cmꢂ1 1H NMR
.
(300 MHz, CHCl3):
d
¼7.68 (dd, J¼7.7, 1.6 Hz, 1H, H-3 CNePh), 7.62
(td, J¼7.7, 1.6 Hz,1H, H-5 CNePh), 7.51 (s, 1H, H-20), 7.39 (ddd, J¼6.8,
7.9, 2.3 Hz, 1H, H-5 Ph), 7.33 (dd, J¼6.8, 2.3 Hz, 1H, H-3 Ph), 7.32 (td,
J¼7.7, 2.1 Hz,1H, H-4 CNePh), 7.29 (td, J¼6.8,1.3 Hz,1H, H-4 Ph), 7.25
(br d, J¼7.7 Hz, 1H, H-6 CNePh), 7.21 (dd, J¼7.9, 1.3 Hz, 1H, H-6 Ph),
6.08 (s,1H, H-5 pyrim), 3.75 (s, 3H, OCH3), 3.62 (s, 3H, CO2CH3), 2.66
(t, J¼7.4 Hz, 2H, H2-6), 2.28 (t, J¼7.7 Hz, 2H, H2-2), 1.64 (quint,
J¼7.4 Hz, 2H, H2-5), 1.57 (quint, J¼7.7 Hz, 2H, H2-3), 1.30 (m, 2H, H2-
(218.7 mg, 90%) as colourless oil. 1H NMR (300 MHz, CHCl3):
d¼7.27
(d, J¼2.1 Hz, 1H), 7.24 (dd, J¼8.5, 2.1 Hz, 1H), 6.90 (d, J¼8.5 Hz, 1H),
6.76 (br s, 1H), 2.53 (t, J¼7.5 Hz, 2H), 2.22 (t, J¼7.4 Hz, 2H), 1.60
(quint, J¼7.4 Hz, 2H), 1.57 (quint, J¼7.5 Hz, 2H), 1.44 (s, 9H), 1.31 (m,
2H) ppm. HRMS calcd for C17H23NO3 289.1678, found 289.1667.
4) ppm. 13C NMR (75 MHz, CHCl3):
d¼179.06 (COOH), 171.95 (C-4
pyrim), 171.36 (C-6 pyrim), 169.96 (C-2 pyrim), 167.61 (CO2Me),
160.92 (C-20), 154.38 (C-1 CNePh), 150.36 (C-1 Ph), 133.95 (C-5
CNePh), 133.38 (C-3 CNePh), 132.74 (C-3 Ph), 129.15 (C-5 Ph),
126.05 (C-2 Ph), 125.70 (C-4 CNePh), 125.63 (C-4 Ph), 122.85 (C-6
CNePh),121.88 (C-6 Ph),115.40 (CN),107.11 (C-2 CNePh),106.86 (C-
10), 88.76 (C-5 pyrim), 62.01 (OCH3), 51.65 (CO2Me), 38.18 (C-6),
33.77 (C-2), 28.31 (C-4), 27.06 (C-5), 24.40 (C-3) ppm. MS (EI): m/z
(%)¼518 (Mþþ1, 24), 517 (Mþ, 89), 502 (37), 486 (98), 458 (52), 430
(41), 402 (77), 398 (83), 386 (95), 359 (57), 326 (46), 267 (20), 223
(37), 191 (47), 176 (45), 145 (47), 119 (63), 102 (65), 91 (100), 75 (68).
HRMS (EI): calcd for C28H27N3O7 517.1849, found 517.1843.
