Chemical Senses Vol. 29 No. 9 © Oxford University Press 2004; all rights reserved
An Odorant Derivative as an Antagonist for an Olfactory Receptor
1 Department of Integrated Biosciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan, 2 Technical Research Center, T. Hasegawa Co., Ltd, 335 Kariyado, Nakahara-ku, Kawasaki 211-0022, Japan and 3 Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Correspondence to be sent to: Kazushige Touhara, Department of Integrated Biosciences, Rm201, The University of Tokyo, Kashiwa, Chiba 277-8562 Japan. e-mail: touhara{at}k.u-tokyo.ac.jp
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Abstract |
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Different odorants are recognized by different combinations of G protein-coupled olfactory receptors, and thereby, odor identity is determined by a combinatorial receptor code for each odorant. We recently demonstrated that odorants appeared to compete for receptor sites to act as an agonist or an antagonist. Therefore, in natural circumstances where we always perceive a mixture of various odorants, olfactory receptor antagonism between odorants may result in a receptor code for the mixture that cannot be predicted from the codes for its individual components. Here we show that stored isoeugenol has an antagonistic effect on a mouse olfactory receptor, mOR-EG. However, freshly purified isoeugenol did not have an inhibitory effect. Instead, an isoeugenol derivative produced during storage turned out to be a potent competitive antagonist of mOR-EG. Structural analysis revealed that this derivative is an oxidatively dimerized isoeugenol that naturally occurs by oxidative reaction. The current study indicates that as odorants age, they decompose or react with other odorants, which in turn affects responsiveness of an olfactory receptor(s).
Key words: antagonist, isoeugenol, odorant, olfactory receptor
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The detection of thousands of volatile odorous compounds is mediated by the G protein-coupled olfactory receptor (OR) superfamily, which are expressed by olfactory neurons in the olfactory epithelium (Buck, 1996
; Firestein, 2001; Touhara, 2002; Mombaerts, 2004). Functional studies of ORs have demonstrated that individual odorants are recognized by multiple ORs, whereas an OR can recognize multiple odorants. Therefore, odorant identities are determined by a combinatorial receptor code wherein different odorants are recognized by specific sets of ORs (Malnic et al., 1999; Touhara et al., 1999; Kajiya et al., 2001). Structure–activity relationship studies suggest that the ligand binding site of ORs can discriminate subtle differences in chemical structures of odorants, but also tolerate large changes in other molecular features (Zhao et al., 1998; Touhara et al., 1999; Wetzel et al., 1999; Araneda et al., 2000; Kajiya et al., 2001; Gaillard et al., 2002).
In natural circumstances, we always perceive a mixture of various odorants. According to the combinatorial receptor code hypothesis, a receptor code for an odorant mixture is expected to be the simple sum of codes for its components. However, psychophysical experiments have demonstrated that the perceived intensity of an odorant mixture tends to be smaller than the sum of the intensities of its components, a process called counteraction (i.e. reduction of odor intensity) or masking (i.e. modification of perceived odor) (Arctander, 1960; Cain and Drexler, 1974). The recent discovery of antagonism between odorants at the receptor level has revealed a molecular mechanism for odorant mixture counteraction (Oka et al., 2004). Thus, odorants not only stimulate some ORs, but they also inhibit or antagonize other ORs (Araneda et al., 2000; Spehr et al., 2003; Oka et al., 2004). In other words, individual odorants appear to have dual functions as OR agonists and antagonists, thus competing with each other at odorant binding sites in the olfactory epithelium.
The mOR-EG, a mouse OR encoded by the MOR174-9 gene (Zhang and Firestein, 2002) has been shown to recognize eugenol (EG), a main odorous compound in clove oil, and other structurally similar odorants (Kajiya et al., 2001). We recently showed that methyl isoeugenol and isosafrol inhibited EG-induced Ca2+ responses of mOR-EG as competitive antagonists in HEK293 cells and in olfactory receptor neurons (Oka et al., 2004). In addition, during the course of screening for antagonists, we found that isoeugenol inhibited EG responses in mOR-EG-expressing HEK293 cells. Freshly purified isoeugenol, however, did not show an inhibitory effect, which let us to speculate that a compound(s) produced in isoeugenol during storage was an antagonist of mOR-EG. Herein we report that this naturally occurring compound is an oxidatively dimerized isoeugenol (OD-IEG) produced from isoeugenol and that it is a potent competitive antagonist for mOR-EG. The current study also points out an important caveat in olfactory research using odorous chemicals: a small amount of an impurity or derivative may have drastic effects on the odorant–OR interaction.
