An N-heterocyclic amine chelate capable of antioxidant capacity and amyloid disaggregation

Abstract

Alzheimer's disease is a neurodegenerative disorder characterized by the development of intracellular neurofibrillary tangles, deposition of extracellular amyloid beta (Aβ) plaques, along with a disruption of transition metal ion homeostasis in conjunction with oxidative stress. Spectroscopic, transmission electron microscopy, and scanning electron microscopy imaging studies show that 1 (pyclen) is capable of both preventing and disrupting Cu(2+) induced AB(1-40) aggregation. The pyridine backbone of 1 engenders antioxidant capacity, as shown by cellular DCFH-DA (dichlorodihydrofluorescein diacetate) assay in comparison to other N-heterocyclic amines lacking this aromatic feature. Finally, 1 prevents cell death induced by oxidative stress as shown by the Calcein AM assay. The results are supported using density functional theory studies which show that the pyridine backbone is responsible for the antioxidant capacity observed.

Chart 1

Figure 1

Turbidity assay showing disaggregation of amyloid plaques upon incubation (24 h at 37 °C) with 1–4. Solutions prepared in 15 mM KH₂PO₄/NaCl buffer. [Aβ_1–40] = 200 μM, [CuSO₄] or Zn(OAc)₂ = 400 μM, [chelator] = 800 μM. n = 3 for each sample. When standard error of the mean are not depicted, they were too small to graph.

Figure 2

Addition of ligands 1–4 to aggregated Aβ results in reconstitution of Tyr¹⁰ fluorescence which was decreased due to aggregate formation. Solutions prepared in 15 mM KH₂PO₄/NaCl buffer. [Aβ_1–40] = 200 μM, [CuSO₄] or [Zn(OAc)₂] = 400 μM, [chelator] = 800 μM. n = 3 for each sample.

Figure 3

TEM Images showing (a) copper induced aggregation of amyloid and (b) its dissolution by 1.

Scheme 1. Redox Cycling of Copper in the Presence of Oxygen and Ascorbate to Produce OH·

Figure 4

Fluorescence intensity of 7-hydroxy-CCA after incubation of CCA [100 μM] and ascorbate [300 μM] with Cu^II (■) [40μM]. Compound 1 (◇) and 3 (●) [40 μM] were added at t = 0 s prior to Cu^II. Asc (▲) is a negative control with buffer and ascorbate only. All solutions except Cu(NO₃)₂ (Milli-Q water only) were dissolved and diluted in KH₂PO₄/NaCl [15 mM] buffer containing desferryl [2 μM]. Final volume = 4 mL, n = 3 for each sample.

Figure 5

Calcien AM viability assay of FRDA cells after 48 h exposure to BSO [1 mM] with 1–4 [1 nM final]. n = 4 for each sample. * indicates significance with a p value of <0.05.

Figure 6

DCFH-DA fluorescent response in FRDA cells after 12 h exposure to BSO [1 mM] with 1–4 [4 nM]. n = 4 for each sample. * indicates significance with a p value of <0.05.

Figure 7

DCFH-DA fluorescent response in FRDA cells after 12 h exposure to BSO [1 mM] showing dose dependence with 1. n = 4 for each sample. * indicates significance with a p value of <0.05.

Figure 8

DCFH-DA fluorescent response to chelator 1 in HT-22 cell line. Growth media serves as the positive control and is normalized to 100% while glutamate serves as the negative control. ZYC26 was 1 μM and serves as a positive control for antioxidant ability. *** indicates significance with a p value of <0.001.

Figure 9

Frontier molecular orbitals of 1 and 2 with the spin density of 1· shown (see Supporting Information for methods and data).