Solid-Phase-Supported Approach for the Preparation of Bioresponsive and Multifunctional MRI Probes

The development of bifunctional imaging probes can often be challenging with difficult and time-consuming solution phase chemistry protocols and purification techniques. A solid phase synthetic protocol was therefore utilized to produce a functionalized derivative of a potent bismacrocyclic calcium-responsive contrast agent for magnetic resonance imaging. Through a convenient building block approach, the applicability of this methodology in the preparation and simple future development of multifunctional imaging probes was demonstrated.


General remarks
Commercially available reagents and solvents were used without further purification. Rink amide resin 100-200 mesh was purchased from Merck Millipore. Purification of synthesised compounds was performed using silica gel 60 (0.03-0.2 mm) from Carl Roth (Germany). LC-MS spectra were recorded on an Agilent 1100 series LC/MS system with a Polaris 5 C18-Ether column (250 x 4.6 mm). The LC-MS elution conditions are given in Table S1. High resolution mass spectra were recorded on a Bruker Daltonics APEX II (FT-ICR-MS) with an electrospray ionization source. 1 H and 13 C NMR spectra and relaxometric experiments were performed on a Bruker Avance III 300 MHz spectrometer at 25 °C. Processing was performed using TopSpin 2.1 (Bruker GmbH) and ACD/SpecManager 9.0 (Advanced Chemistry Development, Inc.). The NMR spectra were obtained either in CDCl 3 or D 2 O, using the deuterium lock frequency. The concentration of Gd 3+ in analysed solutions was determined using the bulk magnetic susceptibility shift (BMS). 1 Manual solid phase synthesis was performed with the synthesis 1 apparatus from Heidolph. Reversed-phase HPLC purification was performed on a Varian PrepStar Instrument (Austrailia) with PrepStar SD-1 pump heads. Analytical reversed-phase HPLC was performed with an Atlantis C18 column (4.6 x 150 mm, 5 µm particle size). Semipreparative reversed-phase HPLC was conducted with a Polaris 5 C18-A column (250 x 21.2 mm). Elution conditions are described in Table S2.

Solid phase peptide synthesis
The synthesis of the initial peptide sequence was carried out using the standard Fmoc chemistry strategy on a manual peptide synthesizer. A Rink Amide MBHA resin (0.1 g, substitution 0.78 mmol g -1 ) was used as the solid support. Before the first amino acid was coupled, the resin was allowed to swell in DMF for 1 h and Fmoc deprotection of the resin was carried out using a solution of 20 % piperidine in DMF (3 x 15 min). Prior to each reaction, the resin was allowed to swell in DMF for 1 h. After the coupling of the first amino acid, a capping procedure using an S7 acetic anhydride/pyridine solution (3:2, 4 mL) was performed for 30 min. The resin was then washed with DMF (5 x 3 mL). Each relevant coupling and deprotection procedure was checked by the Kaiser test for completeness. All reactions on solid phase were performed at room temperature. All LC-MS data was provided for the given compounds after removal from the resin and the deprotection of tBu esters and where applicable, the Mtt protecting group.

General amino acid coupling procedure
Fmoc-protected amino acids (3 equiv. relative to the resin substitution) were dissolved in DMF (4 mL) and activated in situ with HBTU (2.9 equiv) and DIPEA (6 equiv). After 10 min of preactivation, the mixture was added to the pre-swelled resin and agitated for 2 h. After, the solution was removed and the procedure was repeated with half the initial amount of amino acid, HBTU and DIPEA for 1 h. After coupling, the resin was washed with DMF (5 x 3 mL) and CH 2 Cl 2 (3 x 3 mL) to remove excess reagents.

General Fmoc deprotection procedure
Fmoc deprotections of the resin and amino acids were carried out with 3 treatments (15 min each) of a 20 % piperidine in DMF solution. After deprotection, the resin was washed with DMF (5 x 3 mL) and prepared for the next procedure.

General procedure for producing analytical data
After each reaction for compounds 6 -11, a micro cleavage of the resin was performed to assess the reaction success by means of LC-MS. Below; the synthetic protocols for the reactions described in the main text are reported. The chemical structures and LC-MS data shown represent that of the same compound after micro cleavage from the resin and removal of protecting groups. For compound L, the procedure for removal of the final ligand from the resin is described.

Compound 10-x (Compound 10 after micro cleavage from the resin)
Resin with compound 9 was exposed to a solution of 40 % piperidine in DMF (3 mL) for 15 min.
This was repeated a further two times to remove the Fmoc protecting group. The resin was then washed with DMF (5 x 3 mL). A mixture of BB3 (130 mg, 0.187 mmol, 2.4 equiv) and DIPEA (68 µL, 0.390 mmol, 5 equiv) in DMF (3 mL) was then added and agitated for 24 h. The solution was then removed and a further portion of BB3 (2.4 equiv) and DIPEA (5 equiv) in DMF (3 mL) S10 was added and allowed to shake for a further 24 h. The resin was then washed with DMF (5 x 3 mL) and CH 2 Cl 2 (3 x 3 mL).