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Materials and methods |
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Odorant compounds
Odorants utilized in this study were kindly provided by T. Hasegawa Co., Ltd or were purchased from Wako Co., Ltd (Tokyo, Japan) or Sigma-Aldrich (Tokyo, Japan). Odorants were kept at 4°C and their purities were checked by thin-layer chromatography (TLC) before experiments. Stored isoeugenol was utilized after storing pure isoeugenol for 
3 years at 4°C. Odorants were diluted to the indicated concentrations in Ringer’s solution (140 mM NaCl, 5.6 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 2 mM sodium pyruvate, 9.4 mM glucose and 10 mM HEPES, pH 7.4).
TLC analysis
Silica-based TLC plates (0.25 mm thickness) used in this study were purchased from Merck Ltd, Tokyo, Japan. Odorant compounds were developed in ethyl acetate:hexane (1:1) and were detected by reaction with phosphomolibdic acid.
Preparation of OD-IEG and guaiacyl isoeugenol
To obtain large amounts of OD-IEG, pure isoeugenol was stirred in flask under an atmosphere of 100% O2. Stirring for 24 h resulted in 10–20% conversion to OD-IEG. The sample was separated by preparative TLC as described for TLC analysis. The OD-IEG was purified by collecting the silica layer of the desired band and extracting it with ethyl acetate. To obtain guaiacyl isoeugenol, equal amounts of guaiacol and isoeugenol were mixed and stirred overnight, and the product was purified in the same way as for OD-IEG. The structure of the compounds was determined by nuclear magnetic resonance (NMR) and liquid chromatography–mass spectrometry (LC–MS).
Ca2+-imaging of HEK293 cells
Ca2+-imaging for mOR-EG-expressing HEK293 cells was performed essentially as described previously (Katada et al., 2003). Briefly, 60–70% confluent HEK293 cells were transfected with 2.0 µg of pME18S-tagged mOR-EG and 1.5 µg of pME18S-G
15 with 2.5 µl lipofectamine 2000/µg DNA (Invitrogen, Tokyo, Japan). Twenty-four to forty-eight hours after transfection, the transfected cells were loaded with 4 µM fura 2-AM for 30 min at 37°C. Odorant solutions were sequentially applied to the cells using a peristaltic pump at a flow rate of 1.5 ml/min for 15 s with a 2.5 min wash with Ringer’s solution between stimulus applications. Intracellular Ca2+ levels were monitored using an AQUA COSMOS Ca2+-imaging system (Hamamatsu Photonics, Shizuoka, Japan). Antagonist screening was performed essentially as described previously (Oka et al., 2004). Briefly, when inhibitory odorants were screened (Figure 1), each odorant (1 mM) was co-applied with EG (100 µM). To perform quantitative pharmacological studies (Figure 3), an antagonist was applied 10 s prior to and during the application of EG (black bar in Figure 3a).
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NMR analysis of OD-IEG
OD-IEG purified by a preparative TLC was dissolved in CDCl3. NMR spectra were acquired at 25°C with a Varian Mercury 300 (1H 300 MHz; Varian, Palo Alto, CA). The chemical shifts were shown on the i-scale in p.p.m. The 1H-NMR spectrum of OD-IEG: 
(300 MHz, CDCl3) 1.01 (3H, dd, J = 3.0, 3.3 Hz), 1.84 (3H, dd, J = 1.5, 5.1 Hz), 3.87 (3H, d, J = 2.1 Hz), 3.90 (3H, d, J = 2.1 Hz), 3.95 (1H, s), 4.06 (1H, dq, J = 6.3, 2.1 Hz), 4.42 (1H, d, J = 8.4 Hz), 5.70 (1H, s), 6.06 (1H, dq, J = 6.6, 9.0 Hz), 6.27 (1H, dd, J = 1.5, 14.1 Hz), 6.58 (1H, d, J = 8.4 Hz), 6.65 (1H, dd, J = 1.8, 6.3 Hz), and 6.80 to 6.91 (4H, m).
