Letters pubs.acs.org/acschemicalbiology
Synthetic versus Natural Receptors: Supramolecular Control of Chemical Sensing in Fish José P. Da Silva,*,† Rajib Choudhury,‡ Mintu Porel,‡ Uwe Pischel,§ Steffen Jockusch,∥ Peter C. Hubbard,*,⊥ Vaidhyanathan Ramamurthy,‡ and Adelino V. M. Canário⊥ †
CIQA, Faculdade de Ciências e Tecnologia and ⊥CCMAR, Universidade do Algarve, Campus de Gambelas 8005-139 Faro Portugal ‡ Department of Chemistry, University of Miami, Coral Gables, Florida 33124, United States § CIQSO-Centro de Investigación en Química Sostenible and Departamento de Ingeniería Química, Química Física y Química Orgánica, Universidad de Huelva, Campus de El Carmen s/n, E-21071 Huelva, Spain ∥ Department of Chemistry, Columbia University, New York, New York 10027, United States S Supporting Information *
ABSTRACT: The encapsulation of odorants by the synthetic receptor cucurbit[7]uril (CB[7]) reduces the response of olfactory receptors in Mozambique tilapia (Oreochromis mossambicus) in vivo. For example, the olfactory receptor response to the odorant adamantan-1-amine, as measured by electro-olfactography, was suppressed by 92% in the presence of CB[7]. A reduction in olfactory response of 88% was observed for pentane-1,5-diamine (cadaverine), an odorant associated with carrion avoidance in some fish. The results reveal how the association constants and the concentrations of natural and synthetic receptors play a determinant role and show that synthetic receptors can be used to remove bioactive molecules from fish olfaction.
A
have been used to compete with enzymes for their inhibitors,13 to change the effect of polyamines in DNA function,15 and to reverse the effect of neuromuscular blocking agents in vivo.3 Among members of the CB[n] macrocycle family, CB[7] shows the highest solubility in water (20−30 mM)6 and displays association constants with aliphatic and aromatic amines ranging from 105 to 1017 M−1 (Scheme 1).6,8−14 The anatomical accessibility of the fish olfactory epithelium makes the G-protein coupled odorant receptors contained therein a readily accessible class of natural receptors for in vivo studies.16 In fish, olfaction takes place entirely in aqueous solution and allows the detection of a variety of water-soluble odorants, which evoke different types of physiological and behavioral responses, such as food location and identification,17 decaying flesh avoidance,18 social communication,19 and reproductive activity.20,21 Odor perception starts with the binding of the odorant molecules to their receptors in sensory neurons in the olfactory epithelium. This initiates a cascade of transduction events that culminate in the generation of action potentials leading to odor perception in the brain.22 Odorant detection by fish can be followed in vivo by monitoring the negative field potential generated over the epithelia surface upon binding of the odorant molecules to the receptor and subsequent cellular transduction pathways, which results in an
major goal of supramolecular chemistry is the creation of receptor molecules that mimic the natural ligand− receptor interaction.1,2 Water solubility as well as high selectivity and affinity under physiological conditions are desirable properties of synthetic receptors. Several classes of effective water-soluble synthetic receptors have been developed, ranging from acyclic flexible structures3 to preorganized macrocycles such as cyclodextrins4 and cucurbit[n]urils (CB[n]).5,6 Natural cell-surface receptors, such as G-protein coupled receptors, upon ligand (neurotransmitters, hormones, or odorants) binding trigger intracellular molecular signals leading to specific cellular, physiological, and behavioral responses. Their binding sites offer a precise stereochemistry and exhibit efficient and specific recognition processes that, however, are achieved at the expense of high molecular weight and chemical complexity.7 Synthetic receptors with high association constants, capable of sequestering biologically relevant molecules under physiological conditions, could be a useful tool for studying their cellular function. However, while drug−receptor interactions show association constants in the range of 106−109 M−1, with the exception of some CB[7]−amine pairs,8 most synthetic receptors show association constants on the order of 103 M−1.9 Cucurbituril-type receptors are an exceptional family of synthetic receptors showing association constants higher than 106 M−1 for a large variety of molecules.5,6,9−14 Because of their high association constants some cucurbituril-type receptors © XXXX American Chemical Society
Received: March 5, 2014 Accepted: May 19, 2014
A
dx.doi.org/10.1021/cb500172u | ACS Chem. Biol. XXXX, XXX, XXX−XXX
ACS Chemical Biology
Letters
Scheme 1. Chemical Structures of Investigated Synthetic Receptors (CB[6] and CB[7]) and Odorants (Amines 1−8)
influx of calcium and sodium ions.22 The recorded potential over time after introduction of a pulse of odorant molecules in vivo is known as the electro-olfactogram (EOG) and represents the summated generator potential in activated odorant receptor neurons.23 The competition between the odorant receptors of fish and synthetic receptors can, therefore, be examined through electrophysiological experiments by using well characterized aqueous solutions of synthetic receptors. Here we report the first in vivo study of the competition between synthetic cucurbituril receptors and fish olfactory receptors (Mozambique tilapia, Oreochromis mossambicus) for amines (Scheme 1), which are odorants of some teleost fish.17,18 Our results suggest the use of high affinity binders, such as CB[7] and CB[6], as potential tools to remove odorants or other signaling molecules from fish olfactory receptors under physiological conditions. It is well-known that supramolecular interactions between CB[n] macrocycles and guest molecules depend delicately on parameters such as pH and ionic strength.6,12,24 For this reason the binding of amines 1−8 by CB[7] under physiological conditions (artificial fresh water; 100 μM of CaCl2, NaCl, and NaHCO3) was re-evaluated and confirmed by 1H NMR spectroscopy, electrospray ionization-mass spectrometry (ESIMS), and isothermal titration calorimetry (ITC); see Supporting Information, Figures S1−26. The association constants KB (Table 1) for amines 1−8, which are protonated under physiological conditions, were found to be of the same order or higher than the ones reported in the literature.6,10−12 The differences were ascribed to the lower ionic strength of our
solutions compared to the buffer solutions used in preceding studies12,24 (Table S1, Supporting Information). Having established the association strength between cucurbituril receptors and amines 1−8 under physiological conditions (protonated amines), we proceeded to evaluate the binding interactions between these odorants and olfactory receptors in the tilapia fish. Amines 1−8 are odorants of some fish species17,18 but have never been tested in tilapia. We followed the successful odorant-natural receptor binding by EOG (Figure 1) and used it to measure the detection thresholds, the independency of receptors for distinct amines, and the response−concentration curves.
Figure 1. Electro-olfactogram (EOG) setup and typical EOG traces. (a) Detail of the EOG setup scheme showing the localization of the recording and reference electrodes and of the stimulus tube (see Figure S30, Supporting Information, for a more detailed scheme of the experimental setup). (b) EOG traces for odorant 1 when free in solution (5 μM, blue trace) and in the presence of CB[7] (5 μM, 1:1, red trace).
Table 1. Odorant−CB[7] Association Constants (KB), Detection Thresholds of Olfaction, and Normalized Olfactory Responses of Odorants in the Presence of CB[7], (SCB[7]/S0) × 100
1 2 3 4 5 6 7 8
threshold of detection (M)
KB (M−1)
odorant 1.5 9.9 3.2 2.2 3.7 1.3
>108 (±0.3) × (±0.2) × (±0.1) × (±0.2) × (±0.1) × (±0.2) ×
