Mode of action of the anthelmintics morantel,pyrantel and levamisole on muscle cell membrane of the nematode Ascaris suum

Intracellular current and voltage clamp techniques were used to investigate the mode of action of the anthelmintics, morantel, pyrantel and levamisole applied to the bag region of Ascaris suum muscle cells. Microperfusion of the anthelmintics and of O-acetylcholine (ACh) increased the input conductance and depolarised the membrane potential of the muscle bags. The relative potencies of these drugs were determined from dose–conductance relationships and found to be: morantel = pyrantel > levamisole > ACh. High doses (>10μM) of morantel caused antagonism of ACh responses. ACh-induced currents were measured under voltage clamp (over the range ?80 to +10mV). At membrane potentials between ?80 and 0 mV, microperfusion of ACh induced a voltage-dependent inward current. The current–voltage relationship was linear for membrane potentials in the range ?30 to +10mV. The reversal potential was measured directly and found to be about +10mV. The relationship became non-linear at membrane potentials more negative than ?30 mV, and the degree of non-linearity was dependent upon the concentration of ACh. The current–voltage relationships for morantel, pyrantel and levamisole also possessed both linear (?30 to 0mV) and non-linear components. The reversal potential for each agonist, determined by extrapolation of the linear component of the current–voltage relationship, was approximately +10mV, indicating the same cation channels were activated both by ACh and the anthelmintics. Evidence for competition between ACh and pyrantel for the same membrane receptor was obtained using iontophoretic delivery of each agonist from a double-barrelled micropipette. It is concluded that the anthelmintics, morantel, pyrantel and levamisole act as potent agonists at ACh receptors on muscle bag membranes of A. suum.     

Actions of quinolizidine alkaloids on Periplaneta americana nicotinic acetylcholine receptors

BACKGROUND: Botanical insecticides do not play a major role as crop protectants, but they are beneficial in some applications. The authors investigated the actions of naturally occurring alkaloids on insect nicotinic acetylcholine (ACh) receptors (nAChRs) by evaluating their abilities to inhibit specific binding of [³H]imidacloprid (IMI) to nerve‐cord membranes from Periplaneta americana L. Two alkaloids were also tested for their actions on nAChRs expressed by cockroach neurons using patch‐clamp electrophysiology. RESULTS: Four natural quinolizidine alkaloids (matrine, sophocarpine, cytisine and aloperine) exhibited more than 50% inhibition of [³H]IMI binding at 10 µM , although other compounds were found to have no or low inhibitory activity. The rank order of potency based on concentration–inhibition curves was cytisine > sophocarpine ≥ aloperine ≥ matrine. Patch‐clamp analysis indicated that sophocarpine and aloperine were not agonists of nAChRs expressed in P. americana neurons, yet, at 10 µM , aloperine, but not sophocarpine, suppressed ACh‐induced inward currents significantly. CONCLUSION: Three of the four natural alkaloids tested possess structural moieties that are necessary for interaction with P. americana nAChRs. Aloperine, which possesses a unique structure and showed a distinctive dose–response curve, was found to act as an antagonist. Appropriate modifications of these alkaloids might result in novel insecticidal nAChR ligands. Copyright © 2008 Society of Chemical Industry     

Comparison of symptomatic and neurophysiological activities of enantiomers of the insecticide 3-phenoxybenzyl 1-(4-ethoxyphenyl)-2,2-dichlorocyclopropane-1-carboxylate

The insecticidal activities of optical isomers of 3-phenoxybenzyl 1-(4-ethoxyphenyl)-2,2-dichlorocyclopropanecarboxylate and related compounds were measured with American cockroaches and their knockdown activities were evaluated with house flies. The activities of the S(?)-isomer of the dichlorocyclopropanecarboxylate were higher than those of the R(+)-isomer. The effects of the compounds on the inward membrane currents induced by a stepdepolarizing pulse in crayfish axonal membranes were examined under voltage clamp conditions by the sucrose gap method. The compounds induced a tail current upon step repolarization of the membrane. The tail current decayed to zero in each record, but developed with time after the start of application of the compound until a steady level was reached. The rate of decay of the tail current observed in axonal membranes treated with the S(?)-isomer was slower than with the R(+)-isomer. The rates of development of the tail current induced by the two isomers were not very different.     