LC–MS analysis
The HPLC system consisted of model 1100 pumps, a tunable absorbance detector (Agilent Technologies, Palo Alto, CA) and an XterraTM MS C18 column (C18, 3.0 x 100 mm; 3.5µm; Waters, Milford, MA). A 5–20 µl sample was injected and eluted with a gradient of H2O (A) and methanol (B) at room temperature and at a flow rate of 0.5 ml/min using the following program: t = 0 min, 20% B; t = 8 min, 100% B; t = 8–11.4 min, 100% B. MS was performed in the ESI positive ion detection mode using a Thermo Electron LCQ ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA). The ionization source temperature was 270°C and the ionization voltage was 4.5 kV.
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Results |
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Screening of an odorant that inhibits mOR-EG activity
Stimulation of mOR-EG-expressing HEK293 cells with its cognate ligand, EG, elicits an increase in intracellular Ca2+ level via G15 coupling to the mOR-EG, followed by activation of an inositol phosphate-mediated signaling cascade (Kajiya et al., 2001; Katada et al., 2003). Using this assay system, we previously identified methyl isoeugenol and isosafrol as antagonists of mOR-EG (Oka et al., 2004). In addition to these compounds, further screening revealed that stored isoeugenol (see Materials and methods) significantly inhibited the response of mOR-EG in HEK293 cells to EG (Figure 1a, upper panel). The average response in the presence of both EG and stored isoeugenol (1:10 ratio) was 16.0 ± 3.3% (mean ± SE; n = 8) of that elicited by EG alone. However, when the same amount of column-purified isoeugenol (pure isoeugenol) was utilized in the experiments, no inhibition was observed (Figure 1a, lower panel). Thus, the average response in the presence of pure isoeugenol was 97.7 ± 3.3% (n = 8) of that elicited by EG alone. Furthermore, although pure isoeugenol is a colorless oil, it gradually becomes yellow and viscous during storage. Thus, we speculated that an isoeugenol derivative(s) produced during storage was responsible for the inhibition activity on mOR-EG.
To examine this possibility, we used TLC to analyze the purity of stored isoeugenol. In addition to isoeugenol (Figure 1b, lane 2), there was one major and some other minor compounds in the stored sample (Figure 1b, lane 1). We purified the major product (arrowhead in Figure 1b) by preparative TLC (Figure 1b, lane 4) and we found that it significantly inhibited EG responses, whereas the other compounds in the stored isoeugenol sample (Figure 1b, lanes 3 and 5) had little effect (Figure 1c). These results suggested that a derivative in the isoeugenol sample that was produced during storage acted as a mOR-EG antagonist.
Structural determination of the antagonist in stored isoeugenol
We carried out structural analysis of the compound that exhibited an inhibitory activity on mOR-EG by NMR and LC–MS. The NMR spectrum of the compound suggested that it possessed two isoeugenol moieties (Figure 2a). LC analysis showed that isoeugenol eluted at retention time of 7.83 min (Figure 2b, left upper), whereas the major product (arrowhead in Figure 1b, lane 4) was found at 8.32 min (Figure 2b, left bottom). The stored isoeugenol contained a significant amount of this compound (10%; Figure 2b, left middle). The product ion mass spectrum of the sample eluting at 8.32 min had a base peak corresponding to (M+Na)+ at m/z 367 (Figure 2b, right), which was consistent with the mol. wt calculated for the structure deduced by NMR analysis. Because this compound (4-(2-hydroxy-1-[2-methoxy-4-(prop-1-en-1-yl)phenoxy] propyl)-2-methoxyphenol) has not previously been reported, we have named it oxidatively dimerized isoeugenol (OD-IEG) for convenience.
