An Automated and Putatively Versatile Approach for Labeling Peptides with Fluorine-18 Using the FastlabTM Synthesizer; Exemplified with Glu-the CO-Lys Pharmacophore

Background: Noninvasive molecular imaging using peptides and biomolecules labelled with positron emitters has become important for detection of cancer and other diseases with PET (positron emission tomography). The positron emitting radionuclide fluorine-18 is widely available in high yield from cyclotrons and has favorable decay (t1/2 109.7 min) and imaging properties. 18F-Labelling of biomolecules and peptides for use as radiotracers is customarily achieved in a two-step approach, which can be challenging to automate. 6-[18F]Fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester ([18F]F-Py-TFP) is a versatile 18F-prosthetic group for this purpose, which can be rapidly be produced in an one-step approach on solid support. This work details an automated procedure on the cassette-based GE FASTlabTM platform for the labeling of a peptidomimetic, exemplified by the case of using the Glu-CO-Lys motif to produce [18F]DCFPyL, a ligand targeting the prostate specific membrane antigen (PSMA). Results: From fluorine-18 delivery a fully automated two-step radiosynthesis of [18F]DCFPyL was completed in 56 min with an overall end of synthesis yield as high as 37% using SPE purification on the GE FASTlabTM platform. Conclusions: Putatively, this radiolabeling methodology is inherently amenable to automation with a diverse set of synthesis modules, and it should generalize for production of a broad spectrum of biomolecule-based radiotracers for use in PET imaging.

A Purification Method of 18F-FP-(+)-DTBZ via Solid-Phase Extraction With Combined Cartridges

Background: To optimize [18F] 9-fluoropropyl-(+)-dihydrotetrabenazine (18F-FP-(+)-DTBZ) purification via solid-phase extraction (SPE) with combined cartridges to facilitate its widespread clinical application.Methods: A modified SPE purification method, employing Sep-Pak PS-2 and Sep-Pak C18 cartridges, was used for the preparation of 18F-FP-(+)-DTBZ. This method was compared to the purification method of high-pressure liquid chromatography (HPLC) and SPE with one cartridge, following quality control test and positron emission tomography (PET) imaging in healthy volunteers and patients with parkinsn's disease (PD).Results: A SPE purification method integrating Sep-Pak PS-2 and Sep-Pak C18 cartridges was implemented successfully. The retention time of 18F-FP-(+)-DTBZ purified by HPLC, SPE with Sep-Pak PS-2, SPE with Sep-Pak C18, and SPE with combined use of Sep-Pak PS-2 and Sep-Pak C18 cartridges was 8.7, 8.8, 8.7, and 8.9 min, respectively. Fewest impurity peak was detected in 18F-FP-(+)-DTBZ purified by the SPE with combined use of Sep-Pak PS-2 and Sep-Pak C18 cartridges. This modified SPE purification method provided a satisfactory radiochemical yield of 29 ± 1.8% with radiochemical purity >99% and shortened synthesis time to 27 min. The brain uptake of 18F-FP-(+)-DTBZ purified by the modified SPE was comparable to that purified by HPLC in both healthy volunteers and PD patients.Conclusions: A SPE method integrating Sep-Pak PS-2 and Sep-Pak C18 cartridges for purification of 18F-FP-(+)-DTBZ may be highly suited to automatic synthesis for routine clinical applications, as it provides excellent radiochemical purity, high yield as well as operational simplicity.

Automated synthesis of the 16α-[18F]fluoroestradiol ([18F]FES): minimization of precursor amount and resulting benefits

The 16α-[18F]Fluoroestradiol ([18F]FES) is an established PET radiotracer for estrogen positive (ER+) breast cancer. Although the radiosynthesis is well-described, the majority of the published methods suffer from modest or irreproducible yields and time-intensive purification procedures. In view of the considerable clinical applications, development of a more efficient and faster synthesis of [18F]FES still remains a task of a significant practical importance. [18F]FES was produced by a direct nucleophilic radiofluorination of 3-O-methoxymethyl-16,17-O-sulfuryl-16-epiestriol (MMSE), followed by acidic hydrolysis using HCl/CH3CN. [18F]Fluoride retained on a QMA carb cartridge (46 mg) was eluted by solution of 1.2 mg of tetrabutylammonium tosylate (TBAOTs) in EtOH. After fluorination reaction (0.3 mg MMSE, 1 ml of CH3CN/100 °C, 5 min) [18F]FES was isolated by single-cartridge SPE purification using OASIS WAX 3cc, elution accomplished with aqueous ethanol of different concentrations. On а GE TRACERlab FX N Pro automated module [18F]FES (formulated in normal saline with 5% EtOH) was obtained in 33 ± 3% yield (n = 5, non-decay corrected) within 32 min. Reduction of precursor amount, exclusion of azeotropic drying step and simplification of purification make the suggested method readily adaptable to various automated synthesizers and offers significant cost decrease.

Oligonucleotide Preparation Approach for Assembly of DNA Synthons

An effective oligonucleotide preparation approach for the thermodynamically balanced, inside-out (TBIO) PCR-based assembly of long synthetic DNA molecules (synthons) is described in the current work. We replaced the necessity to purify individual oligonucleotides with just one purification procedure per approximately 500 base pairs (bp) of duplex DNA. So for an enhanced green fluorescent protein (EGFP) gene of 717 bp, we synthesized 24 oligonucleotides with a length of 50 bases and performed just two solid-phase extraction (SPE) purification procedures. It was found that the capacity of ZipTip microextractors, usually used for sample desalting in proteomics, perfectly corresponds to the gene synthesis scale (40–60 pmol). The robustness of the approach was validated with a 65-mer oligonucleotide design of the same gene. The modification of the oligonucleotide concentration gradient from the original TBIO scheme substantially increased the purity of the PCR product. We proposed a mechanism for the formation of supramolecular structures, which often occur during TBIO assembly. By using the proposed workflow, any laboratory with a standard facility for molecular biology manipulation, a 16-channel oligonucleotide synthesizer, and a conventional thermocycler has the ability to prepare one gene with a length of about 700 bp per day.

Comparison of an HPLC-MS/MS Method with Multiple Commercial ELISA Kits on the Determination of Levels of 8-oxo-7,8-Dihydro-2'-Deoxyguanosine in Human Urine

Introduction: Analysis of 8-oxodG is usually conducted by either chromatography-based methods or by immunochemical methods commonly used based upon their low cost and high-throughput. However, concern regarding the accuracy of ELISA methods has complicated their use. We directly compare the levels of urinary 8-oxodG obtained by HPLC-MS/MS with three commercially available ELISA kits in this report. Methods: In the current study, a total of 9 human urine samples were analyzed by LC-MS/MS and three commonly used commercial available ELISA kits. Results: We found that urinary 8-oxodG levels analyzed by HPLC-MS/MS [1.4 ± 0.3 nmol/mmol creatinine) were 7.6- to 23.5-fold lower than those detected by ELISA. Overall, the correlations between ELISA and HPLC-MS/MS were poor but were improved after SPE purification for kits from ENZO (P = 0.2817 without SPE; P = 0.0086 with SPE) and Abcam (P = 0.0596 without SPE; P = 0.0473 with SPE). Discussion and conclusion: While we confirmed that SPE purification can improve the correlation between the selected ELISA kits and HPLC-MS/MS, HPLC-MS/MS is still the method of choice to accurately assess the levels of 8-oxodG in human urine.