Rapid ¹⁸F-radiolabeling of peptides from [¹⁸F]fluoride using a single microfluidics device

Abstract

To date the majority of ¹⁸F-peptide radiolabeling approaches are multi-step, low yielding and time-consuming processes. Given the short half-life of ¹⁸F (109.8 min), it is critical that methods are developed to increase the efficiency of this process with simpler, higher yielding and faster reactions that can be rapidly translated into clinical use. Here, we demonstrate the first microfluidic synthesis of the [¹⁸F]F-Py-TFP prosthetic group with radiochemical yields of up to 97% and a synthesis time of 3 min. In addition, we utilized a single microfluidics device to prepare the [¹⁸F]F-Py-YGGFL peptide using [¹⁸F]F-Py-TFP, from [¹⁸F]fluoride in a two-step, fully automated approach. The model peptide NH₂-YGGFL was radiolabeled with [¹⁸F]F-Py-TFP in up to 28% overall radiochemical yield within 8 minutes starting from anhydrous [¹⁸F]fluoride.

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Introduction

Synthesis of ¹⁸F-peptide based radiotracers are multi-step, complex, low yielding and often require a preparation time extending beyond one hour.¹ This presents a challenge for radiolabeling with ¹⁸F due to its short half-life (109.8 min) in addition to the low molarity of radioisotope (fM to nM) and the small amount of reagents used. These type of radiosyntheses benefit greatly from automation, in particular miniaturization.^2,3 Automated devices have proven to be excellent tools for speeding up overall radiotracer synthesis times, improving synthesis reproducibility and manipulations of small volumes, as well as limiting radiation exposure to the radiochemist.⁴ Examples of automated devices that are commercially available for the preparation of clinical radiotracers are the GE FASTlab®,⁵ the Siemens Explora® and Eckert and Ziegler's Modular-Lab®.⁶ These devices have demonstrated improved synthesis of many useful radiotracers and labeling agents, including [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG),⁷ [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO),⁸ [¹⁸F]fluorothymidine ([¹⁸F]FLT),⁹ and N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB).⁵

Trends towards miniaturization of automated devices have led to the development of microfluidic technology for radiotracer syntheses.^3,10 In particular microfluidic technology has demonstrated advantages as a platform to address some of the low yielding and time consuming syntheses challenges associated with radiotracer development.¹¹ The small reaction volumes and high surface area to volume ratio achievable with microfluidics result in fast and high yielding reactions.³ Several microfluidic devices have been developed utilizing both chip-based and flow through chemistry reactors. Van Dam et al. developed a chip-based microfluidic technology, in which liquids are manipulated with electric potentials through the electrowetting-on-dielectric (EWOD) mechanism.¹² This chip-based technology allows for fast and efficient chemistry on a small scale (2–12 μL), and has enabled a wide range of radiotracer synthesis using one EWOD chip. Chen et al. demonstrated the synthesis of [¹⁸F]FDG, [¹⁸F]FLT, [¹⁸F]SFB, and [¹⁸F]fallypride all on one single microfluidic chip.¹² The Advion NanoTek® is a commercially available microfluidic flow chemistry device that is specifically designed for radiochemistry, and is highly adaptable for a variety of syntheses.¹³ The Advion NanoTek® is a three-pump flow chemistry system in which reactions take place in reactors composed of crystal quartz capillary tubing. The Advion NanoTek® has been used in both the optimization process and batch production of several radiotracers, including [¹⁸F]fluoroazomycin arabinoside ([¹⁸F]FAZA),¹⁴ [¹⁸F]altanserin,¹⁵ [¹⁸F]fallypride¹⁶ and [¹⁸F]fluoroethyltyrosine [¹⁸F]FET.¹⁷ Further, the Advion NanoTek® has shown initial promise as a tool for the production of ¹⁸F-radiolabeled peptides.^18–20