4.1.3.5. tert-Butyl 6-(3-cyano-4-(6-(2-((E)-1-(methoxycarbonyl)-2-
methoxyvinyl)phenoxy) pyrimidin-4-yloxy)phenyl)hexanoate (32). A
mixture of 6-chloropyrimidine 31 (122.4 mg, 0.382 mmol), Cs2CO3
(134.2 mg, 0.412 mmol) and phenol 30 (119.4 mg, 0.412 mmol) was
dissolved in dry DMF (2.1 mL) at 85 ꢀC under nitrogen. The mixture
was stirred at this temperature for 3 h, then cooled down to rt,
poured into water and worked up as usual using EtOAc as the ex-
traction solvent. Chromatographic purification, eluting with hex-
ane/EtOAc (8:2), afforded the compound 32 (199 mg, 91%) as
a yellowish oil. 1H NMR (300 MHz, CHCl3):
d¼8.40 (s, 1H), 7.49 (br s,
2H), 7.45 (dd, J¼8.6, 2.2 Hz, 1H), 7.41 (ddd, J¼7.7, 8.4, 2.3 Hz, 1H),
7.35 (dd, J¼7.7, 2.3 Hz, 1H), 7.30 (td, J¼7.7, 1.0 Hz, 1H), 7.22 (dd,
J¼8.4, 1.0 Hz, 1H), 7.18 (d, J¼8.6 Hz, 1H), 6.39 (s, 1H), 3.74 (s, 3H),
3.63 (s, 3H), 2.65 (t, J¼7.6 Hz, 2H), 2.22 (t, J¼7.6 Hz, 2H), 1.65 (quint,
J¼7.6 Hz, 2H), 1.63 (quint, J¼7.6 Hz, 2H), 1.44 (s, 9H), 1.37 (m, 2H)
ppm. HRMS (EI): calcd for C32H35N3O7 573.2475, found 573.2485.
4.1.3. Synthesis of hapten AZc6 (4).
4.1.3.1. 2-Hydroxy-5-iodobenzonitrile (27). Ag2SO4 (4.978 g,
15.967 mmol) was added in small portions to a stirred solution
of 2-cyanophenol (16, 1.902 g, 15.967 mmol) and I2 (4.457 g,
17.547 mmol) in dry CH2Cl2 (40.5 mL). The reaction mixture was
stirred for 2 days at rt and filtered to removed the solids. The filtrate
and washings (CHCl3) were successively washed with a 5% aqueous
solution of sodium thiosulfate and brine, and dried over anhydrous
Na2SO4. Evaporation of the solvent under reduced pressure gave
almost pure the iodo-phenol 27 (3.258 g, 84%) as a white solid,
which showed the same physical and spectroscopic data as those
previously reported.32
4.1.3.6. 6-(3-Cyano-4-(6-(2-((E)-1-(methoxycarbonyl)-2-methoxy-
vinyl)phenoxy)pyrimidin-4-yloxy) phenyl)hexanoic acid (hapten AZc6,
4). A solution of tert-butyl ester 32 (32 mg, 0.055 mmol) in dry
CH2Cl2 (160 mL) was treated with CF3CO2H (160 mL) under nitrogen.
The mixture was stirred at rt for 10 min and concentrated to dryness
in vacuum; the residue was purified by chromatography, using
CHCl3/MeOH (95:5) as eluent, to give hapten AZc6 (4, 25.9 mg, 90%)
as a viscous oil. IR (NaCl) nmax¼3213 (m), 3019 (m), 2234 (m), 1709
(s), 1629 (m), 1591 (s), 1570 (s) cmꢂ1 1H NMR (300 MHz, CHCl3):
.
4.1.3.2. 2-(Benzyloxy)-5-iodobenzonitrile (28). Benzyl bromide
(1.4 mL, 12.013 mmol) was added to a solution of anhydrous K2CO3
(1.66 g, 12.013 mmol) and phenol 27 (2.676 g, 10.921 mmol) in dry
DMF (20 mL). The mixture was stirred for 4 h at rt, then poured into
water and work up as usual with hexane. The residue was purified
by silica-gel column, eluting with hexane/EtOAc (9:1), to afford
benzyl ether 28 (2.77 g, 74%) as a white solid. Mp 75e77ꢀC (from
d
¼8.41 (s,1H, H-2 pyrim), 7.50 (s, 1H, H-20), 7.49 (d, J¼2.3 Hz, 1H, H-2
CNePh), 7.45 (dd, J¼8.7, 2.3 Hz, 1H, H-6 CNePh), 7.41 (ddd, J¼7.7, 8.2,
2.3 Hz,1H, H-5 Ph), 7.35 (dd, J¼7.7, 2.3 Hz,1H, H-3 Ph), 7.30 (td, J¼7.7,
1.0 Hz, 1H, H-4 Ph), 7.21 (dd, J¼8.2, 1.0 Hz, 1H, H-6 Ph), 7.18 (d,
J¼8.7 Hz, 1H, H-5 CNePh), 6.91 (br s, 1H, COOH), 6.38 (s, 1H, H-5
pyrim), 3.75 (s, 3H, OCH3), 3.63 (s, 3H, CO2CH3), 2.66 (t, J¼7.6 Hz, 2H,
H2-6), 2.36 (t, J¼7.4 Hz, 2H, H2-2), 1.68 (quint, J¼7.4 Hz, 2H, H2-3),
1.67 (quint, J¼7.6 Hz, 2H, H2-5), 1.40 (m, 2H, H2-4) ppm. 13C NMR
benzene). 1H NMR (300 MHz, CHCl3):
d
¼7.84 (d, J¼2.2 Hz, 1H), 7.74
(dd, J¼8.9, 2.2 Hz, 1H), 7.38 (m, 5H), 6.77 (d, J¼8.9 Hz, 1H), 5.20 (s,
(75 MHz, CHCl3):
d
¼178.81 (COOH), 171.71 (C-4 pyrim), 170.27 (C-6
2H) ppm. HRMS (EI): calcd for C14H10INO 334.9807, found 334.9801.
