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A Two-Input Fluorescent Logic Gate for Glutamate and Zinc Caixia Yin, Fangjun Huo, Nicholas P. Cooley, David Spencer, Kyle Bartholomew, Charles L. Barnes, and Timothy Edward Glass ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00420 • Publication Date (Web): 03 Mar 2017 Downloaded from http://pubs.acs.org on March 5, 2017
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A Two-Input Fluorescent Logic Gate for Glutamate and Zinc Caixia Yin,*† Fangjun Huo,† Nicholas P. Cooley, ‡ David Spencer,‡ Kyle Bartholomew,‡ Charles L. Barnes,‡ Timothy E. Glass*‡ †
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China. ‡ Department of Chemistry, University of Missouri, 601 S. College Ave., Columbia, MO 65203
[email protected],
[email protected] Supporting Information Placeholder CO 2O Et 2N
N
N
Glu,
Zn2+ Et 2N
N N
N
Zn2+
O O-
2-input AND glu Zn2+
fluor
ABSTRACT: The direct visualization of neurotransmitters is a continuing problem in neuroscience, however, functional fluorescent sensors for organic analytes are still rare. Herein, we describe a fluorescent sensor for glutamate and zinc ion. The sensor acts as a fluorescent logic gate, giving a turn-off response to glutamate or zinc ion alone. The combination of analytes produces a large increase in fluorescence. These types of sensors will aid in the study of neurotransmission; in this case for neurons which copackage high concentrations of zinc and glutamate. Keywords: Fluorescent sensor, logic gate, neurotransmitter, glutamate, zinc
Fluorescence imaging has become a premiere method for unraveling complex biological processing due to easy access to imaging systems and an explosion in the availability of fluorescent proteins, probes, and tags. Although fluorescent sensors have made great advances in recent years, selectivity remains a major challenge in this area given the complexity of the biological medium. One good method to prepare more selective sensors is to have them be sensitive to more than one condition or analyte. While this method requires a more complex sensor design, the resulting sensors will, in principle, be much more selective because activation by multiple analytes is required. These types of sensors can be classified as logic gates, which have been developed over the years for various applications.1,2 ,3 Indeed, there are many fluorescent logic gates in the literature that respond to a variety of inputs, producing a signal based on some type of Boolean logic operation (e.g., AND, OR, NOR, etc.).4,5 We have recently been interested in the preparation of fluorescent sensors for neurotransmitters. 6 Such sensors can be used to visualize neurotransmitters in cells by fluorescence microscopy providing access to experiments that explore the mechanisms of neurotransmission. 7 Currently, many fluorescent probes are used in neurobiology, including sensors for zinc and calcium ions.8,9,10 In addition, neurosecretory vesicles can be fluorescently labeled by probes that stain the vesicle membrane11 or that can be actively loaded into vesicles (i.e., false fluorescent neurotransmitters).12 However, none of these methods actually label the vesicle cargo – the neurotransmitters. We have been interested in the neurotransmitter gluta-
mate, the most abundant neurotransmitter in the brain.13 Glutamate is heavily involved in the processes of memory and thought.10 In particular, there are glutamatergic neurons in the forebrain containing high concentrations of both glutamate and zinc.14 To address the issue of directly imaging neurotransmitters, including glutamate, we have been investigating a series of sensors based on a coumarin-aldehyde scaffold that detect neurotransmitters by formation of a fluorescent iminium ion. Initially, we developed a simple derivative, NS521 (Figure 1a), which had some selectivity for catecholamines such as norepinephrine.15 This sensor can be thought of as a simple 1input logic gate. We then reported ES517, a fluorescent logic gate for amines and the pH jump associated with exocytosis.16 In this case, the sensor has an aldehyde to react with amines as well as an acid group (in green) to respond to pH changes. This sensor can be used to image active neurotransmitter as it is ejected into a synapse. Finally, we developed sensor 1, a 3input logic gate for glutamate (in red), zinc ion (in blue) and pH change (in green) to image active neurotransmitter for synapses using zinc ion and glutamate together.17 Herein, we report sensor 2 (Figure 1b) which is a 2-input logic gate for Zn2+ and glutamate only. Such a sensor can detect static Zn2+ and neurotransmitter in resting cells and would be an advance over sensors that image only zinc,18 because this 2-input logic gate would have negligible affinity for zinc in the absence of high concentrations of glutamate. Thus the sensor would only respond to Zn2+ and glutamate in a secretory vesicle, providing for very low background signal.