¹⁸F-radiolabeled peptides have demonstrated their clinical usefulness as effective in vivo imaging agents in oncology.²¹ This is attributed to their excellent targeting ability, low immunogenic effects, small size and rapid preferential renal excretion.¹ Peptides containing the RGD motif have been used for imaging of the α_vβ₃ integrin, a receptor that is over expressed in many cancers as well as on new blood vessels formed during tumor angiogenesis.²² RGD peptides have been radiolabeled with several radionuclides (^99mTc, ⁶⁴Cu, ⁶⁸Ga, and ¹⁸F) for use in both PET and SPECT.²³ Additionally, our group has done pioneering work in developing the ¹⁸F-radiolabled PEGylated version of A20FMDV for imaging of the α_vβ₆ integrin, a receptor that is overexpressed in many cancers including colon, breast, lung and pancreatic.^24–26 Traditionally, radiolabeling of peptides with fluorine-18 requires the synthesis of an ¹⁸F-prosthetic group followed by its conjugation to the peptide using mild reaction conditions.²⁷ This approach can be time consuming, result in multiple labeled products, and is typically low yielding. Many different prosthetic groups have been extensively used, each with their own advantages and disadvantages.²¹ The most commonly utilized prosthetic group for ¹⁸F peptide radiolabeling is [¹⁸F]SFB. Peptide radiolabeling with [¹⁸F]SFB is a 4-step process that takes up to 3 hours to complete, and is reported in 10–24% overall yields. Attempts have been made to improve this synthesis by decreasing reaction time and improving yields using microfluidics and microwave heating. However, a simpler, faster, automated approach would be deemed valuable in ¹⁸F peptide radiolabeling. Recently, the microfluidic synthesis of [¹⁸F]SFB was reported in an overall synthesis time of 25–60 min and radiochemical yields of 33–55%.^12,28 [¹⁸F]Fluorobenzoic acid has also been used to site specifically radiolabel peptides on solid phase in high purity.²⁹ While successful, radiochemical yields of these reactions are highly variable depending on the peptide sequence and type of solid support used. From our studies, typical reaction conditions for solid-phase peptide radiolabeling result in yields ranging from 18–41%.^30,24,31 Most recently, McBride et al. has developed a chelate approach for ¹⁸F-radiolabeling, involving an NOTA–Al[¹⁸F]F⁻ complex that is conjugated to the peptide.³² This approach has many advantages in that it is simple, fast and high yielding, but sometimes suffers in vivo drawbacks including high renal uptake and retention.^25,33 To address some of the challenges of multistep synthesis, Olberg et al.²⁷ developed a one-step synthesis of the activated ester prosthetic group, 6-[¹⁸F]fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester ([¹⁸F]F-Py-TFP, Fig. 1 II). [¹⁸F]F-Py-TFP can be used to radiolabel peptides bearing free-amines. Several groups have shown the effectiveness of [¹⁸F]F-Py-TFP to radiolabel peptides³⁴ and small molecules³⁵ in fast (10–20 min) and relatively high yielding (40–90%) reactions.

Fig. 1 Reaction scheme of [¹⁸F]F-Py-TFP synthesis and peptide radiolabeling.

Here we report the first microfluidic synthesis of [¹⁸F]F-Py-TFP and its subsequent use in¹⁸F-radiolabeling of the model peptide, NH₂-Tyr-Gly-Gly-Phe-Leu (NH₂-YGGFL) on a microfluidic platform. The process is fully automated and allows for the rapid microfluidic synthesis of ¹⁸F radiolabeled peptides from [¹⁸F]fluoride, on a single microfluidic device (Advion Nanotek®).

Experimental

General

All reagents were purchased from Sigma Aldrich (Milwaukee, WI), and VWR (Radnor, PA, USA) and used without further purification, unless otherwise stated. Peptides were assembled on rink amide resin (Novabiochem, Darmstadt, Germany) using traditional solid-phase 9-Fluorenylmethoxycarbonyl (Fmoc) chemistry.³⁶ [¹⁸F]fluoride was purchased from PETNet Solutions (Sacramento, CA, USA) and delivered on a solid ion cartridge (PS-HCO₃, ABX GmbH, Radeberg, Germany). All solvents used for microfluidic synthesis were filtered prior to use with a Millex syringe driven 0.42 μm filter (Millipore, Bedford, MA, USA).

Microfluidic device description and operation

Radiolabeling reactions were performed on an Advion NanoTek® microfluidic flow chemistry device (Fig. 2). The Advion NanoTek® is a three-pump microfluidic flow chemistry device that is specifically designed for radiochemistry. The Advion NanoTek® is comprised of three main modules: a concentrator module, pump module, and reactor module. The concentrator module is comprised of a syringe pump, a 6-way valve, and a heating block that is designed to hold a 3 mL V-vial, and is used for [¹⁸F]fluoride drying and resolubilization. The pump module consists of 2 high pressure syringe pumps with attached storage loops, pump 1 (used to hold [¹⁸F]F-Py-TFP precursor, I) and pump 2 (used to hold peptide). The reactor module consists of 1 syringe pump, pump 3, and is used to transfer solvent through the system. Typical configuration of the device allows for a one or two-step reaction to be performed. The three-pump device can be loaded with three different reagents into pump 1 (P1), pump 2 (P2), and pump 3 (P3) (Fig. 2). The first reaction consists of reagent one (from P1) and reagent 3 (from P3) combining in reactor one, and the second reaction (if applicable) involves the combination of the bolus of reaction 1 with reagent 2 (from P2) in reactor 2.