pyrim),167.52 (CO2Me),160.84 (C-20),157.89 (C-2 pyrim),152.00 (C-4
CNePh), 150.10 (C-1 Ph), 140.81 (C-1 CNePh), 134.36 (C-6 CNePh),
133.06 (C-2 CNePh),132.74 (C-3 Ph),129.18 (C-5 Ph),125.98 (C-2 Ph),
125.92 (C-4 Ph), 122.83 (C-5 CNePh), 122.01 (C-6 Ph), 115.34 (CN),
106.92 (C-3 CNePh), 106.85 (C-10), 92.27 (C-5 pyrim), 61.02 (OCH3),
4.1.3.3. tert-Butyl 6-(4-(benzyloxy)-3-cyanophenyl)hex-5-ynoate
(29). A solution of alkyne 11 (670 mg, 3.99 mmol) in anhydrous DMF
(4.2 mL) and Et3N (4.2 mL) were consecutively added to a mixture of

J. Parra et al. / Tetrahedron 67 (2011) 624e635
633
51.65 (CO2Me), 34.63 (C-6), 33.66 (C-2), 30.51 (C-5), 28.33 (C-4),
24.38 (C-3) ppm. MS (EI): m/z (%)¼518 (Mþþ1, 6), 517 (Mþ, 20), 502
(42), 486 (15), 458 (100), 440 (14), 414 (4), 343 (3), 145 (4), 75 (5).
HRMS (EI): calcd for C28H27N3O7 517.1849, found 517.1849.
pressure (balloon) for 1 h. The catalyst was remove by filtration
though a pad of Celite and rinsed with EtOAc. The solvent was re-
moved under reduced pressure to give a clear yellow oil. Purification
by column chromatography using hexane/EtOAc (from 9:1 to 7:3)
gave the phenol 37 (368 mg, 84%) as an oil. 1H NMR (300 MHz,
4.1.4. Synthesis of hapten AZo6 (5).
CHCl3):
d
¼7.67 (s,1H), 7.20 (ddd, J¼7.4, 8.4,1.7 Hz,1H), 7.14 (dd, J¼7.4,
1.7 Hz,1H), 6.95 (dd, J¼8.4,1.2 Hz,1H), 6.90 (td, J¼7.4,1.2 Hz,1H), 6.43
(br s,1H), 4.05 (t, J¼6.5 Hz, 2H), 3.75 (s, 3H), 2.20 (t, J¼7.4 Hz, 2H),1.68
(quint, J¼6.5 Hz, 2H), 1.58 (quint, J¼7.4 Hz, 2H), 1.43 (s, 9H), 1.34 (m,
2H) ppm. HRMS (ES): calcd for C20H28O6 364.1886, found 364.1877.