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fluor
∆ pH
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O O S N H
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O O
O
O
HO
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ES517
O N
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Glu, Zn2+ Et2N
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CO2b) Et2N
sponse of the sensor was also tested at pH 5.5 and a very similar response was observed (see the supporting information). The quantum yield of the sensor was calculated to be 0.016 at pH 7.4. Upon binding glutamate and Zn2+, the quantum yield increases to 0.023.
fluor
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2-input AND Glu Zn2+
fluor
2
Figure 1. a) Logic gates for neuronal imaging. b) Sensor 2 binding with Glu/Zn2+. Nor = norepinephrine, Glu = glutamate, fluor = fluorescence output.
Sensor 2 is based on sensor 1, except it uses a pHindependent donor group (diethylamine) at the 7-position and a benzimidazole has been integrated into the system to provide higher affinity for zinc ion, once glutamate has bound. b)
Scheme 1. Synthesis of Sensor 2.
c)
Sensor 2 was synthesized as shown in Scheme 1. The main transformations include a Knoevenagel condensation of intermediate 4 followed by an Ullmann coupling to give the core fluorophore. Selective reduction produced sensor 2, which was characterized by NMR and X-ray crystallography. We further characterized the sensor via titrations with glutamate and zinc. Only small changes in the absorption spectrum were observed when sensor 2 was titrated with Zn2+ or glutamate, likely due to very weak binding of either analyte individually. In fluorescence mode, the sensor showed a small decrease in fluorescence when Zn2+ or glutamate were added alone (Figure 2). However, when Zn2+ was added to sensor 2 in the presence of glutamate, a 4.5-fold increase in fluorescence was observed. Because the interior of secretory vesicles is acidic, the re-
Figure 2. Fluorescence titration of a) sensor 2 (7 µM) with glutamate (7.5- 52.5 mM) in buffer (50 mM HEPES, 120 mM NaCl, pH 7.4, 0.35% methanol), λex= 440 nm; b) sensor 2 (7 µM) in buffer (50 mM HEPES, 120 mM NaCl, pH 7.4, 0.35% methanol), λex= 440 nm) with zinc acetate (0.05mM- 0.35 mM) and c) sensor 2 (7 µM) including 500 mM glutamate in buffer (50 mM HEPES, 120 mM NaCl, pH 7.4, 0.35% methanol), λex= 440 nm) with zinc acetate (8.25- 82.5 µM). Inset is the fit to a one-site binding isotherm along with cuvette samples irradiated with 365 nm light.
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To demonstrate that the fluorescence increase resulted from imine formation as hypothesized, we studied the reaction of sensor 2 and glutamate/Zn2+ by 1HNMR (Figure 3). The experiment was carried out in a mixture of methanol-d4 and D2O due to the limited solubility of sensor 2 in pure water. The sensor did not react to any extent with glutamate, however, upon addition of zinc acetate, clear imine formation is observed in which the aldehyde signal at 10.2 ppm shifted to 8.77 ppm. It is interesting to note that qualitatively both the NMR (Figure 3) and fluorescence spectroscopy (Figure 2) indicate that the sensor has very low affinity for either analyte in the absence of the other. In addition, ESI-MS of a mixture of sensor 2, glutamate, and Zn2+ provided strong evidence of the full complex (Figure S13).
(Zn2+)a
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_d
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n-butylamine GABA
aspartic acid
glutamate glycine a
Apparent binding of sensor-amine complex (7 µM sensor, 500 mM amine, 50 mM HEPES, 120 mM NaCl, pH 7.4) using 10 mM zinc acetate solution. bCorrected binding constants. cRatio of the relative fluorescence intensity of the sensor−amine complex with and without Zn2+. λex = 440 nm. dReliable stability constants for GABA and Zn2+ are not available.
Figure 3. a) 1HNMR spectra of probe in methanol-d (minor peaks are due to small amounts of hemi-acetal formation with the solvent); b) probe (1 equiv.) + glutamate (20 equiv.) in methanold/D2O; c) probe (1 equiv.) + glutamate (20 equiv.) + Zn2+ (10 equiv.) in methanol-d/D2O.
