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. 2022 May;596(10):1279-1289.
doi: 10.1002/1873-3468.14303. Epub 2022 Feb 7.

Improved synthesis of an ergothioneine PET radioligand for imaging oxidative stress in Alzheimer's disease

William J Behof  1   2 , Clayton A Whitmore  1   2 , Justin R Haynes  1   2 , Adam J Rosenberg  1   2 , Mohammed N Tantawy  1   2 , Todd E Peterson  1   2 , Fiona E Harrison  3   4   5 , Robert B Beelman  6 , Printha Wijesinghe  7 , Joanne A Matsubara  7 , Wellington Pham  1   2   4   5   8   9   10   11
Affiliations

Improved synthesis of an ergothioneine PET radioligand for imaging oxidative stress in Alzheimer's disease

William J Behof et al. FEBS Lett. 2022 May.

Abstract

L-ergothioneine (ERGO) is a potent antioxidant with cytoprotective effects. To study ERGO biodistribution and detect oxidative stress in vivo, we report an efficient and reproducible preparation of [11 C]-labeled ERGO PET radioligand based on protecting the histidine carboxylic group with a methyl ester. Overall, this new protection approach using methyl ester improved the chemical yield of a 4-step reaction from 14% to 24% compared to the previous report using t-butyl ester. The [11 C]CH3 methylation of the precursor provided the desired product with 55 ± 10% radiochemical purity and a molar activity of 450 ± 200 TBq·mmol-1 . The [11 C]ERGO radioligand was able to detect threshold levels of oxidative stress in a preclinical animal model of Alzheimer's disease.

Keywords: PET imaging; ROS; alzheimer; ergothioneine; inflammation; oxidative stress.

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Conflict of interest statement

Competing interests. The authors declare no competing interests.

Figures

Figure 1.
Figure 1.
Design and synthesis of an ERGO precursor for [11C]CH3 labeling.
Figure 2.
Figure 2.
Radiolabeling to generate [11C]ERGO radioligand. After labeling, the Boc groups were deprotected in TFA, while the methyl ester was cleaved in 5M NaOH at an elevated temperature. The RP-HPLC chromatogram of the co-injection data of the ERGO compound, which was detected in the UV channel (254 nm) along with [11C]ERGO radioligand, detected in the radiometric channel. The radioactive compound [11C]ERGO elutes at 7.6 min, while the “cold” compound elutes at 7.3 min.
Figure 3.
Figure 3.
A set of representative data of a 30-min dynamic microPET imaging of the distribution and retention of [11C]ERGO radioligand with a focus on the brains of 5XFAD mice (A, B, C,D) versus WT control (E, F,G,H) (n = 4, each). Each animal received 14.8 MBq of [11C]ERGO in 0.1 mL via the lateral tail vein. The uptake was quantified and compared using ImageJ (G). Data are shown as the mean ± SEM for all brain subregions, p<0.05.
Figure 4.
Figure 4.
Representative fluorescence images of the mouse hippocampus stained with activated astrocytes (A,B), microglia (D,E), and OCTN1 (G-J) using anti-GFAP, anti-IBA1 protein markers, and anti-OCTN1/2 antibodies, respectively in WT (A,D,G,H) versus 5XFAD (B,E,I,J). The coronal sections were stained with DAPI (nuclear staining, blue, 465 nm channel) to provide anatomical laminar landmarks. The GFAP expression (green, 517nm channel, scale bar 100μm) is prominent across the hippocampal regions of 5XFAD compared to those from WT counterparts. The GFAP signal was quantified, and the signal distribution was scored on an ordinal scale after thresholding using the Otsu method and presented in the bar graph (C). An asterisk indicates significant differences between WT vs. 5XFAD (*p<0.05). Meanwhile, IBA1 immunohistochemistry (red, 696 nm channel, scale bar 200μm) reveals a significant increase in microglial in 5XFAD vs. WT mice (F) (**p<0.01). The OCTN1 immunohistochemistry (green, 517 nm channel) was observed with a magnification of 20x (G,I; scale bar 50μm) and 63x oil immersion (H,J; scale bar 10μm).
Figure 5.
Figure 5.
Mechanism of acidic catalyzed hydrolysis of (A) a Boc group and (B) t-butyl ester.
See this image and copyright information in PMC

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