Fig. 2 Schematic of Advion NanoTek® microfluidics device.

The entire system is controlled by a standalone laptop computer, which allows for the precise setting of reaction parameters including reaction volume, temperature, flow rate, and reagent ratio. Reactions were done in Discovery Mode of the device. Reactions were carried out in 2 or 4 meter long reactors with 100 μm inner diameter (holding 15.7 μL or 31.4 μL, respectively).

[¹⁸F]fluoride drying procedure

Azeotropic drying of the [¹⁸F]fluoride was done using the concentrator module of the microfluidics device. [¹⁸F]fluoride was delivered on a PS-HCO₃ cartridge and placed into the microfluidics device. [¹⁸F]fluoride was eluted from the cartridge with 450 μL of a tetrabutylammonium bicarbonate solution (30 μL 0.2 M TBA-HCO₃ in 600 μL 1 : 1 acetonitrile : water). [¹⁸F]fluoride/TBA-HCO₃ solution was then azeotropically dried with 3 × 1 mL anhydrous acetonitrile. [¹⁸F]fluoride/TBA-HCO₃ was then resolubilized in anhydrous acetonitrile.

Analysis

Radiochemical reactions were monitored using radio thin layer chromatography (radioTLC) and radio high performance liquid chromatography (radioHPLC) and identity of radiolabeled product was confirmed by co-elution with non-radioactive standards. RadioTLC was performed on a Bioscan 3000 TLC (Washington DC, USA) with a 1 : 1 ethyl acetate : hexanes mobile phase on silica TLC plates (EMD, Darmstadt, Germany). RadioHPLC was performed using a Phenomenex Jupiter 300 Å column (150 × 4.6 mm) 5 μm with HPLC mobile phase solvents 0.05% trifluoroacetic acid in water (v/v; solvent A) and 100% acetonitrile (solvent B). Mobile phase gradient was 9% solvent B isocratic for 2 min, then a linear gradient from 9% to 90% over 12 min with a flow rate of 3.0 mL min⁻¹. All peptides were analyzed by mass spectrometry using an ABI 4700 matrix-assisted laser desorption-ionization time of flight/time of flight (MALDI TOF/TOF) spectrometer (Applied Biosystems, Foster City, CA, USA).

[¹⁹F]-reference standards and precursor synthesis

N,N,N-Trimethyl-5-((2,3,5,6-tetrafluorophenoxy)-carbonyl)pyridine-2-aminium trifluoromethanesulfonate (I) and [¹⁹F]F-Py-TFP reference standard were synthesized as described by Olberg et al.²⁷ For [¹⁹F]F-Py-YGGFL reference standard synthesis, 50 mg of NH₂-YGGFL on TGR resin was combined with 3 equivalents of 6-fluoronicotinic acid, 3 equivalents of 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (HATU), and 6 equivalents of N-diisopropylethylamine (DIPEA) and rotated at room temperature for 1.5 hours. After completed reaction, peptide was cleaved from resin with 95 : 2.5 : 2.5 : 1 v : v : v : v trifluoroacetic acid (TFA) : water : ethanedithiol (EDT) : triisopropylsilane (TIPS) and purified by semi-preparative HPLC. [¹⁹F]F-Py-TFP reference standard was characterized by analytical HPLC (retention time 8.5 min, ESI Fig. 1†) and ESI MS (m/z = 288.2). NH₂-YGGFL peptide was cleaved from resin, purified by semi-preparative HPLC, and characterized by HPLC (retention time 4.5 min) and MALDI-TOF-MS (m/z + H = 555.3). [¹⁹F]F-Py-YGGFL reference standard was characterized by analytical HPLC (retention time 5.5 min, ESI Fig. 2†) and MALDI-TOF MS (m/z + H = 677.4).