This effect is born out in the fluorescence studies as well. In the absence of Zn2+, the binding constant of sensor 2 for glutamate is 26 M-1, which is quite low. In the absence of glutamate, the affinity of the sensor for Zn2+ is 7500 M-1. However, in the presence of glutamate, the apparent affinity for Zn2+ goes up to 37,000 M-1. Even this number is an underestimate of the true binding constant since the added Zn2+ is largely bound by the glutamate present in solution. It is relatively straightforward to correct the binding constant by calculating the concentration of free Zn2+ in solution using the known stability constants for the Zn2+/glutamate complex. 19 By this method, the corrected binding constant of the sensor/glutamate complex for Zn2+ is 6.3 x 108 M-1. Thus the sensor truly only binds the combination of both Zn2+ and glutamate, whereas either analyte alone do not bind well at all. This type of sensor selectivity is quite unusual, even in the field of logic gates. Typically, logic gates bind to both analytes but only give signal when both are bound. In the case of sensor 2, neither analyte binds to any significant extent in the absence of the other. We hypothesize that this binding mechanism will give rise to very low levels of background activation of the sensor. Table 1. Spectroscopic and binding data for Sensor 2 with various amines. Amine
Log Kapp
Log Kcorr
To examine the mechanism of action of the sensor, we repeated Zn2+ titrations with other amines (Table 1). As above, the stability constants of Zn2+ and the various amines and amino acids20 were used to correct the apparent binding constants for the concentration of free Zn2+. These amines are not physiologically relevant since there are no regions where high concentrations of zinc and any other amine exist together. However, such studies give us insight into the binding properties of the sensor. For amines lacking an α-carboxylate, the fluorescence decreases as the sensor-amine complex is titrated with Zn2+. This result is both surprising and fortuitous. We have seen in other examples, that when a sensor in this series lacks a pendant aryl group, the binding of simple amines leads to a decrease in fluorescence.17 However, we anticipated that Zn2+ binding would rigidify the system providing a turn-on fluorescence response. This effect is observed for sensor 2, but only with the α-amino acids. Surprisingly, butyl amine and GABA both gave fluorescence decreases upon Zn2+ binding. In this case, since Zn2+ binding must induce conformational rigidity, we propose that the α-carboxylate must modulate the Lewis acidity of the metal, providing a fluorescence increase. 21 Among the three α-amino acids, the Zn2+ binding constants were similar, with aspartate being somewhat higher than the other two, although glutamate gave the largest fluorescence increase upon Zn2+ binding (Figure 4). In addition to the analytes presented in Figure 4, no response was observed from sensor 2 in the presence of either cysteine and zinc ion or glutathione and zinc ion (data not shown). Finally, sensor 2 and glutamate were titrated with calcium ion to test for cross reactivity and no similar fluorescence increase was observed (Figure S12). These negative controls speak to the selectivity of this sensor.
IZn2+/I0 c
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Figure 4. Ratio of the fluorescence intensity of the sensor−amine complex with and without Zn2+. λex = 440 nm.
In conclusion, we have developed a novel Zn2+/glutamate sensitive fluorescent sensor. The sensor mechanism ensures that neither Zn2+, nor glutamate bind with any significant affinity in the absence of the other. However, in the presence of both analytes, a significant increase in fluorescence is observed. Because of this novel mechanism, we anticipate that this sensor will provide excellent selectivity for imaging Zn2+ in secretory vesicles, over other pools of free Zn2+, since only in secretory vesicles is Zn2+ found in the presence of large amounts of free amino acids (i.e., glutamate). Cell studies to test this sensor will be carried out in due course. Supporting Information Uv/Vis and fluorescence spectra, synthetic procedures, 1H and 13C NMR spectra, and X-ray data. The Supporting Information is available free of charge on the ACS Publications website. Author Contributions: CY: Synthesis and analysis of sensor 2; FH: design and strategy; NPC: synthesis of intermediates; DS: synthesis of intermediates; KB synthesis of intermediates; CLB: X-ray diffraction of single crystals and analysis; TEG: project design and manuscript preparation.
ACKNOWLEDGMENT We thank the National Institutes of Health (EB020415) for financial support and Shanxi University funds for study aboard 2014.
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