For fluorescence off on control

For fluorescence off/on control of the molecules, we optimized the TokyoGreen structure of the fluorescent glucosamine conjugate based on the PeT mechanism. The developed platform, 2-Me-4-OMe TGG, showed stronger fluorescence in the anionic form than in the neutral form of its xanthene moiety. 2-Me-4-OMe TGG also has a more appropriate pKa for the intracellular environment (pH 7.4) compared to the acidic state (pH ∼ 6.5) seen in the extracellular environment of tumors.25, 26 Further optimization of its pKa should lead to the development of very useful activatable glucose analogues. It would also be weakly fluorescent in the extracellular environment of gastric cancers, because the environmental pH in the stomach is ∼1.5. Moreover, this platform could be developed to target intracellular enzymatic activities such as esterases with an O-acetyl group, β-galactosidase (for ovarian cancers) with a O-β-galactopyranosyl group, and bioreductases with a 4-nitrobenzyl or 4-nitrofuryl O-protecting group, as shown in Fig. 5a. As a proof of concept, we designed and synthesized 2-Me-4-OMe TGG masked with an O-β-galactopyranosyl group (Scheme S7, Figs. S8 and S9) and applied it to the fluorescence imaging of MIN6 cells. The probe was fluorescently activated by β-galactosidase in the Lesinurad (Figs. S10 and S11) and the cells could be visualized without any washout process of excess probe (Fig. S12), though some improvement of the uptake efficiency may still be needed. Such activatable fluorescent glucose analogues would be useful to detect cancers without the need for any washout process to remove excess analogues, and might be applicable for the rapid detection of cancers with high S/N during endoscopy and/or surgical operation. The appropriate target intracellular enzyme (or the target pH change) would depend upon the target cancer, and further investigations of biological applications of 2-Me-4-OMe TGG are in progress.
Experimental section
Acknowledgments MIN6 cells were kindly provided by Dr. Junichi Miyazaki, Osaka University. This research was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (JP16H05099 and JP18H04609 to K.H., and JP16H06574 to T.U.), SENTAN, JST to K.H, and Hirosaki University Institutional Grant to K.Y. K.H. was also supported by a grant JSPS Core-to-Core program, A. Advanced Research Networks.
Introduction Radical S-adenosylmethionine (SAM) enzymes are a large superfamily of enzymes that utilize radical chemistry to catalyze diverse reactions through a similar mechanism for radical initiation. The radical SAM enzymes utilize a [4Fe–4S] cluster that is coordinated to the enzyme via a conserved cysteine motif, most commonly CX3CX2C, that coordinates three of the four irons of the cluster. The fourth iron of the cluster is then free to bind SAM through its amino and carboxylate moieties (Fig. 1). In the reduced state, the [4Fe–4S]+ cluster transfers an electron to SAM, resulting in homolytic cleavage of SAM to produce methionine and the highly reactive 5′-deoxyadenosyl radical (dAdo·) intermediate. The dAdo· abstracts a hydrogen atom from substrate to produce 5′-deoxyadenosine (dAdoH) and a substrate radical (Fig. 2, blue arrow) which can be the product of the reaction or can undergo further transformation [1], [2], [3]. In addition to a common mechanism, the radical SAM enzymes exhibit a conserved fold, with the [4Fe–4S] cluster bound within a partial (α/β)6 or full (α/β)8 triosephophate isomerase (TIM) barrel (Fig. 3) [1]. Other variations of the cluster binding motif [4], [5] and enzyme fold [6], [7] have been indentified in radical SAM enzymes or radical SAM-like enzymes. SAM has also been reported to undergo alternative cleavage reactions: in a radical SAM-like enzyme [6], [7] as well as one GRE–AE [8], cleavage of the SC(γ) bond has been reported (Fig. 2, green arrow), while the radical SAM enzyme TsrM cleaves the SC(methyl) bond of SAM but in a non-radical mechanism [9]. This review will focus on the radical SAM enzyme pyruvate formate lyase activating enzyme (PFL-AE) as well as other radical SAM enzymes that utilize SAM to abstract a hydrogen atom from a protein glycine residue, placing them in a group known as glycyl radical enzyme activating enzymes (GRE–AEs).