Design, synthesis and use of chiral pheromone-based probes to study pheromone enantiomer discrimination in the pheromone binding proteins from the gypsy moth, Lymantria dispar

The gypsy moth is a widespread and harmful pest causing extensive damage to the Canada’s forest and orchard ecosystems. It uses (+)-disparlure as a sex pheromone. Discovery of the pheromone, including its absolute configuration, has enabled monitoring of gypsy moth populations. Disparlure of low enantiopurity is not attractive to the moths and, for this reason, enantiopure (+)-disparlure has been a synthetic target for many years. To access (+)-disparlure of high enantiopurity we have used a diastereoselective nucleophilic addition reaction with the enantiopure α-chloroaldehyde (2-chlorododecanal) that yields a stereocontrolled access to the 1,2-anti chlorohydrin core. The (+)-disparlure was prepared through a series of transformations that include a Mitsunobu inversion. We have successfully completed the synthesis of (+)-disparlure in 5 steps as compared to Iwaki’s first synthesis in 12 steps and Sharpless’s widely used synthesis in 6 steps. The same approach was used to produce 18-hydroxydisparlure enantiomers, which were coupled to a linker with an alkyne moiety at the end. The alkyne was then coupled to azide-based commercial fluorescent probes, to furnish fluorescent disparlure-based probes for physical studies. The gypsy moth has two different pheromone binding proteins, LdisPBP1 and LdisPBP2. Previously, our group has addressed the enantiomer selectivity of these two PBPs and found that PBP1 binds (-)-disparlure more strongly than (+)-disparlure, while PBP2 binds (+)-disparlure more strongly. Despite several binding assays, the interaction and discrimination of gypsy moth PBPs towards disparlure enantiomers are not fully understood due to lack of binding interaction and kinetic studies, which are technically demanding, due to the hydrophobicity of the pheromone. In this thesis, we have studied the binding interaction of deuterium-labelled (+)-disparlure and (-)-disparlure with LdisPBPs by 2H NMR spectroscopy. The results from NMR studies were correlated with the results from docking simulations of (+)-disparlure and (-)-disparlure bound to one internal site and multiple external sites of LdisPBP1 and LdisPBP2. These results indicated that (+)-disparlure and (-)-disparlure adopt different conformations and orientations in the binding pockets of LdisPBP1 and LdisPBP2. Most of the reported work on PBPs focuses on the pheromone binding affinities of PBPs. However, the pheromone-PBP interactions require more than half an hour to establish equilibrium, whereas male moths respond to female pheromones in milliseconds. Therefore, the interactions between pheromones and olfactory components such as PBPs and pheromone receptors may not be under thermodynamic control. In this thesis, we aimed to provide a dynamic perspective of pheromone-PBP interactions and to link these to the functions of PBPs. We have studied thermodynamic (Kd) and kinetic properties (kon and koff) of LdisPBPs-disparlure enantiomer interaction by fluorescence binding assays and kinetic experiments using fluorophore-tagged disparlure enantiomers. The result indicated that the binding preference of disparlure enantiomers to LdisPBPs. Based on the kinetic data of LdisPBPs with fluorophore-tagged disparlure enantiomers, we propose a kinetic model that includes a two-step binding process. Each of these two steps may contribute to a different function of the LdisPBPs

Synthesis of Isotopically Labelled Disparlure Enantiomers and Application to the Study of Enantiomer Discrimination in Gypsy Moth Pheromone‐Binding Proteins

To study the binding mechanism of disparlure (7,8)-epoxy-2-methyloctadecane enantiomers with pheromone-binding proteins (PBPs) of the gypsy moth, oxygen-17 or 18 and 5,5,6,6-deuterium labelled disparlure enantiomers were prepared in an efficient, enantioselective route. Key steps involve the asymmetric α-chlorination of dodecanal by SOMO catalysis and Mitsunobu inversion of a 1, 2-chlorohydrin. The pheromone, (+)-disparlure (7R, 8S), was tested in two infested zones, demonstrating that it is very attractive towards male gypsy moths. Studies of the binding of (+)-disparlure and its antipode to gypsy moth PBPs by 2H &17O NMR at 600 MHz are reported. Chemical shifts, spin-lattice relaxation times and transverse relaxation times  of deuterium atoms of disparlure enantiomers in 2H NMR show that binding of disparlure enantiomers to PBP1 differs from binding to PBP2, as expected from their opposite binding preferences (PBP1 binds (-)-disparlure, and PBP2 binds (+)-disparlure more strongly). Models of the disparlure enantiomers bound to one internal binding site and two external binding sites of both PBPs were constructed. The observed chemical shift changes of deuterated ligand signals, from non-bound to bound, T1 and T2 values are correlated with results from the simulations. Together these results suggest that the disparlure enantiomers adopt distinct conformations within the binding sites of the two PBPs and interact with residues that line the sites

Can We Disrupt the Sensing of Honey Bees by the Bee Parasite Varroa destructor?