4.1.4.1. tert-Butyl 6-((E)-2-(methoxycarbonyl)vinyloxy)hexanoate
(34). PBu3 (72 mL, 0.291 mmol) was added to a solution of tert-butyl
6-hydroxyhexanoate33 (33; 331.5 mg, 1.763 mmol) in anhydrous
CH2Cl2 (19.3 mL) under nitrogen. The mixture was cooled to 0 ꢀC
and methyl propiolate (162
m
L, 1.938 mmol) was then added drop
4.1.4.5. tert-Butyl 6-((E)-2-(methoxycarbonyl)-2-(2-(6-chloropy-
rimidin-4-yloxy)phenyl)vinyloxy) hexanoate (38). A solution of
phenol 37 (94 mg, 0.257 mmol) in anhydrous DMF (1 mL) was added
to a solution of 4,6-dichoropyrimidine (38.5 mg, 0.257 mmol) and
Cs2CO3 (84 mg, 0.257 mmol) in DMF (1 mL) at 0 ꢀC under nitrogen.
The mixture was stirred at 0 ꢀC for 45 min, then poured into water
and worked up as usual with EtOAc. Chromatographic purification of
the residue obtained, using hexane/EtOAc (9:1) as eluent, gave the
chloropyrimidine 38 (85.5 mg, 70%) as a yellowish oil. 1H NMR
wise via a syringe over 5 min. The reaction mixture was allowed to
reach rt and stirred for 30 min, during which time the initially
colourless solution turned to yellow and finally to deep red. The
reaction mixture was exposed to the air and stirred for 20 min in
order to oxidize and facilitate the separation of the PBu3. The
resulting black mixture was concentrated at reduced pressure and
the residue was purified by chromatography, using hexane/EtOAc
(8:2) as eluent, to give acrylate derivative 34 (445 mg, 93%) as an oil.
1H NMR (300 MHz, CHCl3):
d
¼7.56 (d, J¼12.6 Hz, 1H), 5.16 (d,
(300 MHz, CHCl3):
d
¼8.57 (d, J¼0.6 Hz, 1H), 7.50 (s, 1H), 7.40 (ddd,
J¼12.6 Hz, 1H), 3.81 (t, J¼6.4 Hz, 2H), 3.67 (s, 3H), 2.20 (t, J¼7.3 Hz,
2H),1.69 (quint, J¼6.4 Hz, 2H),1.59 (quint, J¼7.3 Hz, 2H),1.41 (s, 9H),
1.38 (m, 2H) ppm. HRMS (ES): calcd for C14H24NaO5 [MþNa]þ
295.1521, found 295.1527.
J¼7.2, 8.0, 3.0 Hz, 1H), 7.34 (dd, J¼7.2, 3.0 Hz, 1H), 7.31 (td, J¼7.2,
1.2 Hz, 1H), 7.16 (br d, J¼8.0 Hz, 1H), 6.75 (d, J¼0.6 Hz, 1H), 3.92 (t,
J¼6.9 Hz, 2H), 3.58 (s, 3H), 2.19 (t, J¼7.4 Hz, 2H),1.59 (quint, J¼6.9 Hz,
2H), 1.56 (quint, J¼7.4 Hz, 2H), 1.43 (s, 9H), 1.29 (m, 2H) ppm. HRMS
(ES): calcd for C24H2935ClN2O6 476.1714, found 476.1702.
4.1.4.2. tert-Butyl 6-((Z)-2-(methoxycarbonyl)-2-iodovinyloxy)
hexanoate (35). A solution of 34 (211 mg, 0.77 mmol) in CH2Cl
(2 mL) was added to N-iodosuccinimide (235 mg, 1.044 mmol) at rt
4.1.4.6. tert-Butyl 6-((E)-2-(methoxycarbonyl)-2-(2-(6-(2-cyano-
phenoxy)pyrimidin-4-yloxy)phenyl) vinyloxy)hexanoate (39). A
solution of chloropyrimidine 38 (85.5 mg, 0.180 mmol), Cs2CO3
(58.4 mg, 0.18 mmol) and 2-cyanophenol (31.3 mg, 0.18 mmol) in
dry DMF (1.2 mL) was stirred at 85 ꢀC for 5 h under nitrogen. The
mixture was cooled down to rt and worked up as usual with EtOAc.
