7‐Coumaryl permethrate and its cis‐ and trans‐isomers as new fluorescent substrates for examining pyrethroid‐ cleaving enzymes

Abstract

New esterase substrates were synthesized using cis-, trans- and cis-trans-permethrinic acid chloride and then used to measure pyrethroid-cleaving enzymes in insects. The new substrates, namely cis-, trans- and cis-trans-7-coumaryl permethrates (7-CP), show a structure very similar to permethrin insecticide and yield fluorescent products on hydrolysis. These substrates were hydrolyzed by a commercial porcine preparation that provided esterase-specific activity, and were stable at different pH values (5.2-7.8). Studies made with house fly, Musca domestica (L.), homogenates showed that these compounds are appropriate for determining pyrethroid hydrolysis activity on individual insects. The measured activity of house fly esterase was 870 relative fluorescence units (RFU) min(-1) with cis-7-CP as substrate, 1117 RFU min(-1) with trans-7-CP as substrate and 1423 RFU min(-1) with cis-trans-7-CP as substrate. The fluorescent substrates for pyrethroid-cleaving esterases described in this paper have advantages over methods already given in the literature. They are substrates with structures very similar to pyrethroids, the cleavage of which can be followed by an increase in fluorescence emission at 440 nm; it takes only about 5 min to measure the reaction, and moreover the high sensitivity of the fluorescence technique allows the quantification of esterase activity on individual insects.

Activity of increased specific and non-specific esterases and glutathione transferases associated with resistance to permethrin in pediculus humanus capitis (phthiraptera: pediculidae) from Argentina

“…All solvents and general reagents were analytical grade. The synthesis of 7-coumaryl (1R,S)-cis-trans-3-(2,2-dichloro vinyl)-2,2-dimethyl cyclopropane carboxylate (7-coumaryl permethrate) was carried out according to Santo Orihuela et al (2006). (1R,S) cis-trans-(2,2-dichloro vinyl)-2,2-dimethyl cyclopropane carboxylate acid chloride (cis-, trans-and cistrans-permethrinic acid chloride) was from Chemotecnica, Buenos Aires, Argentina, 7-OHC was from Sigma, USA, toluene was from Merck and triethylamine from Aldrich, USA.…”

“…Thus, in order to obtain a specific substrate for pyrethroid esterases, we synthesized 7-coumaryl permethrate (7-CP), an ester structurally similar to permethrin and with the same acid moiety. The hydrolysis of this ester yields fluorescent 7-hydroxycoumarin (7-OHC) that allows using microfluorometric methods to quantify esterase activity (Santo Orihuela et al 2006).…”

Relative Contribution of Monooxygenase and Esterase to Pyrethroid Resistance in Triatoma infestans (Hemiptera: Reduviidae) from Argentina and Bolivia

“…Esterase activity was determined by the hydrolysis of 7-CP, a new ßuorescent substrate appropriate for determining pyrethroid hydrolysis activity on individual insects (Santo Orihuela et al 2006). For this, the insects were cooled, and each nymph was homogenized in 220 l of phosphate buffer (0.05 M), pH 7.2, by using a plastic mortar and pestle.…”

“…cis-trans (43.8% cis; 56.2% trans)-Permethrinic acid was supplied from Chemotecnica (Buenos Aires, Argentina), and thionyl chloride (Cl 2 SO) (99%) and triethylamina (ϩ99%) were purchased from Aldrich Chemical (Milwaukee, WI). 7-Coumaryl permethrate (7-CP) was synthesized in our laboratory by the method of Santo Orihuela et al (2006). Topical Application Bioassays.…”

“…Moreover, the study showed a signiÞcant difference in nonspeciÞc esterase activity measured as phenyl-thioacetate and ␣-naphtyl acetate activities between the susceptible and Salta populations. Later, a new ßuorescent substrate for examining pyrethroid-cleaving esterases was reported by Santo Orihuela et al (2006). The high sensitivity of this ßuorescent technique allowed the quantiÞcation of pyrethroid esterase activity on individual insects, an important tool to assess the distribution of esterase activity in individual from susceptible and resistant populations.…”