Background The ectoparasitic mite, Varroa destructor, is considered to be one of the most significant threats to apiculture around the world. Chemical cues are known to play a significant role in the host-finding behavior of Varroa. The mites distinguish between bees from different task groups, and prefer nurses over foragers. We examined the possibility of disrupting the Varroa – honey bee interaction by targeting the mite\u27s olfactory system. In particular, we examined the effect of volatile compounds, ethers of cis 5-(2′-hydroxyethyl) cyclopent-2-en-1-ol or of dihydroquinone, resorcinol or catechol. We tested the effect of these compounds on the Varroa chemosensory organ by electrophysiology and on behavior in a choice bioassay. The electrophysiological studies were conducted on the isolated foreleg. In the behavioral bioassay, the mite\u27s preference between a nurse and a forager bee was evaluated. Principal findings We found that in the presence of some compounds, the response of the Varroa chemosensory organ to honey bee headspace volatiles significantly decreased. This effect was dose dependent and, for some of the compounds, long lasting (>1 min). Furthermore, disruption of the Varroa volatile detection was accompanied by a reversal of the mite\u27s preference from a nurse to a forager bee. Long-term inhibition of the electrophysiological responses of mites to the tested compounds was a good predictor for an alteration in the mite\u27s host preference. Conclusions These data indicate the potential of the selected compounds to disrupt the Varroa - honey bee associations, thus opening new avenues for Varroa control

Synthesis of Enantiopure Alicyclic Ethers and Their Activity on the Chemosensory Organ of the Ectoparasite of Honey Bees, <i>Varroa destructor</i>

The preparation of enantiopure conformationally restricted alicyclic ethers and their inhibitory activities on the chemosensory organ of the <i>Varroa destructor</i>, a parasite of honey bees, are reported in this article. We tested the effect of enantiopure ethers of <i>cis</i>-5-(2′-hydroxyethyl)­cyclopent-2-en-1-ol on the <i>Varroa</i> chemosensory organ by electrophysiology, for their ability to inhibit the responses to two honey bee-produced odors that are important for the mite to locate its host: nurse bee head space odor and (<i>E</i>)-β-ocimene, a honey bee brood pheromone. Previous work with the racemic compounds showed that they suppress the mite’s olfactory response to its bee host, which led to incorrect host choice. Based on a structure–activity relationship, we predicted that the two most active compounds<i>cis</i>-1-butoxy-5-(2′-methoxyethyl)­cyclopent-2-ene, <b>cy</b>{4,1}, and (<i>cis</i>-1-ethoxy-5-(2′ethoxyethyl)­cyclopent-2-ene, <b>cy</b>{2,2}could have opposite active enantiomers. Here we studied the enantiomers of both ethers, whose preparation involved enzymatic resolution of racemic diol <i>cis</i>-5-(2′-hydroxyethyl)­cyclopent-2-en-1-ol using Lipase AK with vinyl acetate. The racemic diol was prepared from commercially available 2,5-norbornadiene. We observed that the responses of the chemosensory organ to honey bee head space volatiles were significantly decreased by both enantiomers of <b>cy</b>{4,1} and <b>cy</b>{2,2}, but that responses to (<i>E</i>)-β-ocimene were decreased significantly only by (+)-<b>cy</b>{4,1} (1<i>R</i>,5<i>S</i>) and (−)-<b>cy</b>{2,2} (1<i>S</i>,5<i>R</i>) and not by their respective enantiomers. The importance of this result is that the racemates could be used to inhibit olfactory detection of bee odors by mites, without a loss in activity relative to the more expensive enantiopure compounds

Active space and structure-activity of host choice alteration activity.