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OD-IEG appears to be produced via oxygen-assisted reactions during storage. Stirring of pure isoeugenol for 24 h in the presence of 100% oxygen resulted in
10–20% conversion to OD-IEG, suggesting that this compound was easily generated by exposure to air. Therefore, this compound was likely produced via the reactions shown in Figure 2c. This intermolecular reaction of isoeugenol appears to be accelerated in the presence of oxygen. Antagonistic activity of OD-IEG on mOR-EG
To determine whether the reduction of Ca2+ responses was derived from competitive inhibition, we examined the effects of various concentrations of OD-IEG on EG-induced responses of HEK293 cells expressing mOR-EG (Figure 3a). OD-IEG was prepared from isoeugenol by stirring overnight in the presence of oxygen and was purified by preparative TLC. The purified OD-IEG was then utilized immediately for experiments. In HEK293 cells expressing mOR-EG and G15, the Ca2+-responses elicited by EG were inhibited by OD-IEG in a dose-dependent manner with an IC50 of 10 µM (Figure 3b). It should be of note that the IC50 value was much lower than for methyl isoeugenol (66 µM; see Oka et al., 2004), which had previously been the most potent antagonist of mOR-EG. A Ca2+-response was not observed when OD-IEG was applied alone, even at 1 mM, suggesting that OD-IEG was not an agonist or a partial agonist of mOR-EG (data not shown). In addition, the Ca2+-responses elicited by isoproterenol via endogenous ß-adrenergic receptors were not affected by OD-IEG, excluding the possibility that the inhibitory activity was due to non-specific effects on signaling downstream of G15 (data not shown). These results demonstrated that OD-IEG is a potent antagonist of the mOR-EG and that it inhibits the response to EG by competing at the receptor ligand binding site.
Identification of OD-IEG in natural fragrant oils
Although OD-IEG has not previously been reported as a natural compound, our results indicated that it might exist in natural fragrant oils containing isoeugenol. Clove oil is known to contain 1% isoeugenol, although the content depends on its origin. We separated clove oil by preparative TLC and extracted the compounds that migrated around the same Rf value as OD-IEG. LC analysis showed a peak that eluted at 8.28 min and that contained a compound with an m/z = 367 on MS, demonstrating that OD-IEG is present in clove oil (Figure 4a). We also found that mace oil, which is made up of 0.6% IEG, contained OD-IEG. MS/MS analysis confirmed that the m/z 367 compounds in clove and mace oil were identical to OD-IEG (Figure 4b). These results indicate that OD-IEG is naturally produced in fragrant oils that contain a modest amount of isoeugenol.
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Discussion |
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In the current studies, we identified a novel, naturally occurring compound that is oxidatively produced by the intermolecular reaction of two isoeugenol molecules. This compound, OD-IEG, is a potent antagonist for the mouse olfactory receptor mOR-EG. This is the first evidence of a natural derivative of an odorant that affects the responsiveness of a particular OR. Our results also reveal a caveat in experiments using odorous chemicals: a small amount of an impurity or a derivative may cause drastic effects on the odorant-OR interaction.
OD-IEG appears to be a novel natural compound that had not previously been identified. OD-IEG was probably overlooked in earlier studies because it is easily pyrolysed in a GC column at high temperature, producing isoeugenol, vanillin and 4-hydroxy-3-methoxy phenylacetate (data not shown). Exposure to oxygen accelerates the coupling reaction of isoeugenol, suggesting that the generation of OD-IEG in the stored isoeugenol sample proceeds via an oxygen-assisted reaction. Isoeugenol appears to be highly reactive with alcohols at low temperature in the presence of oxygen due to the formation of a peroxide transition state. Indeed, when isoeugenol and guaiacol were mixed under 100% oxygen, the isoeugenol peroxide reacted with an alcohol group of guaiacol, resulting in the coupling product of isoeugenol and guaiacol, which we named guaiacyl isoeugenol (structure shown in Figure 5:2). It appears that OD-IEG was detected in natural fragrant oils, such as clove and mace oils, because isoeugenol is reactive in natural conditions.
