Accelerated 19F·MRI Detection of Matrix Metalloproteinase-2/-9 through Responsive Deactivation of Paramagnetic Relaxation Enhancement

Paramagnetic gadolinium ions (GdIII), complexed within DOTA-based chelates, have become useful tools to increase the magnetic resonance imaging (MRI) contrast in tissues of interest. Recently, “on/off” probes serving as 19F·MRI biosensors for target enzymes have emerged that utilize the increase in transverse (T 2 ∗ or T 2) relaxation times upon cleavage of the paramagnetic GdIII centre. Molecular 19F·MRI has the advantage of high specificity due to the lack of background signal but suffers from low signal intensity that leads to low spatial resolution and long recording times. In this work, an “on/off” probe concept is introduced that utilizes responsive deactivation of paramagnetic relaxation enhancement (PRE) to generate 19F longitudinal (T 1) relaxation contrast for accelerated molecular MRI. The probe concept is applied to matrix metalloproteinases (MMPs), a class of enzymes linked with many inflammatory diseases and cancer that modify bioactive extracellular substrates. The presence of these biomarkers in extracellular space makes MMPs an accessible target for responsive PRE deactivation probes. Responsive PRE deactivation in a 19F biosensor probe, selective for MMP-2 and MMP-9, is shown to enable molecular MRI contrast at significantly reduced experimental times compared to previous methods. PRE deactivation was caused by MMP through cleavage of a protease substrate that served as a linker between the fluorine-containing moiety and a paramagnetic GdIII-bound DOTA complex. Ultrashort echo time (UTE) MRI and, alternatively, short echo times in standard gradient echo (GE) MRI were employed to cope with the fast 19F transverse relaxation of the PRE active probe in its “on-state.” Upon responsive PRE deactivation, the 19F·MRI signal from the “off-state” probe diminished, thereby indicating the presence of the target enzyme through the associated negative MRI contrast. Null point 1H·MRI, obtainable within a short time course, was employed to identify false-positive 19F·MRI responses caused by dilution of the contrast agent.


S2: Synthesis details
DMF (peptide synthesis grade) was supplied by Rathburn Chemicals Ltd, anhydrous DMF was purchased from Sigma Aldrich ® ; all reagents were purchased from either Sigma Aldrich ® , Alfa Aesar ® , Merck Chemicals Ltd, or Strem Chemicals Inc; HPLC-grade solvents purchased from Fisher Scientific ® were used for all reactions Chemical shifts (δ) are given in parts per million (ppm) and J values in Hertz (Hz).
All 19 F NMR spectra were recorded on a Bruker TM AV(III)400, or DPX 400 spectrometer at 376 MHz and at ambient temperature. Assignments were based on DEPT 90, DEPT 135, and HMQC spectra. High field NMR work was performed on a AV(III)600 MHz. High field 19 F NMR was performed solely on the AV(III)600 MHz which was fitted with an fluorine probe.
High resolution mass spectroscopy (HRMS) was recorded on a Bruker TM microTOF, an orthogonal Time of Flight instrument with electrospray ionisation (ESI, both positive and negative ion) sources as indicated. Values of mass to charge ratio (m/z), are given to four decimal places. The mass of the counter ions are H + 1.0078, and Na + 22.9898.
Infared spectroscopy was recorded on either a Thermo Scientific NICOLET IR200 FT-IR infared spectrometer, with samples prepared as a nujol mull on NaCl discs or as KBr discs. All HPLC were run on an Agilent 1200 series system. Analytical HPLC was performed using an Agilent Eclipse XDB-C18 analytical column 4.6 x 150 mm, with a 5 µm pore size.
Semi-preparative HPLC was performed using either an Agilent Eclipse XDB-C18 semipreparative column 9.4 x 150 mm, with a 5 µm pore size, or an Agilent Eclipse XDB-C18 semi-preparative column 9.4 x 250 mm, with a 5 µm pore size. Solvent A was 0.1% formic acid in milli Q water, and solvent B was 0.1% formic acid in HPLC grade acetonitrile.
Once at ambient temperature the solvent was removed in vacuo to yield the desired product as

S3: General solid state peptide synthesis (SPPS) methodology
General procedure 1 -Resin loading 2-Chlorotrityl chloride resin was suspended in dichloromethane (2 mL) and very slowly stirred (to avoid resin grinding). Fmoc-AA-OH (1.2 eq.) was added followed by diisopropylethylamine (2.0 eq.); once no more HCl gas was observed the resin was stirred for 3 hours. Excess methanol (2 mL) was then added and the mixture stirred for a further 20 minutes. The resin was then filtered and washed with dimethylformamide (3 mL x 2), dichloromethane (5 mL x 2), hexane (5 mL x 2), dimethylformamide (3 mL x 2), dichloromethane (5 mL x 2) and finally hexane (5 mL x 2). The resin was then dried in vacuo and a sample removed for testing. Once tested, the resins were soaked overnight in dichloromethane / dimethylformamide (3 mL, 1:1).

General procedure 2 -Loading testing
Loaded resin (~10 mg) was stirred in piperidine/ dimethylformamide (3 mL, 2:8) for 2 hours, the absorbance at 290 nm was then measured indicating Fmoc presence, and therefore amino acid loading was estimated according to the Beer-Lambert equation. The remaining resin was transferred to a peptide synthesiser column [4].
General procedure 3 -Fmoc deprotection The resin was washed by a steady flow of dimethylformamide, followed by a piperidine / dimethylformamide (2:8) mix until the absorbance at 290nm was reduced to starting point, and followed by an additional dimethylformamide wash.
General procedure 6 -"Click" washings The resin bound product was successively washed with water, methanol, acetonitrile and dichloromethane.
General procedure 7 -Cleavage The loaded resin was then washed with cleavage mixture (TFA: TIPS: H 2 O, 9: 0.5: 0.5, 20 mL) and the solution slowly stirred for 6 hours before being concentrated in vacuo. The cleaved mixture was then washed with diethyl ether and filtered. The collected peptide was then dried in vacuo.