pH-controlled release of auxin plant hormones from cucurbituril macrocycle
© Nuzzo et al.; licensee Springer. 2014
Received: 17 October 2013
Accepted: 2 January 2014
Published: 13 March 2014
The influence of pH on the formation of host-guest complexes between the cucurbituril (CB) macrocyclic host and three auxin plant hormones, namely indole-3-acetic acid (IAA), 2-naphthalene acetic acid (2-NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D), was studied by 1H NMR and relaxation experiments.
Only protonated auxins formed inclusion complexes with CB, exhibiting preferential encapsulation of the aromatic part inside the host cavity, and orientation of the carboxyl group towards the carbonyl-laced portals of CB. At pH values above the auxin pKa values, the guest molecules were negatively ionized and were no longer retained within the macrocyclic host, suggesting that a pH-controlled release of auxin guests from the CB host is possible.
The development of a technology based on the use of cucurbit[n]urils for the pH-controlled release of auxin molecules in plant systems represents an opportunity to exploit these macrocyclic compounds in a variety of agricultural applications.
Food security is an incumbent social problem, exacerbated by an ever-increasing population, decreasing arable land, and ecological adversities such as soil erosion and climate change. Chemical and biochemical technologies to support crop production could therefore play a vital role towards food security in the coming years. In this context, a technology based on host-guest complexation of bioactive compounds by macrocyclic host molecules in aqueous media may represent a controlled release system that may achieve efficient and balanced regulation of plant growth and increase of crop yields . Such an innovative technology may become important in sustainable agriculture to substitute inefficient application practices of agrochemicals resulting in their hazardous and expensive dissipation in the environment .
Auxin is the generic name for a class of plant hormones active in coordinating many growth processes in the life cycle of plants. Indole-3-acetic acid (IAA) is the most abundant and potent native auxin active in plants . 2-Naphthalene acetic acid (2-NAA) is used as a mimic for 1-naphthalene acetic acid synthetic auxin, which is commonly applied to stimulate the rooting potential of plant cuttings or to prevent fruit drop in orchards. The widely used 2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic plant growth regulator stimulating responses similar to those of natural auxins. Its auxin activity is mostly relevant at low concentrations (20 to 40 mg L−1), while it becomes phytotoxic at relatively high concentrations .
Results and discussion
1H NMR chemical shifts
IAA, 2-NAA, and 2,4-D are weak acids with only one dissociation constant in aqueous solution. Values of pKa in water for IAA and 2-NAA are 4.7 and 4.2, respectively, thus leaving these molecules fully protonated and uncharged at pH ≤ 2.0 and as negatively charged anions only at pH ≥ 7.0. Conversely, the pKa value of 2.7 for 2,4-D renders the molecule totally ionized and negatively charged already at pH ≥ 5.0.
Complexation with CB is reported to shift the pKa values of encapsulated guests [26, 27]. Guests comprising suitable chromophores allow the direct spectrophotometric determination of complexation in the host through shifts in the pKa values (i.e., the pKa of guest before and after complexation) [28, 29]. With the exception of the IAA-CB complex at pH 1, the lack of distinct absorption maxima in the UV-vis region for complexes involving CB and the other auxin guests prevented the determination of pKa shifts, by UV-vis spectroscopy under pH changes. Therefore, the pKa values of the carboxylic group of auxins, before and after CB addition, were determined by a 1H NMR study following the variation with pH of chemical shifts for protons in the alkyl groups next to the carboxylic moieties .
No significant pKa shifts were observed for the auxin guests upon addition of CB (results not shown), most likely because the hormone carboxylic acid moieties, which are positioned outside the macrocyclic host, were not affected by changes in the electronic environment. Indeed, it is reported in the literature that when a similar position is adopted by carboxylic acid groups during the encapsulation of weak acids by CB, no pKa shifts can be observed [31–33]. Conversely, an increase of the pKa values, upon complexation with the CB[n] hosts, has been invariably reported at low pH values for guests holding protonated nitrogen-containing functional groups, [34–37] presumably due to stabilization of the protonated moieties by interaction with carbonyl portals in the CB host. This interaction represents an additional binding strength over the hydrophobic forces which already keep the guest neutral forms inside the CB even at higher pH.
The pH titrations of IAA, 2-NAA, and 2,4-D by 1H NMR spectroscopy showed the influence of solution pH on binding interactions between both protonated and anionic forms of auxin molecules and CB. 1H NMR spectra were used to determine the portion(s) of guest molecules located within the hydrophobic cavity, as opposed to those positioned adjacent to the polar carbonyl-laced portals of the cucurbit[n]urils [4, 5, 7]. An upfield shift of a guest proton resonance (∆δlim = δfree − δbound) indicates its average position within the shielding hydrophobic cavity, whereas a downfield shift suggests that the guest proton is near one of the deshielding carbonyl-laced portals .
1 H NMR limiting chemical shifts (∆ δ lim ) for protons in IAA, 2-NAA, and 2,4-D
Since host-guest interactions are very sensitive to structural features, the negative charge produced on the guest molecule by deprotonation of the terminal auxin carboxyl group at high pH may disrupt the stability of the IAA-CB complex. In fact, at high pH values, the IAA protons experienced negligible or downfield shifts (Figure 3, spectrum II) after addition of the macrocycle to the guest solution, thus suggesting that the auxin was outside of the CB cavity and that no more intermolecular interactions occurred between the host and IAA. The absence of binding was likely due to the electrostatic repulsion between the IAA carboxylate negative charge and the carbonyl oxygens on the two electron-rich portals of CB, which overcomes any stabilization provided by the CB hydrophobic cavity for the aromatic indole moiety.