Purification by chromatography eluting with hexane/EtOAc (from
8:2 to 7:3) afforded the compound 39 (77.5 mg, 80%) as an oil. 1H
under nitrogen. Then AcOH (80
mixture was stirred for 17 h. After this time, the red-wine solution
formed was treated with Et3N (347 L, 2.49 mmol) and then stirred
mL, 1.386 mmol) was added and the
m
for 22 h, poured into water, and extracted with EtOAc. The com-
bined organic extracts were washed with 10% aqueous solution of
Na2S2O3 and brine, and dried over anhydrous Na2SO4. Chromato-
graphic purification of the residue left after evaporation of the
solvent, using hexane/EtOAc (from 9:1 to 8:2) as eluent, afforded, in
order of elution, first the vinyl iodide 35 (172 mg, 74% based on
recovered starting material) as a yellowish oil, followed by the
starting compound 34 (54 mg). Spectral data for 35: 1H NMR
NMR (300 MHz, CHCl3):
d¼8.38 (s, 1H), 7.71 (dd, J¼7.8, 1.6 Hz, 1H),
7.66 (td, J¼7.8, 1.6 Hz, 1H), 7.55 (s, 1H), 7.40 (ddd, J¼6.9, 8.0, 2.3 Hz,
1H), 7.36 (td, J¼7.8, 1.1 Hz, 1H), 7.34 (dd, J¼6.9, 2.3 Hz, 1H), 7.293 (br
d, J¼7.8 Hz, 1H), 7.290 (td, J¼6.9, 1.1 Hz, 1H), 7.21 (dd, J¼8.0, 1.1 Hz,
1H), 6.391 (s, 1H), 3.95 (t, J¼6.7 Hz, 2H), 3.61 (s, 3H), 2.18 (t,
J¼7.3 Hz, 2H), 1.61 (quint, J¼6.7 Hz, 2H), 1.56 (quint, J¼7.3 Hz, 2H),
1.42 (s, 9H), 1.31 (m, 2H) ppm. HRMS (ES): calcd for C31H33N3O7
559.2319, found 559.2313.
(300 MHz, CHCl3):
d
¼7.71 (s, 1H), 4.16 (t, J¼6.5 Hz, 2H), 3.77 (s, 3H),
2.23 (t, J¼7.3 Hz, 2H), 1.74 (quint, J¼6.5 Hz, 2H), 1.62 (quint,
J¼7.3 Hz, 2H), 1.43 (s, 9H), 1.42 (m, 2H) ppm. HRMS (ES): calcd for
C14H23INaO5 [MþNa]þ 421.0488, found 421.0496.
4.1.4.7. 6-((E)-2-(Methoxycarbonyl)-2-(2-(6-(2-cyanophenoxy)
pyrimidin-4-yloxy)phenyl)vinyloxy) hexanoic acid (hapten AZo5,
5). Trifluoroacetic acid (0.5 mL) was added to a solution of tert-
butyl ester 39 (40 mg, 0.072 mmol) in dry CH2Cl2 (0.5 mL) at 0 ꢀC.
The mixture was stirred at rt for 20 min under nitrogen, the mix-
ture was diluted with benzene and then concentrated under vac-
uum without heating. The residue obtained was purified by silica
gel chromatography, using CHCl3/MeOH (90:1) as eluent, to give
hapten AZo6 (5, 33.1 mg, 92%) as a yellowish viscous oil. IR (NaCl)
4.1.4.3. tert-Butyl 6-((E)-2-(methoxycarbonyl)-2-(2-(benzyloxy)
phenyl)vinyloxy)hexanoate (36). A mixture of vinyl iodide 35
(89 mg, 0.223 mmol), 2-(benzyloxy)phenylboronic acid (23,
79.7 mg, 0.349 mmol), K3PO4 (148.38 mg, 0.699 mmol) and Pd
(PPh3)4 (17 mg, 0.015 mmol) in a previously degassed mixture of
dioxane (1.15 mL) and water (233 m
L) was heated at 90 ꢀC with
stirring under nitrogen for 7 h. The cooled mixture was poured
into water and worked up with EtOAc in the usual manner to yield
an oily residue. Purification by chromatography gave compound
nmax¼3200 (m), 3071 (m), 1708 (s), 1632 (s), 1591 (s), 1567 (s) cmꢂ1
.