Pharmacological differentiation of responses to dopamine,octopamine and noradrenaline recorded from a cockroach motoneurone

Recordings have been made from the soma of a cockroach common inhibitory motoneurone. Locally applied dopamine, octopamine or noradrenaline all depolarised the neurone and produced a modest increase in membrane conductance. Under voltage-clamp, currents evoked by these amines showed considerable voltage dependence. The current-voltage relationships for dopamine and octopamine responses were similar, but differed from that for noradrenaline. The unusual voltage-dependent characteristics of these responses suggest that these compounds may serve a modulatory function in the insect CNS. The effects of a range of pharmacological antagonists indicate that dopamine acts upon a class of receptors distinct from those mediating responses to octopamine, noradrenaline and acetylcholine (ACh). Dopamine currents were abolished by SCH 23390 or cis-(Z)-flupenthixol, which did not depress responses to the other agonists. Octopamine and noradrenaline currents were selectively blocked by metoclopramide and ergometrine respectively, while β-bungarotoxin and gallamine preferentially suppressed ACh responses. At the present time the ionic basis of dopamine responses is unclear. Dopamine-evoked currents were totally abolished by application of Cd2²⁺ or verapamil or when the preparation was bathed in Ca²⁺ free external solutions. Changes in the external concentrations of Na⁺, K⁺ or Cl^?, however, also had some effect upon dopamine currents.     

Recent studies on the effects of DDT and pyrethroid insecticides on nervous activity in cockroaches

A summary is given of the results of three sets of electrophysiological experiments on the American cockroach (Periplaneta americana). The effects of DDT and S-bioallethrin [bioallethrin (S)-cyclopentenyl isomer] on single giant axons were studied using the voltage-clamp technique. Whereas both molecules induced long tails of inward (sodium ion) current, the voltage and time-dependency of these tails differed. With DDT, the tail was proportional to the activation of the peak current and decreased with the duration of the pulse, suggesting that the molecules were bound to open sodium channels and delayed their closing. With S-bioallethrin, the voltage dependency of the tail was different from that of the peak current, and the tail current increased exponentially with the duration of the depolarisation, suggesting that the pyrethroid insecticide modified resting (or silent) sodium channels into slowly activating channels. Modified action potentials, mimicking those produced by the two molecules, were computed on the basis of these results. Deltamethrin, one of the most potent pyrethroid insecticides, was applied topically on a leg mechanoreceptor and was found to have little effect on the local ‘receptor potential’ but to inhibit action potential production. The effects of topical applications of deltamethrin on the dorsal part of the abdomen, on nervous activity in the abdominal connectives, were studied under different experimental conditions. The results suggest that the insecticide molecules diffused rapidly through the cuticle, were concentrated in the haemolymph, and eventually reached the central nervous system, where they inhibited nerve activity.     

新烟碱类杀虫剂毒理学研究进展

以吡虫啉和啶虫脒为代表的新烟碱类杀虫剂具有卓越的内肖活性及很高的残留活性。放射配基结合实验表明，新烟碱类杀虫剂对多种昆虫乙酰胆碱受体具有高亲和力，乙酰胆碱是其竞争性结合抑制剂，电生理学研究表明，该类杀虫剂与乙酰胆碱类似，可诱导瞬时内向电流，并可作用于药理学性质不同的昆虫烟碱型乙酰胆碱受体（nAChR)亚型，分子生物学研究进一步表明，该类杀虫剂主要作用于昆虫nAChRα亚基。     

Correlations of the electrophysiological activity of neonicotinoids with their binding and insecticidal activities

The electrophysiological actions of various neonicotinoids, including substituted benzyl derivatives, against recombinant Drosophila SAD/chicken beta2 hybrid nicotinic acetylcholine receptor (nAChR) were measured to analyze the relationships between the in vivo (insecticidal) and in vitro (binding and agonist) activities. Most of the neonicotinoids tested were capable of inducing inward currents by activating the hybrid nAChRs expressed in Xenopus laevis oocytes, whereas some compounds had no agonist activity and only blocked the acetylcholine-induced currents. Variations in the agonist activity were well correlated with those in the binding potency evaluated using [3H]imidacloprid as well as insecticidal activities.     

