1 Introduction
The "click chemistry" could be carried out in high yields under mild and tolerable conditions of neutral pH and room temperature in aqueous media within a reasonable reaction time[1,2]. Due to these favorable aspects with click chemistry, the use of this strategy for making 18F-labeled biomolecules has been reported [3,4,5,6,7,8,9,10]. And now it has been a mature method for the labeling of 18F[11,12]. Recently, the organometallic precursor fac-[188Re(CO)3(H2O)3]+ was shown to be an ideal candidate agent for labeling biomolecules[13] because of the high stability of the three carbonyl groups and the substitution liability of the three water molecules[14,15]. And for fac-[188Re (CO)3]+ labeling, many research groups have reported the use of "click to chelate" for compounds labeling or SPECT imaging[16,17,18,19].
Our group focuses on the preparation of the organometallic precursor fac-[188Re(CO)3(H2O)3]+[20] and the labeling method of this organometallic precursor[21]. We have labeled an RGD-containing peptide with fac-[188Re(CO)3(H2O)3]+[22] and obtained encouraged results. In this paper six triazoles were obtained via click chemistry, and the excellent radiochemical yields and stability in phosphate-buffered saline and new-born calf serum have been shown to be an extraordinarily ideal method for fac-[188Re (CO)3]+ labeling.
2 Materials and methods
2.1 General
Pyridine-2-methylamine, Bis(pyridin-2-ylmethyl)ami-
ne and L-Propargylglycine were purchased from Aldrich Co., Ltd; c(RGDfk)-N3 was synthesized by China Tech Peptide Co., Ltd. 188Re-perrhenate was eluted from 188W/188Re generator (Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China) using 0.9% saline; Plus QMA Sep-Pak cartridges were manufactured by Waters Corporation (Massachusetts, USA). All reagents were analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai). γ counter (SN-697, Shanghai Rihuan Photoelectronic Instrument Co., Ltd., Shanghai, China).
Plus QMA Sep-Pak cartridges were produced by Waters Corporation (Ma, USA); A Dionex P680 pump equipped a PDA-100 ultraviolet detector and a radiometric detector system with a Macherey-Nagel C-18 reversed phase column (5 μm, 150×4.6 mm) were used to perform HPLC; (HPLC method: the flow rate was set at 1 mL/min, with the mobile phase starting from 95% B (0.1% trifluoroacetic acid in water) and 5% A (0.1% trifluoroacetic acid in acetonitrile) to 5% B and 95% A at 30 min. Thin layer chromatography (TLC) analysis was performed using silica gel plates (silica gel 60 GF254, mobile phase: 99% CH3OH and 1% concentrated HCl) on a Bioscan system AR-2000 with Winscan software of Version 3.09 (Beijing, China).
2.2 Preparation of fac-[188Re(CO)3(H2O)3]+
As described in the literature[20], 5 mg BH3·NH3 and 5 mg K2[H3BCO2] were placed in a 10 mL glass vial, to which the mixture of 188Re-perrhenate eluate and concentrated H3PO4, flushed with nitrogen for 20 min were injected and then the glass vial was incubated at 75℃ for 15 min. The reaction was ended by cooling in ice bath. In addition, the QMA Sep-Pak cartridge was used to purify the product. The chelating efficiency was determined by TLC.
2.3 Radiolabeling of small organic molecules
100 µL of triazole analog solution (dissolved in methanol, 0.01 mol/L) was mixed with 900 µL of freshly prepared 188Re tricarbonyl complex solution (37 MBq/mL) and incubated at 75℃ for 1 hour. The radiolabeling efficiency was determined by HPLC.
2.4 Radiolabeling of c(RGDfk)-N3 peptide
100 µL of c(RGDfk)-N3 peptide (1 mg) solution was mixed with 900 µL of freshly prepared 188Re tricarbonyl complex (37 MBq/mL) and incubated at 75℃ for 30 min. The radiolabeling efficiency was determined by HPLC.
2.5 Octanol–water partition coefficient
Approximately 111 kBq of conjugation compounds in 500 μL of PBS (pH=7.4) were added to 500 μL of octanol in an Eppendorf microcentrifuge tube. The mixture was vigorously vortexed for 1 min at room temperature and centrifuged at 12 500 rpm for 5 min. After centrifugation, 200 μL aliquots of both layers were measured using a γ-counter. The experiment was carried out in triplicate. And the octanol–water partition coefficient (logP) was obtained by the following formula:
2.6 Stability in vitro
188Re-labeled triazole analogs were mixed with phosphate-buffered saline or new-born calf serum for the stability test. The admixtures were incubated at 37℃ for 24 hours. Stability was determined at various time points (0, 1, 4, 8 and 24 h) by HPLC.
3 Results and discussion
3.1 Radiolabeling of c(RGDfk)-N3 peptide and benzyl azides
The chemical structure of c(RGDfk)-N3 peptide was shown in Fig.1. The radiolabeled efficiencies of the labeled compounds were 93%, 94%, 95%, 95%, 91% and 92% respectively and the retention times (tR) were 9 min, 19 min, 8 min, 10 min, 14 min and 10 min respectively according to the radio-high-performance liquid chromatography (Fig.2). The shoulder peaks on the main peaks of compound 1 and compound 5 to be exported were determined and the optical isomers were produced.
3.2 Octanol–water partition coefficient
The octanol-water partition coefficients (logP) for the six labeled compounds were illustrated in Table 1. The data indicate that the tracers containing c(RGDfk)-N3 peptide are slightly more hydrophilic than containing benzyl azides.
3.3 Stability in vitro
The stability of the 188Re-labeled compounds at 37℃ in the presence of phosphate-buffered saline or newborn calf serum was monitored by radio-HPLC. After 24 hours incubation, the radiochemical purity was more than 90% in both selected conditions, which was shown in Fig.3.
| Entries | logP |
|---|---|
| 1 | 0.84±0.02 |
| 2 | -2.35±0.03 |
| 3 | 0.75±0.04 |
| 4 | -2.06±0.02 |
| 5 | 0.97±0.05 |
| 6 | -1.74±0.04 |
4 Conclusion
Six 188Re-labeled compounds were successfully prepared using a simple click chemistry method. The main role of the click chemistry was to synthesis the bifunctional chelating agents containing triazole rings.
The use of click chemistry in compounds 1, 2, 3 and 4 was "conjugation-chelating" and in compounds 5 and 6 was just "conjugation" . The well chemical yields and ideal stability in vitro of 188Re-labeled c(RGDfk)-N3 peptide compounds forebode the further in vivo research even the tumor SPECT imaging and radiotherapy.
