Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

Summary

A facile, one-pot synthesis of N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30).

Abstract

Biomolecules, including peptides,^1-9 proteins,^10,11 and antibodies and their engineered fragments,^12-14 are gaining importance as both potential therapeutics and molecular imaging agents. Notably, when labeled with positron-emitting radioisotopes (e.g., Cu-64, Ga-68, or F-18), they can be used as probes for targeted imaging of many physiological and pathological processes.^15-18 Therefore, significant effort has devoted to the synthesis and exploration of ¹⁸F-labeled biomolecules. Although there are elegant examples of the direct ¹⁸F-labeling of peptides,^19-22 the harsh reaction conditions (i.e., organic solvent, extreme pH, high temperature) associated with direct radiofluorination are usually incompatible with fragile protein samples. To date, therefore, the incorporation of radiolabeled prosthetic groups into biomolecules remains the method of choice.^23,24

N-Succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB),^25-37 a Bolton-Hunter type reagent that reacts with the primary amino groups of biomolecules, is a very versatile prosthetic group for the ¹⁸F-labeling of a wide spectrum of biological entities, in terms of its evident in vivo stability and high radiolabeling yield. After labeling with [¹⁸F]SFB, the resulting [¹⁸F]fluorobenzoylated biomolecules could be explored as potential PET tracers for in vivo imaging studies.¹ Most [¹⁸F]SFB radiosyntheses described in the current literatures require two or even three reactors and multiple purifications by using either solid phase extraction (SPE) or high-performance liquid chromatography (HPLC). Such lengthy processes hamper its routine production and widespread applications in the radiolabeling of biomolecules. Although several module-assisted [¹⁸F]SFB syntheses have been reported,^{29-32, 41-42} they are mainly based on complicated and lengthy procedures using costly commercially-available radiochemistry boxes (Table 1). Therefore, further simplification of the radiosynthesis of [¹⁸F]SFB using a low-cost setup would be very beneficial for its adaption to an automated process.

Herein, we report a concise preparation of [¹⁸F]SFB, based on a simplified one-pot microwave-assisted synthesis (Figure 1). Our approach does not require purification between steps or any aqueous reagents. In addition, microwave irradiation, which has been used in the syntheses of several PET tracers,^38-41 can gives higher RCYs and better selectivity than the corresponding thermal reactions or they provide similar yields in shorter reaction times.³⁸ Most importantly, when labeling biomolecules, the time saved could be diverted to subsequent bioconjugation or PET imaging step.^28,43 The novelty of our improved [¹⁸F]SFB synthesis is two-fold: (1) the anhydrous deprotection strategy requires no purification of intermediate(s) between each step and (2) the microwave-assisted radiochemical transformations enable the rapid, reliable production of [¹⁸F]SFB.

Protocol

1. Initial preparations

1. A V-vial (5-mL) RV1 (with stirring bar) is used as the main reaction vessel for performing microwave synthesis. It is connected to a PEEK adaptor with seven inlet/outlet ports connects and placed inside the microwave cavity (see Figure 2). RV2 is connected to SPE cartridge (I) to collect the crude [¹⁸F]SFB. RV3 is connected to SPE cartridge (II) for collecting the final [¹⁸F]SFB solution. It can be placed in a warm water bath (40°C) to concentrate the corresponding solution before reconstituting in PBS buffer, especially for the downstream radiolabeling of biomolecules.
2. Setup for collecting crud....

Discussion

This simplified three-step, one-pot radiosynthesis of the ¹⁸F-acylation reagent [¹⁸F]SFB is developed based on non-aqueous chemistry. This process has excellent reproducibility and could be used reliably for the production of [¹⁸F]SFB in automated radiochemistry modules, owing to two key modifications described as followings: 1. We employ a deprotection/saponification step in anhydrous KOtBu/DMSO system to replace the common aqueous basic or acidic solution. Our non-aqueous deprotection .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
acetic acid in aqueous solution (5%, v/v)	Fisher Scientific	A38-500	Prepared in our lab
Acetonitrile	Sigma-Aldrich	75-05-8
Diethyl ether	Sigma-Aldrich	14775
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich	472301
Ethyl 4-(N,N,N-trimethylammonium) benzoate triflate	Prepared in Lab
4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K222)	Sigma-Aldrich	29,111-0
O-(N-succinimidyl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TSTU)	Sigma-Aldrich	105832-38-0
Potassium carbonate in aqueous solution (1M)	Sigma-Aldrich	209619	Prepared in our lab
Potassium tert-butoxide	Sigma-Aldrich	156671