Mechanism of resistance to spinosyn in the tobacco budworm, Heliothis virescens

Topical laboratory selection of tobacco budworm larvae, Heliothis virescens, with technical spinosad for multiple generations resulted in larvae 1068-fold resistant to topical applications of the insecticide and 316.6-fold resistant to insecticide treated diet as compared to the parental strain. The penetration of 2′-O-methyl[¹⁴C]spinosyn A across the cuticle of the susceptible (parental) and selected (resistant) tobacco budworms increased with time 3-12 h after application. A trend of reduced penetration in the resistant strain was found but the differences were not statistically significant. 2′-O-methyl[¹⁴C]spinosyn A when injected into the hemocoel was not metabolized 96 h after treatment in both the susceptible and resistant strain, suggesting that a change in metabolism was not the mechanism of resistance. Electrophysiological studies indicated that dose-dependent spinosyn A-induced currents occurred in neurons from spinosyn resistant and susceptible (adult) tobacco budworms. At both 10 and 100 nM spinosyn A, however, the amplitude of these currents in the resistant insects was significantly smaller than the amplitude of currents observed from neurons from susceptible tobacco budworm adults. This suggests that neurons from resistant insects have decreased sensitivity to spinosyn A. However, the reduced inward currents in the resistant strain may or may not be related to the mode of action of the spinosyns. No statistically significant cross-resistance was noted for the spinosad resistant tobacco budworms for topical applications of permethrin (Pounce^®), profenofos (Curacron^®), emamectin benzoate (Denim^®), or indoxacarb (Steward^®). A statistically significant reduction in susceptibility to acetamiprid (Mospilan^®) in artificial diet as determined from a resistance ratio of 0.482 was found.     

Insecticidal activity of Sikimi extract and its modulation of the GABAA receptor channel

Sikimi plant (also known as Japanese star anise), Illicium anisatium, is toxic to mammals. Extracts of Sikimi were studied for their insecticidal activity against the larvae of mosquito, Culex quinquefasciatus, and for their mechanism of action on ion channels. Crude methanol extract and its ethyl acetate-soluble fractions were insecticidally active, with EC₅₀ values of 63·0 μg ml^-1 and 43·7 μg ml^-1, respectively. The ethyl acetate-soluble fraction was perfused through the bathing solution and the current induced by a brief (10 ms) application of GABA by pressure ejection through pipette electrode was recorded by the whole-cell patch clamp technique. The extract suppressed GABA-induced currents irreversibly with an EC₅₀ value of 0·42 μg ml^-1. The time constant of current fitted to the single exponential function was shortened by the ethyl acetate-soluble fraction at concentrations ranging from 0·1 μg ml^-1 to 10 μg ml^-1 in a concentration-dependent manner. It was concluded that Sikimi extracts decreased the affinity of GABA for its binding site on the GABA receptor, thereby suppressing GABA-induced currents. © 1998 SCI     

Effects of 22,23-dihydroavermectin B1a on locust (Schistocerca gregaria) muscles may involve several sites of action

Ibotenic acid [2-(3-hydroxyisoxazol-5-yl)glycine] induced a dose-dependent increase in chloride ion conductance in locust muscle fibres which was not sensitive to 4-aminobutyric acid (GABA). This ibotenate response became rapidly desensitised and appeared to be due to activation of extrasynaptic glutamate H receptors. 22-23-Dihydroavermectin B_1a (DHAVM) (500 pg to 1 μg ml^?1) induced irreversible increases in permeability to the chloride ion and abolished ibotenate responses. DHAVM responses were not altered when glutamate H receptors were desensitised by glutamate (1 mM) or ibotenate (100 μM). Irreversible changes in input conductance caused by DHAVM were not affected by penicillin G (1 mM) or bicuculline (1 mM), but picrotoxin (1 mM) and zinc chloride reduced DHAVM responses by 23.7 and 52.6%, respectively. It is concluded that DHAVM has a number of sites of action on locust muscle that include effects on the glutamate H receptor–chloride ion channel complex, in addition to effects on the GABA receptor–chloride ion channel previously described.     