Microfluidic synthesis of [¹⁸F]F-Py-TFP

N,N,N-Trimethyl-5-((2,3,5,6-tetrafluorophenoxy)-carbonyl)pyridine-2-aminium trifluoromethanesulfonate (I) was dissolved in 1 : 1 tBuOH : MeCN (10 mg mL⁻¹) and loaded into pump 1 (P1, Fig. 2) and dried [¹⁸F]F⁻/tetrabutylammonium bicarbonate (TBA-HCO₃) (∼5–30 mCi) in 1 : 1 tBuOH : MeCN was loaded into pump 3 (P3, Fig. 2). Pumps 1 and 3 were combined in reactor 1 (2 meter reactor) for the production of II. Temperature (40–110 °C), flow rates (15–60 μL min⁻¹), and reagent volume ratios (0.2–2, P1 : P3) were varied. Pump 3 volume was set at 20 μL for each optimization reaction. Radioactivity ranged from 5–400 μCi for each reaction.

Microfluidic synthesis of [¹⁸F]F-Py-YGGFL

[¹⁸F]F-Py-YGGFL synthesis was done in a two-step process. Following [¹⁸F]F-Py-TFP production on the microfluidics, the [¹⁸F]F-Py-TFP was transferred into the second microfluidic reactor (4 meter reactor) and reacted with NH₂-YGGFL (3 mg mL⁻¹), in 0.2 M NaHCO₃ buffer at pH 8.5. Temperature (30–80 °C), flow rate (10–30 μL min⁻¹) and reagent ratio (0.5–3 P2 : Rxn1) were varied. Optimization reactions were done with P3 pump volume of 20 μL. After optimized conditions were found, a larger 50 μL P3 volume reaction was done for scale up purposes.

Results and discussion

Microfluidic synthesis of [¹⁸F]F-Py-TFP

Previous work in the one-step synthesis and application of [¹⁸F]F-Py-TFP has shown the utility of this amine reactive prosthetic group as an alternative to the three step [¹⁸F]SFB approach. The sensitivity of [¹⁸F]SFB to changes in pH can have an detrimental effect on radiochemical yield even with very small shifts in pH.³⁷ In contrast, [¹⁸F]F-Py-TFP tends to be less affected by pH, while retaining the same reactivity as [¹⁸F]SFB. Currently [¹⁸F]F-Py-TFP is synthesized manually with radiochemical yields of 40–90% in 10–20 minutes.^27,35,34

Here, we present a fast, high yielding method for the synthesis of [¹⁸F]F-Py-TFP using microfluidics. For the microfluidic synthesis of [¹⁸F]F-Py-TFP, temperature, flow rate, and reagent ratio by volume were separately varied in order to maximize radiochemical incorporation yield. As seen in Fig. 3, for [¹⁸F]F-Py-TFP synthesis, temperature was varied from 40–110 °C, with highest radiochemical incorporation yield observed at 80 °C. Following temperature optimization, flow rate variation was investigated while other parameters were kept constant (temperature at 80 °C, reagent ratio at 0.5). Flow rate was varied from 20–40 μL min⁻¹, and highest radiochemical incorporation yield was achieved at 30 μL min⁻¹. After temperature and flow rate ratios were optimized, reagent ratio was varied and optimized for best results, keeping temperature (80 °C) and flow rate (30 μL min⁻¹) fixed. Reagent ratio was varied from 0.2–3 (40–600 μg of I) P1 : P3 by volume and highest radiochemical incorporation yield of 93 ± 4% was found at a reagent ratio of 0.5 (Fig. 3, n = 3–5 for each condition).

Fig. 3 Temperature, flow rate, and reagent ratio variation for II synthesis (each n = 3–5).

Overall radiochemical incorporation yields of [¹⁸F]F-Py-TFP as high as 97% were achieved within 3 min. Unlike conventional approaches using [¹⁸F]F-Py-TFP, no purification of the reaction mixture was performed prior to peptide coupling. Our approach improves yields and significantly reduces synthesis time of [¹⁸F]F-Py-TFP, and establishes an automated approach for its production.