The association/dissociation behavior of auxin complexes with CB as a function of pH is in line with the supramolecular chemistry of cucurbit[n]urils previously reported [4, 5, 10]. The adducts of the protonated auxins with CB are likely stabilized by hydrogen bonds between the host carbonyl portals and the auxins' protonated carboxyl groups. Moreover, the hydrophobic effect that directed the aromatic part of auxins into the hydrophobic cavity of CB was also an important supramolecular driving force for binding in aqueous solution. However, the high pH deprotonation of the carboxylic acid led to an electrostatic repulsion that overcame the hydrophobic affinity of auxins to CB. These findings revealed a completely reversible and pH-switchable binding between important auxin guest molecules and CB.
1H NMR relaxation times
T 1 and T 2 relaxation time values for protons of IAA, 2-NAA, and 2,4-D
Cucurbituril was prepared as documented previously . IAA, 2-NAA, and 2,4-D were used as received (Sigma-Aldrich Corporation, St. Louis, MO, USA). The auxin molecules were dissolved separately to reach 0.4 mM in solutions containing 10% (v/v) of deuterated water (99.8% D2O/H2O; Armar Chemicals, Döttingen, Switzerland) and buffers to ensure the following pH values of 1 (hydrochloric acid/sodium chloride (0.05 M)), 3 (citric acid/sodium citrate (0.05 M)), 5 (sodium acetate/acetic acid (0.05 M)), 7 (sodium dihydrogen phosphate/disodium hydrogen phosphate (0.05 M)), 9.5 (ammonia/ammonium chloride (0.05 M)), and 12 (sodium hydroxide (0.05 M)). Then, CB was added to its final concentration of 0.4 mM, to form auxin-CB complexes. All samples were incubated for 48 h to reach complexation equilibrium. Samples were transferred into 5-mm NMR tubes, and the solutions were degassed gently by N2 flux for 5 min before NMR analysis.
A 400-MHz Bruker Avance (Rheinstetten, Germany) spectrometer, equipped with a 5-mm Bruker broadband observe (BBO) probe, working at 1H frequency of 400.13 MHz, was employed to conduct all liquid-state NMR measurements at a temperature of 298 ± 1 K. 1H NMR spectra were acquired for all samples with 22 s of thermal equilibrium delay, 90° pulse length ranging between 13.05 and 14.35 μs, 32,768 time domain points, and 64 transients.
The 1H longitudinal (spin-lattice) relaxation time constants (T1) of auxin proton signals were measured at pH 1 and 12 by applying an inversion recovery pulse sequence, with 16 increments and variable delays from 0.5 to 25 s. The transverse (spin-spin) relaxation time constants (T2) were obtained using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with 16 increments and 2 (2 ms) to 2,000 (5,000 ms) spin-echo repetitions, with a constant 0.5-ms spin-echo delay. A time domain of 32,768 points and 22 s of thermal equilibrium delay were set for all relaxation experiments.
The 1H spectral width had a range of 16 ppm (6,410.5 Hz), and the residual water signal was removed from 1H NMR spectra by pre-saturation technique. All spectra were baseline-corrected and processed by Bruker Topspin Software (v.2.1). No zero filling, as well as 0.2- and 0.5-Hz line broadenings, was adopted to Fourier transform the free induction decays (FID) of spectra deriving from conventional 1D proton acquisitions and relaxation experiments, respectively. Relaxation times of auxin molecules were calculated using MestReC NMR Processing (v. 18.104.22.168) and Origin (v.6.1) software.
Assignment of proton signals was achieved through two-dimensional (2D) experiments: homonuclear 1H-1H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser enhancement spectroscopy (NOESY), and heteronuclear 1H-13C heteronuclear single-quantum correlation (HSQC), and heteronuclear multiple bond coherence (HMBC). Homonuclear and heteronuclear 2D experiments were acquired with 48 and 80 scans, respectively, 16 dummy scans, a time domain of two k points (F2), and 256 experiments (F1). In detail, TOCSY and NOESY experiments were conducted with a mixing time of 80 and 900 ms, respectively, while HSQC and HMBC experiments were optimized for 145-Hz short-range and 8.5-Hz long-range JCH couplings. All 2D experiments were gradient-enhanced, except for TOCSY.
We report for the first time the supramolecular host-guest interactions of the macrocycle CB with bioactive auxin molecules IAA, 2-NAA, and 2,4-D. NMR spectroscopy showed that, for auxins dissolved in acidic aqueous solutions, the addition of CB led to changes in both the shape and chemical shifts of auxin resonances and to a decrease of relaxation times. Conversely, when auxins were negatively charged (i.e., deprotonated), the presence of CB did not induce any significant changes in chemical shifts and relaxation times. We conclude that cucurbituril is capable of hosting the protonated forms of the investigated plant growth molecules within its hydrophobic cavity, whereas the anionic forms of the auxins are released from the macrocycle, thereby suggesting pH control over the sequestration and release of auxin molecules within CB. Such a system has the advantage of being readily triggered at any desiderable time by simply and reproducibly adjusting the soil medium pH, a parameter that is easily controlled. These findings may provide a new approach to the host-guest chemistry of cucurbit[n]urils for the development of a controlled release technology of weakly acidic agrochemicals to plant systems.
AN conducted this work in partial fulfillment of a PhD degree within the Doctorate School “Valorizzazione e Gestione delle Risorse Agroforestali” of the Università di Napoli Federico II. AN gratefully acknowledges hospitality by OAS and his research group in Melville Laboratory for Polymer Synthesis of University of Cambridge.
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