Design and Synthesis of Fluorophore-Tagged Disparlure Enantiomers to Study Pheromone Enantiomer Discrimination in the Pheromone-Binding Proteins from the Gypsy Moth, Lymantria Dispar.

Fluorescent analogues of the gypsy moth sex pheromone (+)-disparlure ( 1 ) and its 25 enantiomer (-)-disparlure ( ent - 1 ) were designed, synthesized and characterized. The 26 fluorescently labelled analogues 6-FAM (+)-disparlure 1a 6-FAM (-)-disparlure ent - 1a 27 were prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC) of disparlure 28 alkyne and 6-FAM azide. These fluorescent disparlure analogues 1a ent - 1a were used to 29 measure the disparlure binding to two pheromone-binding proteins from the gypsy moth, 30 Ldis PBP1 and Ldis PBP2. The fluorescence binding assay using 6-FAM disparlure 31 enantiomers 1 a and ent - 1a showed that the Ldis PBP1 and Ldis PBP2 have different binding 32 affinities with 1a and ent - 1a . The Ldis PBP1 has stronger affinity for 6-FAM (-)-disparlure 33 ent - 1a , whereas Ldis PBP2 has stronger affinity for 6-FAM (+)-disparlure 1a , consistent 34 with the findings from previous study with disparlure enantiomers. The 6-FAM disparlure 35 enantiomers appeared to be much stronger ligands for Ldis PBPs, with the binding constant 36 ( K d ) in nanomolar range, compared to the fluorescent reporter such as 1-NPN (which had 37 K d values in micromolar range). The fluorescence competitive binding assays were used to 38 determine the displacement constant ( K i ) for the disparlure enantiomers in competition 39 with fluorescent disparlure analogues binding to Ldis PBP1 and Ldis PBP2. The K i data 40 showed that disparlure enantiomers can effectively displace the fluorescent disparlure from 41 the binding pocket of Ldis PBPs. disparlure enantiomers, enantioselective synthesis, cis epoxide, Fluorescence binding assays, click reaction by slow addition of enantiopure α -chloroaldehyde 22 to afford 1,2- anti chlorohydrin 24 . 1 H NMR data of the crude reaction mixture revealed a diastereomeric 559 ratio (d.r) of ~ 20:1 ( anti : syn ). The crude reaction mixture was subjected to column 560 chromatography yielding the 1,2- anti chlorohydrin 24 as pale-yellow oil in 68% yield.


Introduction
are highly conserved among Lepidoptera PBPs: Phe12, Phe36, Phe76, Phe119 (PBP1) and 75 Phe120 (PBP2). These residues interact with the hydrophobic region (hydrocarbon chains) 76 of the ligands when the PBP is in the B form (Sandler et al. 2000;Honson et al. 2003; 77 Sanes and Plettner 2016). Furthermore, the binding site residues that vary between 78 LdisPBPs were found to be: Asn35, Ala73, Leu91, and Ala135 in LdisPBP1, whereas in 79 LdisPBP2 these residues were substituted with Asp35, Thr73, Ile91 and Leu136 (Sanes at its internal binding site very slowly, whereas PBP1 has much faster association and 87 dissociation kinetics (Gong et al. 2009;2010). We have shown that in PBP2, the 88 enantiomers of disparlure differ in the rate at which they bind to the internal binding site 89 (Gong et al. 2009). However, due to their higher rates, the enantioselectivities of the 90 external binding events in both PBPs and of the internalization of ligands in PBP1 have not 91 yet been studied. To understand the mechanism by which PBPs recognize different ligands, 92 it is necessary to study the kinetics of the association and dissociation processes.  Ligand-binding experiments between pheromone binding proteins (PBPs) and 97 hydrophobic ligands (e.g. pheromones) can be performed in two general ways: 1) the ligand 98 and protein are equilibrated in buffer, and the protein-bound ligand is then separated from 99 the free ligand using filtration  or 2) the protein is titrated with a  (Ban et al. 2002). 109 However, this approach needs the availability of a fluorescent reporter equipped with good 110 binding strength for the PBP under study, whose fluorescence emission is significantly 111 increased when the reporter binds inside the PBP's binding pocket. 112 The disadvantage of the use of fluorescent reporters in ligand-binding assays is that 113 various compounds differ in their ability to displace the reporter (due to kinetic factors and 114 incomplete equilibration between aliquot additions during these experiments), rather than pheromones. 126 We have covalently linked 6-carboxyfluorescein (6-FAM) to the gypsy moth sex 127 pheromone (+)-disparlure (1) and to its enantiomer (-)-disparlure (ent-1) (Fig. 1) by adding 128 a linker with a terminal alkyne moiety to disparlure and performing a click reaction of the 129 alkyne and 6-carboxyfluorescein azide (6-FAM azide). We choose 6-carboxyfluorescein 130 (6-FAM) as a fluorescent reporter because of its high fluorescence quantum yield (0.93), 131 good water solubility and its derivatives, such as 6-FAM azide, are commercially available.

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In addition to the high quantum yield, it has excellent absorption and emission properties 133 (Sjöback et al. 1995;Zhang et al. 2014). This is the first report describing the synthesis of 134 a fluorophore-tagged insect pheromone. We expect that these fluorophore-tagged 135 pheromones will provide researchers with a viable alternative to the radiolabeled 136 pheromones and fluorescent probes such as 1-NPN that are used in PBP-pheromone 137 binding assays. To date, the use of fluorophore tagged pheromones as a fluorescent reporter 138 in the study of pheromone binding protein interactions has not been reported.

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In this paper, we report the synthesis (Scheme 1) and spectroscopic characterization 140 of fluorophore-tagged disparlure enantiomers 6FAM (+)-disparlure (1a) and 6FAM (-)-141 disparlure (ent-1a) (Fig. 1), and their binding affinities to two pheromone binding proteins  The 1 H NMR spectra were obtained on Bruker DRX 400 and 500 MHz 166 spectrometers in CDCl3. Chemical shifts and coupling constants were reported in parts per 167 million (ppm) and hertz (Hz) respectively. 1 H NMR data was reported as follows: chemical 168 shift values (ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = 169 multiplet). 13 C NMR spectra were recorded in CDCl3 by using a Bruker DRX 400 MHz or 170 DRX 500 MHz. 13 C NMR data are reported as chemical shift values (ppm). IR spectra were 171 obtained with a Perkin-Elmer Spectrum One FT-IR spectrometer and samples were directly 172 placed on the KBr plates. High-resolution mass spectra (HRMS) were obtained by using     MHz, CDCl3) δ: 3.66-3.62 (t, J = 6.6 Hz, 2H), 3.61-3.57 (t, J = 6.6 Hz, 2H), 1.60-1.48 (m,    The procedure was the same for all three compounds; the one for 21 is given in detail. To  1.517 mmol) and Na2S2O8 (2.16 g, 9.103 mmol). The reaction mixture was stirred at 5 o C 258 until dodecanal had been completely consumed (as determined by 1 H NMR spectroscopy).

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After this time, the t-BuOH was removed, and the resulting residue was diluted with water 412 and dichloromethane. The aqueous layer was extracted with 10% MeOH/DCM (4×10 mL).

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The pooled organic layers were washed with saturated sodium chloride solution, dried over   in Scheme S1 (supporting information).  Table 2).