Microfluidic synthesis of [¹⁸F]F-Py-YGGFL

Previously, two groups have described the synthesis of ¹⁸F-radiolabeled peptides using the Advion NanoTek® device. Bouvet et al.²⁰ demonstrated effective microfluidic radiolabeling of peptides using ¹⁸F-fluorodeoxyglucose ([¹⁸F]FDG). In this approach, [¹⁸F]FDG was used to radiolabel two different peptides, an aminooxy functionalized TATE peptide was radiolabeled in a one-step reaction via chemoselective oxime formation, and the glutathione (GSH) peptide was radiolabeled in a two-step reaction using a modified [¹⁸F]FDG-maleimide-hexyloxime (MHO) as a prosthetic group. This approach required the separate production of [¹⁸F]FDG on another platform prior to loading onto the microfluidics device and Bouvet et al. concluded that their results varied from batch to batch, and were greatly dependent on the quality of the starting [¹⁸F]FDG. Richter et al.¹⁸ demonstrated a similar approach as Bouvet et al., using [¹⁸F]SFB as a prosthetic group for radiolabeling of phosphopeptide-cell-penetrating peptide dimers. Here, the [¹⁸F]SFB was produced using a different platform and loaded onto the microfluidics device. This work showed an improvement of ¹⁸F-peptide radiolabeling yield from 2–4% using a traditional approach to up to 26% using the microfluidic approach. Selivanova et al.¹⁹ demonstrated direct ¹⁸F-radiolabeling of three modified bombesin peptides bearing either a trimethylammonium leaving group or a triarylsulfonium moiety. Direct ¹⁸F labeling of peptides required modification with a leaving group prior to loading onto the microfluidic device and high temperatures were required for the fluorination.

While all of these approaches have shown success, further investigation of microfluidic radiolabeling of peptides with other ¹⁸F-labeled prosthetic groups using mild reaction conditions are warranted. The goal of this work was therefore to radiolabel the NH₂-YGGFL model peptide with the [¹⁸F]F-Py-TFP prosthetic group on a single microfluidic device, from [¹⁸F]fluoride. For peptide radiolabeling using [¹⁸F]F-Py-TFP synthesis (Fig. 4) temperature was varied from 30–100 °C, flow rate was varied from 8–18 μL min⁻¹ and reagent ratio was varied from 1–3:1 P2 : reaction 1 bolus by volume. Following a systematic approach for the conjugation of [¹⁸F]F-Py-TFP, optimal conditions were found to be at 50 °C, with a flow rate of 12 μL min⁻¹ and a P2 : reaction 1 bolus volume ratio of 2. Overall these conditions gave peptide radiolabeling yields up to 28%. Specific activity was calculated to be to 1–50 mCi μmol⁻¹ and can be readily improved by the addition of higher amounts of starting radioactivity.

Fig. 4 Temperature, flow rate, and reagent ratio variation for synthesis of radiolabeled peptide. (Each n = 3–5).

After optimized conditions were found, a larger scale reaction was performed to assess scale up capabilities. P3 was set at 50 μL, and all other reaction parameters were kept the same. Product was purified by HPLC to give an overall non-decay corrected yield of 9%, in an overall time of 32 min. Additionally, to compare the microfluidic approach vs. the traditional labeling of NH₂-YGGFL, a test vial reaction was done using [¹⁸F]F-Py-TFP prosthetic group in a manual synthesis technique, according to literature.²⁷ The, manual synthesis resulted in a 6.2% radiochemical incorporation yield in a total time of 60 min.

In comparison to previously reported microfluidic peptide radiolabeling using [¹⁸F]FDG, [¹⁸F]SFB, or leaving group-modified peptides, our approach demonstrates improvement by utilizing only one automated device, and can be used to radiolabel any peptide bearing a free-amine functional group.

Conclusion

We have demonstrated the first microfluidic synthesis and subsequent peptide radiolabeling with the [¹⁸F]F-Py-TFP prosthetic group. Our approach establishes a simple, overall 2-step process to radiolabel a peptide on one microfluidic device from [¹⁸F] fluoride. To our knowledge, this is the first report of a 2-step continuous microfluidic ¹⁸F-peptide radiolabeling using a prosthetic group, from [¹⁸F]fluoride. This approach offers an improvement in radiochemical yield, decreased reaction time from 1 hour to 8 min from anhydrous [¹⁸F]fluoride, and implements an attractive automated approach for ¹⁸F radiolabeling of peptides. This can be seen as a method for ¹⁸F-radiolabeling of amine-bearing peptides serving as a platform for radiolabeling of clinically relevant peptides in a rapid and automated fashion. Radiolabeling of other peptides is currently underway. Future work will focus on the radiolabeling of multiple peptides simultaneously, with the ultimate goal of developing a high-throughput, automated synthesis method for radiolabeling peptides. Additionally, we will focus on incorporation of solid-phase purification into the microfluidic platform, to further expedite the overall production of ¹⁸F-radiolabeled peptides for molecular imaging.

Acknowledgements

The authors would like to acknowledge Lee Collier for technical advice on Advion NanoTek®, Meghann Radcliffe of PETNet Solutions for help with radioactivity deliveries, and Ryan Davis for ESI confirmation of precursor. This research was supported by the NIH grant 1RC4EB01 2836-01 and the Office of Science, United States Department of Energy under the award # DE-SC0002061.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra10520f

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