烟碱乙酰胆碱受体及其与新烟碱类相互作用的研究进展

对烟碱乙酰胆碱受体(nAChRs)的结构与功能、配体结合部位、门控机理以及与新烟碱类的相互作用进行了综述,并对nAChRs亚基基因突变和敲除对新烟碱类和多杀菌素敏感性的影响进行了讨论。nAChRs在脊椎动物和昆虫的胆碱能突触的快速神经传递中起着重要作用,其在昆虫中仅存在于中枢神经系统(CNS)中,而在脊椎动物中同时存在于CNS和神经肌肉连接处。nAChRs是新烟碱类杀虫剂、多杀菌素和杀螟丹的作用靶标。肌肉和CNS中的nAChRs是一个由两个α和三个非α(β,γ和δ)亚基组成的异数五聚体,该受体主要有三部分:一个在细胞外发现的区域(胞外区)、一个位于膜内的区域(跨膜区),另一个是位于细胞内的区域(胞质区)。每个亚基(从N-C端)都具有一个包含乙酰胆碱(ACh)结合部位的细胞外结构域;4个跨膜结构域(M1~4),其中M2的大部分氨基酸位于离子通道的内壁;一个胞质噜扑(loop)和一个胞外C端。通道门位于孔道内的疏水区。ACh结合部位位于天然和功能受体的两个亚基的界面,是由一个亚基的3个噜扑(A-C)和另一个亚基的3个噜扑(D-F)构成。每当受体与ACh(或其他激动剂)分子结合时,M2 α螺旋体的构象发生改变,使通道开启,处于阳离子传导状态,直至一个或两个激动剂分子从结合口袋解离,通道才关闭。如果激动剂一直存在,并反复结合,则通道处于脱敏状态。nAChRs与新烟碱类的各种选择性作用取决于新烟碱类的结构以及nAChRs的亚基组成。     

Boric acid toxicity to the German cockroach, Blattella germanica: Alterations in midgut structure, and acetylcholinesterase and glutathione S-transferase activity

Oral toxicity of boric acid, an inorganic insecticide, was evaluated on German cockroach, Blattella germanica L. (Dictyoptera, Blattellidae). Newly emerged adults were exposed to various concentrations of boric acid incorporated into the diet. Results showed that treated insects exhibited toxic symptoms with a dose-dependent mortality. Histological study of midgut revealed alterations in the epithelial cells and a significant increase in the epithelium thickness. In a second series of experiments, the compound was investigated on the activities of acetylcholinesterase (AChE) and glutathione S-transferases (GSTs). Data showed that the compound induced GSTs and reduced the activity of AChE. From this experiment, it may be concluded that ingested boric acid caused death of insects perhaps ultimately by starvation via alterations of the midgut. Additionally, the compound seemed to present a neurotoxic action as evidenced by the symptoms of poisoning and the reduction in AchE activity.     

Effects of metaldehyde and acetaldehyde on specific membrane currents in neurones of the pond snail Lymnaea stagnalis

Bath application of metaldehyde or acetaldehyde led to tonic depolarization and bursting activity in identified neurones in the isolated central nervous system of Lymnaea stagnalis. This activity was sometimes accompanied by paroxysmal depolarizing shifts in membrane potential. Those neurones which possessed type 2 action potentials showed marked spike broadening in response to metaldehyde, but little increase in action potential duration in the presence of acetaldehyde. Under voltage clamp, metaldehyde caused a reduction in the delayed rectifier and A-currents, both of which are outward potassium currents. Acetaldehyde also reduced the delayed rectifier but had no effect on the A-current. In type 2 neurones, voltage-dependent calcium current appeared to increase in the presence of metaldehyde, but was decreased by acetaldehyde. The action of metaldehyde and acetaldehyde on firing activity, as well as their differential effects on action potential duration, may be partly explained by alterations in these specific membrane currents.     

甲酸乙酯对米象乙酰胆碱酯酶和羧酸酯酶的影响

报道了甲酸乙酯对米象[Sitophilus oryzae(Linnaeus)]乙酰胆碱酯酶(AChE)和羧酸酯酶(CarE)的活性影响,甲酸乙酯对米象表现出很高的毒力,25℃下熏蒸处理24 h的LC₅₀为28.65μL/L。亚致死剂量的甲酸乙酯可以使米象成虫体内AChE的比活力显著下降,用20μL/L的浓度处理24 h,AChE的比活力由15.684 nmol/mg.min下降至9.530 nmol/mg.min;酶动力学研究表明,甲酸乙酯可使米象成虫体内AChE的K_m值明显增加。对于羧酸酯酶,在活体条件下表现为先抑制后激活的规律,离体条件下则主要表现为抑制作用,且随着药剂浓度的增大抑制率也增大。     

Pharmacological properties of presynaptic muscarinic receptors in the sixth abdominal ganglion of the cockroach,Periplaneta americana L.