36 (82.1 mg, 81%) as an oil. 1H NMR (300 MHz, CHCl3):
d
¼7.56 (s,
1H NMR (300 MHz, CHCl3):
d
¼8.40 (s, 1H, H-2 pyrim), 7.71 (dd,
1H), 7.32 (m, 5H), 7.23 (ddd, J¼7.5, 8.1, 1.7 Hz, 1H), 7.21 (dd, J¼7.5,
1.7 Hz, 1H), 6.97 (td, J¼7.5, 1.0 Hz, 1H), 6.94 (dd, J¼8.1, 1.0 Hz, 1H),
5.07 (s, 2H), 3.97 (t, J¼6.7 Hz, 2H), 3.64 (s, 3H), 2.16 (t, J¼7.5 Hz,
2H), 1.63 (quint, J¼6.7 Hz, 2H), 1.54 (quint, J¼7.5 Hz, 2H), 1.43 (s,
9H), 1.30 (m, 2H) ppm. HRMS (ES): calcd for C27H34O6 454.2355,
found 454.2377.
J¼7.8, 1.6 Hz, 1H, H-3 CNePh), 7.66 (td, J¼7.8, 1.6 Hz, 1H, H-5
CNePh), 7.56 (s, 1H, C-10), 7.41 (ddd, J¼7.7, 7.8, 2.7 Hz, 1H, H-4 Ph),
7.36 (td, J¼7.8, 1.0 Hz, 1H, H-4 CNePh), 7.34 (dd, J¼7.7, 2.7 Hz, 1H, H-
6 Ph), 7.29 (br d, J¼7.8 Hz, 1H, H-6 CNePh), 7.30 (td, J¼7.7, 0.8 Hz,
1H, H-5 Ph), 7.21 (dd, J¼7.8, 0.8 Hz, 1H, H-3 Ph), 6.38 (s, 1H, H-5
pyrim), 3.97 (t, J¼7.2 Hz, 2H, H2-6), 3.62 (s, 3H, CO2CH3), 2.33 (t,
J¼7.2 Hz, 2H, H2-2), 1.62 (quint, J¼7.2 Hz, 4H, H2-5, H2-3), 1.36 (m,
4.1.4.4. tert-Butyl 6-((E)-2-(methoxycarbonyl)-2-(2-hydroxyph-
enyl)vinyloxy)hexanoate (37). Benzyl ether 36 (548 mg, 1.205
mmol) was dissolved in EtOAc (5.3 mL),10% Pd/C (26 mg) was added
to the solution and the mixture was hydrogenated at 1 atm of H2
2H, H2-4) ppm. 13C NMR (75 MHz, CHCl3):
d
¼177.92 (COOH), 171.82
(C-6 pyrim), 170.12 (C-4 pyrim), 167.62 (CO2Me), 159.86 (C-10),
157.88 (C-2 pyrim), 154.08 (C-1 CNePh), 150.21 (C-2, Ph), 134.19 (C-
5 CNePh), 133.59 (C-3 CNePh), 132.78 (C-6 Ph), 129.18 (C-4 Ph),

634
J. Parra et al. / Tetrahedron 67 (2011) 624e635
126.25 (C-1 Ph), 126.10 (C-4 CNePh), 125.93 (C-5 Ph), 123.03 (C-6
CNePh),121.83 (C-3 Ph),115.18 (CN),107.27 (C-2 CNePh),106.54 (C-
20), 92.44 (C-5 pyrim), 74.95 (C-6), 51.60 (CO2Me), 33.68 (C-2), 29.23
(C-5), 24.76 (C-4), 24.19 (C-3) ppm. MS (EI): m/z (%)¼504 (Mþþ1, 5),
503 (Mþ, 16), 444 (58), 388 (100), 329 (62), 301 (20), 176 (19), 172
(17), 145 (11), 102 (8), 69 (25), 55 (19). HRMS (ES): calcd for
C27H25N3O7 503.1693, found 503.1718.
immobilized immunoreagent (OVA conjugate) and the immu-
noreagent in solution (antiserum). Sigmoidal curves were mathe-
matically fitted to a four-parameter logistic equation using the
SigmaPlot software package from SPSS Inc. (Chicago, IL). The anti-
serum titre was defined as the reciprocal of the dilution that results
in a maximum absorbance value (Amax) around 1.0 reached at the
zero dose of analyte. Antibody affinity or IC50 was estimated as the
concentration of analyte at the inflection point of the fitted curve,
typically corresponding to a 50% reduction of the Amax if the
background signal approaches to zero. Cross-reactivity (CR) was
calculated according to the formula:
4.2. Immunochemistry
Procedure details and equipment for protein conjugation and
immunoassay performance are given as Supplementary data.