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Because the mol. wt of OD-IEG (344) is near the upper limit of odorous compounds, OD-IEG may not be volatile. Therefore, in physiological conditions, it may not enter the nasal cavity to antagonize mOR-EG-expressing neurons. Thus, our findings may not be physiologically relevant; however, they clearly illustrate the following: (i) ORs can be a target of non-volatile molecules that are present in the mucus, thereby affecting olfactory sensitivity or perception and (ii) it is conceivable that, after odorants activate an OR repertoire specific to each odorant, they are easily degraded or converted in the mucus into forms that act as agonists or antagonists of other ORs.
Because the IC50 value of OD-IEG for mOR-EG was 10 µM, a concentration of 30–100 µM was high enough to completely inhibit the response of the mOR-EG to EG. Given that the old isoeugenol contained 10% of OD-IEG, it was a reasonable that it showed antagonistic activity at 1 mM in the initial antagonist screening. This implies that contaminants or decomposition products often present in commercially available compounds may greatly affect the results of odorant–OR experiments. Indeed, it is widely known that most odorants undergo decomposition, resulting in the generation of many derivatives (Arctander, 1960). For example, limonene and linalool, the most frequently incorporated fragrance chemicals in scented products, are easily oxidized in air during handling or storage (Skold et al., 2002; Matura et al., 2003). Most aldehyde compounds are also degradable unless they are handled properly. Therefore, it is important that odorants are stored at low temperature under argon without oxygen and that the purity is checked prior to use.
Several studies on odorant-OR interaction have revealed that an individual OR recognizes multiple structurally-related compounds with distinct ligand specificity (Touhara et al., 1999; Araneda et al., 2000; Kajiya et al., 2001; Spehr et al., 2003). Identification of multiple antagonists for mOR-EG enabled us to further investigate the structure–activity relationship of an OR. Figure 5 shows a structure–activity matrix for odorants based on the EG structure that have different functional groups at three positions on the benzene ring. Agonists for mOR-EG (35, 1214: gray) possess an allyl or aldehyde group at the R1 position, whereas a propenyl group was always found at this position in antagonists (8, 10, 11: dark gray). This suggests that the R1 position is a major factor in determining whether an odorant is an agonist or antagonist for mOR-EG. Consistent with this rule, guaiacyl isoeugenol (Figure 5: 2), which has the same structure as OD-IEG except that it lacks the propenyl group, was not an antagonist (data not shown). In contrast, a subtle difference at the R3 or R4 positions leads to a change in agonist and antagonist potency, indicating that these positions are determinants of ligand specificity for mOR-EG. For example, ethyl isoeugenol (9) was neither an antagonist nor an agonist, suggesting that ORs can discriminate small differences in an odorant molecule. This information will be of great help in the analysis of the mode of agonist and antagonist binding to an OR and the molecular dynamics involved in transition of an OR from an inactive to an active state.
Odor perception appears to be regulated by complex interactions between odorants and ORs that are based on a combinatorial coding strategy and antagonism between odorants. We identified a novel, naturally occurring compound that was oxidatively produced from isoeugenol and that was a potent antagonist for an OR. Thus, degradation or conversion of an odorant can affect odorant recognition by ORs. Furthermore, we identified a previously unrecognized chemical reaction between odorants that occurs under natural conditions that must be considered when examining molecular recognition in olfaction. These studies demonstrate that odorant recognition by ORs and subsequent sequestration of odorant compounds in the olfactory mucus, where the components are largely unknown, might involve chemistry much more complex than previously imagined.
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We thank T. Wada, T. Kawanaka and their colleagues for helping with NMR analysis and technical advice and H. Kataoka for support. Special thanks are due to people in T. Hasegawa Co., Ltd for helpful discussion. This work was supported in part by grants from the Japan Society for the Promotion of Science (JSPS) and the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) Japan. K.T. is recipient of grants from Uehara Memorial Foundation, Kato Memorial Bioscience Foundation, The Naito Foundation and The Mochida Memorial Foundation.
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Accepted September 27, 2004
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