The effects of muscarinic agonists on acetylcholine (ACh) release in the cercal-afferent giant-interneuron synapses of the cockroach have been studied using the single-fibre oil-gap method. Decrease in amplitude of the cEPSP was induced by pressure ejection of arecoline (ARE), carbachol (CCh) and oxotremorine free base (OXO) within the sixth abdominal (A6) ganglion. This depressive effect was dose-dependent without any effect at the post-synaptic site in the range of concentrations used. The concentration for 50% of cEPSP inhibition of the most commonly used muscarinic agonists was determined. The rank order of potencies was: ARE > CCh > OXO > McN-A-343 > bethanechol (BET). Plateau levels of 56%, 54% and 68% were reached when increasing the concentration of ARE, CCh and OXO respectively. McN-A-343 and BET had an inhibitory effect on ACh release but higher doses also acted at the postsynaptic level. Furthermore, muscarinic agonists were able to evoke uIPSP and a smaller inhibitory effect of ARE and OXO was observed in presence of picrotoxin. It is concluded that the ACh release in the cockroach A6 ganglion is modulated by presynaptic muscarinic receptors which are possible target sites for insecticides. The presence of putative muscarinic receptors on inhibitory GABAergic interneurons, involved in pre- and postsynaptic inhibition is suspected.     

Acaricidal activity against Panonychus citri of a ginkgolic acid from the external seed coat of Ginkgo biloba

An acaricidal substance extracted from the external seed coat of Ginkgo biloba L. was identified by UV (ultraviolet), IR (infrared), EI-MS (electron impact ion source mass spectrometry), (1)H NMR (nuclear magnetic resonance) and (13)C NMR as 6-[(Z)-10-heptadecenyl]-2-hydroxybenzoic acid (compound 1). Laboratory bioassay on citrus red mite, Panonychus citri (Mcg), showed that compound 1 possessed the following properties. (i) Powerful contact toxicity with an LC(50) of 5.2 mg litre(-1) after 24 h that was similar to that of pyridaben (LC(50) = 3.4 mg litre(-1)) and significantly superior to that of omethoate (LC(50) = 122 mg litre(-1)). Furthermore, its LC(90) was 13.4 mg litre(-1) after 24 h, which is significantly superior to both pyridaben (LC(90) = 69.6 mg litre(-1)) and omethoate (LC(90) = 453 mg litre(-1)). (ii) Quick-acting acaricidal activity. At identical concentrations, compound 1 was much faster-acting than pyridaben or omethoate. (iii) Compound 1 had strong corrosive action on the cuticle of P. citri but no phytotoxicity to plants.     

Pyrethroid action on the nicotinic acetylcholine receptor/channel

The interactions of natural pyrethrins and nine pyrethroids with the nicotinic acetylcholine (ACh) receptor/channel complex of Torpedo electric organ membranes were studied. None caused significant reduction in [³H]ACh binding to the receptor sites, but all inhibited [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) binding to the channel sites in presence of carbamylcholine. Allethrin inhibited [³H]H₁₂-HTX binding noncompetitively, but [³H]imipramine binding competitively, suggesting that allethrin binds to the receptor's channel sites that bind imipramine. The pyrethroids were divided into two types according to their actions: type I, which included pyrethrins, allethrin, bioallethrin, resmethrin, and tetramethrin, was more potent in inhibiting [³H]H₁₂-HTX binding and acted more rapidly (i.e., in <30 sec). Type II, which included permethrin, fluvalinate, cypermethrin and fenvalerate, was less potent and their potency increased slowly with time. Also, inhibition of the initial rate of [³H]H₁₂-HTX binding by type I compounds increased greatly by the presence of the agonist carbamylcholine, but this was not so with type II compounds. The receptor-regulated ⁴⁵Ca²⁺ flux into Torpedo microsacs was inhibited by pyrethrins and pyrethroids, suggesting that their action on this receptor function is inhibitory. There was very poor correlation between the potencies of pyrethrins and pyrethroids in inhibiting [³H]H₁₂-HTX binding and their toxicities to house flies, mosquitoes, and the American cockroach. However, the high affinities that several pyrethroids have for this nicotinic ACh receptor suggest that pyrethroids may have a synaptic site of action in addition to their well known effects on the axonal channels.     

