Single‐Sided Competitive Axial Coordination of G‐Quadruplex/Hemin as Molecular Switch for Imaging Intracellular Nitric Oxide

Abstract

Axial coordinationi sacrucial biological process to regulate biomolecules' functions in natural enzymes. However,i ti sagreat challenge to determine the single or dual axial interaction between the metal centero fe nzymes and the ligand.I nt his work, ac ontrollable axial coordination system was developed based on G-quadruplex/ hemin complexb yd esigning as eries of fluorescent derivatives. The mechanism on axial coordination of G-quadruplex/hemin with coumarin-imidazolel igandsw as proposed to be single-sided, and led to fluorescenceq uenching of ligands. Upon addition of nitric oxide,t he fluorescence of ligands was recovered through competitive axial coordination, providinga"signal on" strategyf or signal transduction. More significantly, the fluorescent imaging of intracellular nitric oxide was achieved after conjugating with gold nanoparticles. Also, the proposed protocol provided as mart strategy to monitort he relationship between nitric oxide and p53 protein activity in living cells.In naturale nzymes,t he axial coordination between the active site of metalloenzyme and the small biomolecule ligandp lays as ignificant role in regulating biological functions. [1] Inspirationally,c hemists prepared pyridine or imidazole derivatives to axially coordinatet he Fe centero fm etalloporphyrins. Such coordinations tabilizedt he low-spin state of the center metal configuration to imitate the natural enzyme structure. [2] The axial ligands, including chloride, cyanide and nitrate, wereu su-ally coordinated to hemin on both axial sites. [3] However,i n natural enzymes,t he thiolatel igand dominated the fifth coordination position of metal center of hemin, while the sixth position remained open. [4] Therefore, the determinationo fs ingle or dual axial interaction between the metal centero fm etalloporphyrin and the ligand is achallenging endeavor,which usually requires complicated chemical modificationso ft he metalloporphyrin to control the single-sided coordination. [5] To address the problem, af our-stranded nucleic acid structure, G-quadruplex( G4), was introduced to bind tightly with hemin to form specific hemin-binding tertiary structure via intramolecular guanineq uadraplexes. [6] The resulting G4/hemin complex provided one unoccupied axial site on the center Fe in hemin due to the end-stackingatthe terminals of G-quadruplexesa gainst the other positions. [7] In addition, the majority of the hemin in the G4/hemin complex was monomeric,w hich resultedi nam ore reactive activation, compared to the aggregated hemin in aqueous solution. [8] The above advantages could bring new opportunities to decode the mechanism on hemin-based axial interaction. Moreover, the application of the single-sided axial coordinationo fG 4/hemin complexa samolecular switch provided greatp otential in biosensing and intracellular function analysis.Due to the significance of axial Fe-NO interactions on the biologically relevant process of ferriporphyrin, [9] nitric oxide (NO) was taken as am odel for competitive binding of the G4/ hemi...

Recent Advances in Molecular Imaging with Gold Nanoparticles

“…Therefore, AuNP conjugated with a fluorescent moiety through a biomolecule-responsive linker allow the specific and local release of the dye for turning on the probe’s signal in fluorescence imaging. This concept has been used to image a wide array of biomolecules with AuNP based nanoprobes, namely, pH, , ROS, , proteins, enzymes, , RNA, − metal ions, , and other metabolites. , Intracellular pH regulation is critical to living cells and can be used as one of the hallmarks of cancer metabolism for early diagnosis . Recently, several studies have demonstrated the possibility of imaging pH variations at the cellular level using engineered AuNP based nanoprobes. − For example, Yu et al constructed a pH-sensitive AuNP functionalized with fluorescent dyes, one responsive to acidic pH and the other to basic pH, and such a system can be used for mapping brain pH variations very finely using fluorescence imaging .…”

“…130 Therefore, AuNP conjugated with a fluorescent moiety through a biomolecule-responsive linker allow the specific and local release of the dye for turning on the probe's signal in fluorescence imaging. This concept has been used to image a wide array of biomolecules with AuNP based nanoprobes, namely, pH, 131,132 ROS, 133,134 proteins, 135 enzymes, 136,137 RNA, 138−140 metal ions, 141,142 and other metabolites. 143,144 Intracellular pH regulation is critical to living cells and can be used as one of the hallmarks of cancer metabolism for early diagnosis.…”

An oxidatively damaged G-quadruplex/hemin DNAzyme

“…2) [5][6][7][8] . The exquisite interaction between G4s and hemin (iron (III)-protoporphyrin IX) results in peroxidasemimicking G4-DNAzyme systems 9 , which have been used as biosensors 10,11 and biodevices 5,12,13 .…”

Mechanochemical Preparation of Protein : hydantoin Hybrids and Their Release Properties

“…Under these conditions, full conversion could be obtained by continuous milling (Figures S1–S5). As for hydrazones 1 and 2 , the mechanochemical preparation of fluoresceine‐tagged aminohydantoin 3 (Scheme 1) presents several advantages compared to similar solution‐based experimental procedures: [3g] (a) the use of a base to generate the more nucleophilic free amine can be avoided (reagent economy), the activation being provided by mechanochemical shocks between the reactants; (b) the reaction kinetics is improved (time economy), with full conversion of the starting materials obtained in only 150 min (instead of 24 h); (c) the experimental set‐up was simplified and no dry conditions were needed; (d) the throughput of the process and its ecological footprint were improved, the recovery of the final product being achieved by simply precipitating in water the thiourea 3 (waste economy), with only water and hydrochloric acid generated as by‐products of the reaction and with higher yield (93 %) compared to solution‐based procedures involving click‐type amine‐isothiocyanate coupling reaction (62–70 %) [40,41] …”