CR ¼ ½IC50ðAZE isomerÞ=IC50ðAZZ isomerÞꢃ ꢄ 100
4.2.1. Bioconjugate preparation. Immunogens were prepared by
coupling the haptens to BSA. In a first step, the carboxylate group of
haptens was activated in DMF using either N-hydroxysuccinimide
and N,N-dicyclohexylcarbodiimide or N,N0-disuccinimidyl carbon-
ate. Next, the coupling reaction was performed in buffer at a hap-
ten-to-protein molar ratio of 44:1. On the other hand, assay coating
conjugates were prepared using OVA as carrier. In this case, the
activation of the carboxylate was accomplished using tributylamine
and isobutyl chloroformate. The initial molar ratio in the coupling
reaction with this protein was 13:1. A brief description of the
followed coupling procedures has been included as Supplementary
data.
Acknowledgements
ꢀ
This work was supported by Ministerio de Educacion y Ciencia
(AGL2006-12750-C02-01/02/ALI) and cofinanced by FEDER funds.
J.P. and J.V.M. were hired by the CSIC, the former under a pre-
doctoral I3P contract and the latter under a Ramon y Cajal post-
ꢀ
doctoral contract, both of them financed by Ministerio de Ciencia e
ꢀ
Innovacion and the European Social Fund.
Limited amounts of the newly described immunoreagents are
available upon request for evaluation.
Supplementary data
4.2.2. Polyclonal antibody production. Animal manipulation was
performed in compliance with the laws and guidelines of the
Spanish Ministry of Science and Innovation (RD 1201/2005 and law
32/2007) and according to the European Directive 2003/65/EC
concerning the protection of animals used for experimental and
other scientific purposes. New Zealand white rabbits were immu-
Supplementary data associated with this article can be found in
the online version, at This Supplementary data contains general
details, experimental biochemical procedures, full spectral data and
assignments for all intermediates of the synthesis of haptens 2e5,
preparation and characterisation data of compounds 40 and 41 and
copies of the 1H NMR spectra of haptens 2e5. Supplementary data
associated with this article can be found in the online version, at
doi:10.1016/j.tet.2010.11.054. These data include MOL files and
InChiKeys of the most important compounds describedin this article.
nized at three-week intervals with 300 mg of BSAehapten conjugate
(see the Supplementary data) following described standard pro-
tocols.34 10 days after the fourth boost the animals were exsangui-
nated, the antisera were separated by centrifugation and
precipitated twice with an ammonium sulfate saturated solution.
References and notes
4.2.3. Competitive ELISAs. 96-well polystyrene ELISA plates were
coated with 100
mL per well of OVA conjugate solution at 1.0 or
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pp 623e679.
5. (a) Matsushita, M.; Hoffman, T. Z.; Ashley, J. A.; Zhou, B.; Wirshing, P.; Janda, K. D.
Bioorg. Med. Chem. Lett. 2001, 11, 87e90; (b) Peterson, E. C.; Gunnell, M.; Che, Y.;
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overnight incubation at rt. Plates were washed four times with
a solution containing 150 mM NaCl and 0.05% (v/v) Tween 20.
Bidimensional competitive assays were carried out in order to find
the limiting concentrations of the immunoreagents. A serial dilution
of azoxystrobin was prepared, in borosilicate glass tubes, with
10 mM phosphate buffer, pH 7.4 containing 140 mM NaCl, and the
antisera were also serially diluted in the same buffer containing
0.05% (v/v) Tween 20. Each plate column received a complete
standard curve of the analyte (50
dilution per column of a given antiserum (50
distribution of the reagents was repeated for each plate with a dif-
ferent coating conjugate concentration. The immunological
reaction took place during 1 h at rt, and plates were washed again as
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mL per well). The same
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