Modulation of sodium channels by the oxadiazine insecticide indoxacarb and its N-decarbomethoxylated metabolite in rat dorsal root ganglion neurons

The effects of the oxadiazine insecticide indoxacarb and its N-decarbomethoxylated metabolite (DCJW) on tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels in rat dorsal ganglion neurons were studied using the whole-cell patch clamp technique. Indoxacarb and DCJW suppressed the peak amplitude of action potentials, and DCJW exhibited a faster time course and higher potency than indoxacarb in the blocking effects. In voltage-clamp experiments, indoxacarb and DCJW suppressed TTX-R sodium currents in a time-dependent manner without a steady-state level of suppression. IC50 values for indoxacarb and DCJW on TTX-R sodium currents were estimated to be 10.7 and 0.8 microM after 25 min of bath application, respectively. DCJW was about 10 times more potent than indoxacarb in blocking TTX-R sodium currents. Although the suppressive effects of indoxacarb were partially reversible after washout with drug-free external solution, no recovery of sodium current was observed in DCJW treated neurons after prolonged washout. In current-voltage relationships, both indoxacarb and DCJW blocked the sodium currents to the same degree in the entire range of membrane potentials. The sodium conductance-voltage curve was not shifted along the voltage axis by indoxacarb and DCJW at 10 microM. In contrast, the steady-state inactivation curves were shifted in the hyperpolarizing direction by indoxacarb as well as by DCJW. Based on these results, it was concluded that indoxacarb and DCJW potently blocked the TTX-R sodium channel in rat DRG neurons with hyperpolarizing shifts of the steady-state inactivation curves, suggesting preferential association of the insecticides to the inactivated state of sodium channels. The small structural variation between indoxacarb and DCJW resulted in clear differences in potency for blocking sodium channels and reversibility after washout.     

Surfactant-enhanced foliar uptake of some organic compounds: Interactions with two model polyoxyethylene aliphatic alcohols

Interactions occurring during the surfactant-enhanced foliar uptake of seven model organic compounds were examined using two homogeneous surfactants, hexaethylene glycol monotridecyl ether (C13E6) and hexadecaethylene glycol monododecyl ether (C12E16). Surfactant–compound and compound–surfactant interactions were detected by measurement of their relative uptake rates following application of c. 0·2 μl droplets of the corresponding radiolabelled formulations. The magnitude of surfactant–compound interaction was found to vary according to the physicochemical properties of both the compound and the surfactant, and was influenced by surfactant concentration and target plant species. Interactive and non-interactive mechanisms, both leading to substantial enhancement of compound uptake, could be identified, but their precise nature could not be elucidated. Although penetration of C13E6 into the site of application appeared to be essential in order to activate the uptake of a compound, substantial absorption of C12E16 was not always required to produce the same effect. The results are discussed in the light of possible sites and modes of action for activator polyoxyethylene surfactant adjuvants.     

The importance of whole plant studies in determining the biochemical mode of action of herbicides,as shown by studies with the experimental compound 6-chloro-2-trifluoromethyl-imidazo(4, 5-b)pyridine

The experimental herbicides 6-chloro-2-trifluoromethyl-imidazo(4,5b)pyridine and 2-t-butyl-6-chloroimidazo(4,5-b)pyridine inhibit the Hill reaction of isolated chloroplasts by 50% at 1.5 and 2.4 pm, respectively. The first compound also uncouples plant and animal oxidative phosphorylation at about 50 pm, whilst the second compound is not an uncoupler, Surprisingly, light does not affect the herbicidal action of either compound on whole plants, so it is unlikely that the herbicidal activity is due to inhibition of the Hill reaction. Since both compounds probably act by the same mechanism, uncoupling of oxidative phosphorylation cannot be the site of herbicidal action, which therefore remains unknown. A study of the metabolism of 6-chloro-2-trifluoromethyl-imidazo(4,5-b)pyridine shows the occurrence of hydroxylation of the pyridine ring in rat and maize and conversion to trifluoroacetic acid in the latter.