Modified EDTA selectively recognized Cu²⁺ and its application in the disaggregation of β-amyloid-Cu (II)/Zn (II) aggregates

Highlights

• A fluorescent compound based on EDTA was synthesized.

• This compound exhibited a specific recognition towards Cu²⁺.

• The chelating affinity was sufficient to capture Cu²⁺ from β amyloid (Aβ)-Cu (II) aggregates.

• An enhanced disaggregation capability was obtained compared with EDTA.

Abstract

The accumulation of the β-amyloid (Aβ) aggregates induced by Cu²⁺/Zn²⁺ in conjunction with toxicity is closely related to Alzheimer's disease (AD). Herein, we intended to improve the efficiency and selectivity of traditional chelator ethylenediaminetetraacetic acid (EDTA) combined with a fluorescent group 4-aminosalicylic acid (4-ASA)to acquire a novel potential chelator 4,4′-((2,2′-(ethane-1,2-diylbis((carboxymethyl)azanediyl))bis(acetyl))bis(azanediyl))bis(2-hydroxybenzoic acid) (EDTA-ASA) capable of disaggregating Aβ-Cu(II)/ Zn(II) aggregates. EDTA-ASA combines 4-ASA as fluorophore and multidentate amino nitrogen, hydroxyl and carboxyl groups to chelate Cu²⁺ from Aβ-Cu (II) aggregates. The specific selectivity of EDTA-ASA towards Cu²⁺ in Tris-HCl buffer solution was investigated by fluorescence measurements. It exhibits high recognition towards Cu²⁺ with no significant interference of other competitive metal ions, which overcomes the deficiencies of EDTA. Importantly, the binding sites and binding mode for Cu²⁺ were clarified through DFT calculations. The thioflavin-T (ThT) fluorescence analyses and transmission electron microscopy (TEM) results have revealed EDTA-ASA exhibited an enhanced disaggregation capability on Aβ-Cu (II)/Zn (II) aggregates in comparison to EDTA. The Cu²⁺ chelating affinity was sufficient for EDTA-ASA to sequester Cu²⁺ from Aβ-Cu (II) aggregates.

Introduction

Alzheimer's disease (AD) is the most common form of dementia in the world and a devastating neurodegenerative disorder, it is incurable so far [1]. The predominant cause of AD is a heated discussion. Currently, though multiple factors mutually influence each other, the most prevailing hypothesis is still the amyloid cascade [2,3]. The amyloid cascade proposes amyloid-β (Aβ) aggregation as the leading cause of the disease. The misfolding of the extracellular Aβ accumulated in senile plaques (SP) has been recognized as the dominant hallmark of AD [4]. Aβ is a typical 39–43 residue polypeptide encompassing a C-terminal hydrophobic domain and an N-terminal hydrophilic sequence. Its interaction with Cu²⁺, Zn²⁺ facilitates Aβ aggregation, Aβ misfolding and reactive oxygen species (ROS) production [[5], [6], [7]]. Especially, high millimolar concentrations range of Cu²⁺ and Zn²⁺ are found in SP [8]. Up to now, the treatment of AD faces the greatest challenges in that there are few competent therapeutic approaches to modulate related metal ions and disaggregate Aβ-Cu (II)/Zn (II) aggregates.

Metal chelators have been applied in AD therapy research due to their metal ion chelating ability and have been proven to be a critical approach [[9], [10], [11], [12]]. In the past few decades, plenty of chelators for metal ions, especially for Cu²⁺ selective recognition, have been reported such as ethylenediaminetetraacetic acid (EDTA) [13], quinolines [14], clioquinol (CQ) [15a], 5,7-dichloro-2-((dimethylamino)methyl) 8-quinolinol (PBT-2) [16], calixarenes [17], curcumins [18], and rhodamines [19,20]. Potential regulation of Cu²⁺-induced Aβ aggregation has been shown in vitro and in vivo by using these metal chelators. Several chelators including CQ and PBT2 have been employed in murine AD models and AD patients. CQ was proved to be capable of dissolving Aβ deposits in transgenic mice and it can slow the cognitive decline associated with AD in some cases, and PBT2 could decrease the levels of Aβ in AD patients. Yet, all these metal chelators have not yielded promising results. The unpredictable side effects of these metal chelators, including drug-resistance and subacute myelo-optic neuropathy, limit their universal clinical applications. Besides, most of these chelators exhibited limited application due to problems such as complicated synthetic procedures, poor water solubility, and high interference by co-existing metal ions [[21], [22], [23], [24]]. Therefore, bearing the above considerations in mind, the functional chelator designs are still novel, and useful methods. It is of great significance and necessary to develop a novel metal chelator which exhibits high selectivity towards Cu²⁺ in aqueous solution via a simply synthetic method.

Interestingly, the well-known chelator, EDTA, has attracted considerable attention in recent years [[25], [26], [27]]. Despite its limitations in clinical studies, its EDTA backbone inspires us to functionalize it further to improve its possible properties. We have started to construct a modified EDTA derivative as a functional metal chelator. 4-aminosalicylic acid (4-ASA) has been widely utilized for the treatment of inflammatory diseases since the 1940s [[28], [29], [30], [31]]. ASA conjugates of EDTA were reported as promising anti-inflammatory prodrugs [32]. The compound 4-ASA possesses a fluorescent moiety which is useful for probing metal ions. Furthermore, 4-ASA conjugates of EDTA derivative as a chelator for selective recognition of Cu²⁺ in AD therapy has not been reported [33,34]. From the above, we have herein proposed to conjugate 4-ASA to EDTA with the amide linkage (-CONH) as a bridging unit to acquire the desired chelator, which will show water solubility improvement, strong fluorescence, and be capable for specific recognition of Cu²⁺ in aqueous solution.

We strategically designed and synthesized a modified EDTA derivative 4,4′-((2,2′-(ethane-1,2-diylbis ((carboxymethyl)azanediyl)) bis(acetyl)) bis(azanediyl))bis(2-hydroxybenzoic acid) (EDTA-ASA) (scheme. 1). It is based on EDTA as receptor and modified with 4-ASA as fluorophore. EDTA-ASA comprising both 4-ASA and EDTA-backbone group could provide excellent coordinating sites, such as electron-rich N (amino‑nitrogen, -NH-) and O (hydroxyl and carboxyl groups, –OH, –COOH) atoms. Both the framework of EDTA and side cores of 4-ASA have the potential to chelate Cu²⁺. EDTA-ASA presents a highly symmetrical structure, which is hoped to provide a special configuration and binding cavity for Cu²⁺. In addition, the groups of –COOH and –OH on 4-ASA could increase the solubility in aqueous solution. The -CONH extends the distance between the two functional groups thereby adjusting the effect of photoinduced electron transfer (PET) of secondary amines of 4-ASA to EDTA group. Furthermore, the –COOH and the –OH group in EDTA-ASA may have electrostatic interactions and hydrogen bonds with the carbonyl groups of N-terminal residues at Aβ. Besides the chelating interaction with Cu²⁺, EDTA-ASA is expected to have other synergistic effects to disaggregate the Cu (II)/Zn (II)-mediated Aβ aggregates. Collectively, EDTA-ASA is anticipated to have a wide potential application as potential chelator agents in the field of AD therapy.