<p><b>A</b>. Overlay of energy minimized conformers of <b>cy</b>{<i>4,1</i>} and <b>3b</b>{<i>2,2</i>}. The Connolly molecular surface of the overlaid molecules is shown in light blue. Hydrogen atoms and lone pairs have been omitted on the structures, but are included in the surfaces. Distances: a∼8.6 Å, b∼10 Å, c∼8.5 Å, d∼6.9 Å, e∼5.9 Å. <b>B</b> and <b>C</b>. Examples of the two structure-activity correlations found. <b>B</b>. Correlation between the highest occupied molecular orbital (HOMO) energy and the difference in short-term inhibition (%) between the “Bee before” and “Bee + compound” treatments (Δ STI (%)). Only the aromatic compounds (<b>3c</b> series, <b>3a</b>{<i>2,2</i>}, <b>3b</b>{<i>2,2</i>} and DEET) are included. <b>C</b>. Correlation between the polar accessible surface area (ASA_P) and the difference in long-term inhibition (%) between the “Bee before” and “Bee after” treatments (Δ LTI (%)).</p

Electrophysiological screening of the compounds.

<p><b>A</b>. Order of the <i>Varroa</i> foreleg stimulations and terminology used for the corresponding responses. The time interval between each stimulus was 30 s, unless otherwise stated. The stimuli were: Air, Headspace of five nurse bees (bee stimulus), Bee stimulus together with the compound (Bee stimulus + comp) or of the hexane control (Bee stimulus + hexane). In italics, below the stimuli, are the names of the values presented in the results. <b>B</b>. Initial screen of the <i>Varroa</i> foreleg electrophysiological response to different stimuli, all loaded at 10 µg in the stimulus cartridge (normalized values against the response to air %, average+SE). For the bee stimuli, the headspace from 5 nurse bees was used. Bars marked by different letters are significantly different, ANOVA repeated measures, p<0.05, n = 10.<b>C</b>. Testing of the individual components of the blend HCO-2169 at 10 µg doses (n = 10). <b>D</b>. Experiment with the three isomers of diethyoxybenzene at 10 µg doses (n = 6).</p

The effect of selected compounds on <i>Varroa</i> ability to reach any host.

<p>Effect of 3 selected compounds on the percentage of mites reaching any of the hosts in the choice bioassay, 180 min from the beginning of the experiment. The data are percentage of viable mites in the presence of hexane (control) or disrupting compound at each of three tested doses (0.01 µg, 0.1 µg, 10 µg) Chi-square test, ns.</p

Compounds used in this study.

<p><b>A</b>. Structures of the dialkoxybenzenes; their codenames are explained in ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106889#pone.0106889-Paduraru1" target="_blank">[17]</a>. <b>B</b>. Structures of the 5(2′-methoxyethyl) cyclopent-2-en-1-alkoxy diethers (<b>cy</b>{<i>R₁,1</i>} compounds). <b>C</b>. Synthesis of the <b>cy</b>{<i>R₁,1</i>} compounds. Abbreviations: rt =  room temperature; TBDMSCl  =  <i>tert</i>-butyl dimethylsilyl chloride; THF  =  tetrahydrofuran.</p

Dose responses of long-term inhibitory compounds cy{<i>4,1</i>}, 3b{<i>2,2</i>} and cy{<i>2,2</i>}.

<p>The responses of the <i>Varroa</i> forelegs to stimulation with different amounts of each compound and with the headspace from 5 nurse bees (normalized values against the response to air %, average+SE). Bars within each dose, marked by different letters, are significantly different, ANOVA repeated measures, p<0.05, n = 7.</p

Detailed evaluation of the long-term inhibitory effect of the most active compounds.

<p>The effect of 0.1 µg <b>cy</b>{<i>4,1</i>} (<b>A</b>) or <b>3b</b>{<i>2,2</i>} (<b>B</b>), with and without a simultaneous stimulus of the headspace volatiles from 5 nurse bees, on the electrophysiological response of the <i>Varroa</i> foreleg. The data are normalized values (%, average+SE): bars marked by different letters are significantly different, ANOVA repeated measures, p<0.05, p<0.05, n = 6. The longevity of the inhibitory effect of 0.1 µg <b>cy</b>{<i>4,1</i>} (<b>C</b>) or 0.1 µg <b>3b</b>{<i>2,2</i>} (<b>D</b>) on <i>Varroa</i> foreleg electrophysiological responses. The time interval between the mixed stimulus (Bee + compound) and the pure bee stimulus was varied. Values are normalized against the response to air (%, average+SE): bars marked by different letters are significantly different, ANOVA repeated measures, p<0.05; n = 6.</p