Enzymatically Shifting Nitroxides for EPR Spectroscopy and Overhauser‐Enhanced Magnetic Resonance Imaging

Abstract

In vivo investigations of enzymatic processes using non-invasive approaches are a long-lasting challenge. Recently, we showed that Overhauser-enhanced MRI is suitable to such a purpose. A β-phosphorylated nitroxide substrate prototype exhibiting keto-enol equilibrium upon enzymatic activity has been prepared. Upon enzymatic hydrolysis, a large variation of the phosphorus hyperfine coupling constant (Δa(P)=4 G) was observed. The enzymatic activities of several enzymes were conveniently monitored by electronic paramagnetic resonance (EPR). Using a 0.2 T MRI machine, in vitro and in vivo OMRI experiments were successfully performed, affording a 1200% enhanced MRI signal in vitro, and a 600% enhanced signal in vivo. These results highlight the enhanced imaging potential of these nitroxides upon specific enzymatic substrate-to-product conversion.

Selective On/Off‐Nitroxides as Radical Probes to Investigate Non‐radical Enzymatic Activity by Electron Paramagnetic Resonance

“…Free radicals such as nitroxides or trityl radicalsa re stable enough in physiological conditions (in vitro and in vivo) to be detected by electronic paramagnetic resonance( EPR). [16] Preliminary investigationsi no ur groups [17][18][19] indicated that nonradicale nzymatic activity can be investigated both by EPR and OMRI using signal changes based either on the linewidthb roadening effect [17,18] or on the shift [19] of hyperfine coupling constant of an itroxide. Unfortunately,d ue to the very fast relaxation of free electron, EPR imaging( EPRI) is at the moments low and insufficiently resolved.…”

“…Consequently,t he reactionw as repeated under different conditions (e.g.,M g(ClO 4 ) 2 ,1 00 8C, ammonium acetate which also failed to improve the yield of the reaction. [19] Enantiomers of 1C were separated by using aC hiralpak-IEc olumn (1aC, t R = 6.21 min, 1bC, t R = 7.64 min in 1:1h eptane/ethanol at 1mLmin À1 flow rate). Finally,1 0mol %o fF eCl 3 was found to be an efficient catalyst for this reaction providing the amino-phosphonate 3 in 87 %y ield.…”

“…Attempts to prepare (R)-3C or (S)-3C using either as tep-bystep growth of the peptidic chain or the coupling between the enolateo f1C and the beforehand-prepared activated tetrapeptide Fmoc-Ala-Ala-Pro-Phe-X failed to yield the aimed compounds.T hus,n itroxide (R)-1C was treated [19] with excesso f LiHMDS at À78 8Ca nd the resulting enolatew as trappedw ith the activated dipeptide Fmoc-l-Pro-l-Phe-O-Piv 6,p repared by treating the commerciallya vailable dipeptide Fmoc-l-Pro-l-Phe-OH with pivaloyl chloride, [30,31] to yield peptide( R)-4C (82 %). 1,8-Diazabicyclo [5.4.0]undec-7-ene( DBU) [32] mediated Fmoc deprotection of (R)-4C,f ollowed by N,N'-dicyclohexylcarbodiimide (DCC) coupling with commercially available dipeptide, Fmoc-Ala-Ala-OH afforded the tetrapeptiden itroxide (R)-5C (13 %f or two steps).…”

Imaging of Enzyme Activity by Electron Paramagnetic Resonance: Concept and Experiment Using a Paramagnetic Substrate of Alkaline Phosphatase

“…Recent work demonstrated the detection of proteases using a β-phosphorylated NR-substrate prototype [11] and an NR-labeled peptide specific to chymotrypsin and cathepsin G. [12] Upon enzymatic hydrolysis of the peptide, the enol ester moiety of the NR is converted into a ketone moiety accompanied by a large 4 to 5 G change of the phosphorus hyperfine splitting (hfs) constant. Note that the measurement of hfs changes provides higher specificity than changes in correlation time or spin exchange, as the latter are sensitive to the local environment.…”

“…[11] One can expect a significant change in the nitrogen hfs upon the change in ring conformation, therefore allowing for discrimination between NRs 1 and 2 that exhibit narrow EPR linewidths due to deuteration.…”