Bong Rae Cho

Amyloid-β (Aβ) tracers have made a significant contribution to the treatment of Alzheimer’s disease (AD) by allowing a definitive diagnosis in living patients. Unfortunately, they also detect tau and other protein aggregates that compromise test accuracy. In AD research, there has been a growing need for in vivo Aβ imaging by two-photon microscopy, which enables deep-brain-fluorescence imaging. There is no suitable neuritic Aβ probe for two-photon microscopy. Here we report PyrPeg, a novel two-photon fluorescent probe that can selectively target insoluble Aβ rather than tau and α-synuclein aggregates in the AD model brain and post-mortem brain. When injected intravenously, PyrPeg detects the neuritic plaques in the brain and olfactory bulb of the AD model. PyrPeg may serve as a useful blood-brain-barrier-penetrating diagnostic tool for optical and functional monitoring of insoluble forms of Aβ aggregates in the living AD brain.

BER-blue and FER-green can detect the endoplasmic reticulum in a live tissue by two-photon microscopy.

We developed Pyr1-infliximab: a two-photon probe for TNF-α. Pyr1-infliximab showed absorption maxima at 280 and 438 nm and an emission maximum at 610 nm in an aqueous buffer and effective two-photon action cross-section values of (520–2830) × 10^-50 cm⁴s/photon in RAW 264.7 cells. After this probe was labeled, it was possible to detect Pyr1-infliximab–transmembrane TNF-α complexes in a live cell and to determine the relative proportion of these complexes in human colon tissues. This proportion among healthy, possibly inflamed, and inflamed tissues of patients with ulcerative colitis was found to be 1.0/4.5/10. This probe may find useful applications for selective detection of transmembrane TNF-α in a live cell or tissue, for quantification of inflammation in human colon tissue or of antidrug antibodies in patients who stop responding to anti-TNF therapy, and for monitoring of the response to this therapy.

Reactions of 2,4‐dinitrophenyl 2‐thiophenecarboxylate (2a–d) with R₂NH/R₂NH₂⁺ in 20 mol % DMSO(aq) have been studied. The reactions are overall second order, first order to the substrates, and first order to the nucleophiles. The Brönsted plots showed downward curves with pK_a⁰ = 9.5, β₁ = 0.22–0.34, and β₂ = 0.85–0.92. The k₁ values increased with a stronger electron‐withdrawing 5‐thienyl substituent and a stronger nucleophile, whereas the k₂/k₋₁ values remained nearly the same for all 5‐thienyl substituents. The influence of 5‐thienyl substituent on the reaction rates showed excellent correlations on the Yukawa‐Tsuno plots with ρ = 1.28–2.16 and r = 0.20–0.60. The ρ value increased and r value decreased with a stronger nucleophile, indicating an increase in the electron density at the carbonyl carbon and a decrease in the resonance demand. From these results, a stepwise mechanism with a change in the rate‐determining step has been proposed.

We have developed blue- and yellow-emitting two-photon probes (BGolgi-blue and PGolgi-yellow) derived from 6-(benzo[d]oxazol-2-yl)-2-naphthalylamine and 2,5-bis(benzo[d]oxazol-2-yl)pyrazine derivatives as the fluorophores and trans-Golgi network peptide (SDYQRL) as the Golgi apparatus targeting moiety. HeLa cells labeled with BGolgi-blue and PGolgi-yellow emitted two-photon excited fluorescence at 462 and 560 nm, respectively, with effective two-photon action cross-section values of 1860 and 1600 × 10^-50 cm⁴s/photon, respectively. The probes can detect the Golgi apparatus in live cells and deep inside live tissue using two-photon microscopy at widely separated wavelength regions with high selectivity and minimum pH interference, as well as being photostable and having low cytotoxicity.

We have developed two-photon (TP) pH-sensitive probes (BH-2 and BHEt-1) that exhibit absorption and emission maxima at 370 and 466 nm, and TP absorption cross-section values of 51 and 61 GM (1 GM = 10-50cm4s/photon), respectively, at 750 nm and pH 3.0 in a universal buffer (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M KCl)/1,4-dioxane (7/3) solution. The TPM images of CCD-18co (a normal colon cell line) and HCT116 cells (a colon cancer cell line) labeled with BH-2 were too dim to be distinguished. When the same cells were labeled with BHEt-1, however, the TPM image of the HCT116 cells was much brighter than that of CCD-18co cells, and the relative proportion of the acidic vesicles (Pacid) of the former was 5-fold larger than that of latter. BHEt-1 could also differentiate HepG2 cells (a human liver cancer cell line) from LX-2 cells (a human hepatic stellate cell line) with a 6-fold larger Pacid value. Human colon cancer tissues labeled with BHEt-1 showed similar results, demonstrating much brighter TPM images and 6-fold larger Pacid values compared to normal tissue. These results suggest the potential utility of BHEt-1 for detecting colon cancer in human tissues using TPM.