Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles.

The presence of circulating tumor cells (CTCs) in a patient's bloodstream is a hallmark of metastatic cancer. The detection and analysis of CTCs is a promising diagnostic and prognostic strategy as they may carry useful genetic information from their derived primary tumor, and the enumeration of CTCs in the bloodstream has been known to scale with disease progression. However, the detection of CTCs is a highly challenging task owing to their sparse numbers in a background of billions of background blood cells. To effectively utilize CTCs, there is a need for an assay that can detect CTCs with high specificity and can locally enrich CTCs from a liquid biopsy. We demonstrate a versatile methodology that addresses these needs by utilizing a combination of nanoparticles. Enrichment is achieved using targeted magnetic nanoparticles and high specificity detection is achieved using a ratiometric detection approach utilizing multiplexed targeted and non-targeted surface-enhanced Raman Scattering Nanoparticles (SERS-NPs). We demonstrate this approach with model prostate and cervical circulating tumor cells and show the ex vivo utility of our methodology for the detection of PSMA or folate receptor over-expressing CTCs. Our approach allows for the mitigation of interference caused by the non-specific uptake of nanoparticles by other cells present in the bloodstream and our results from magnetically trapped CTCs reveal over a 2000% increase in targeted SERS-NP signal over non-specifically bound SERS-NPs.

Expression and function of hexose transporters GLUT1, GLUT2, and GLUT5 in breast cancer-effects of hypoxia.

Elevated growth in breast cancer (BC) activates hypoxia-inducible factor (HIF1α) and downstream, facilitative glucose transporter 1 (GLUT1), which can be visualized with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG). GLUT5 (fructose) and GLUT2 (glucose/fructose) might provide alternative targets for BC imaging as to why effects of hypoxia on GLUT1/2/5 levels and function were examined in human BC models. GLUT1/2/5 and HIF1α mRNA was analyzed in BC patient biopsies. In MCF10A, MCF7, and MDA-MB231 cells, [18F]FDG, 6-deoxy-6-[18F]fluoro-d-fructose (6-[18F]FDF) and [18F]-fluoroazomycin arabinoside were used in radiotracer experiments, whereas GLUT1/2/5 mRNA was analyzed with real-time PCR and protein levels determined via Western blot/immunohistochemistry. Positron emission tomography imaging was performed in MCF7 and MDA-MB231 tumor-bearing mice. Glucose/fructose/cytochalasin B reduced cellular 6-[18F]FDF uptake by 50%, indicating functional involvement of GLUT2. With GLUT5 staining lower than GLUT1, 6-[18F]FDF revealed lower uptake than [18F]FDG [standardized uptake value (SUV)6-[18F]FDF, 120 min 0.77 ± 0.06 vs. SUV[18F]FDG, 120 min 1.08 ± 0.07] in MDA-MB231 tumors and was blocked by 20% with cytochalasin B after 10 min. Whereas correspondence between 6-[18F]FDF uptake and GLUT5 protein was low, high GLUT2 levels were detected in all cell lines and tumor models. Besides GLUT1, GLUT5 seems to be regulated under hypoxia on the molecular and functional level. Additionally, results strongly support a functional involvement of GLUT2 in fructose metabolism, possibly by compensating for the weaker expression and function of GLUT5 in BC.-Hamann, I., Krys, D., Glubrecht, D., Bouvet, V., Marshall, A., Vos, L., Mackey, J. R., Wuest, M., Wuest, F. Expression and function of hexose transporters GLUT1, GLUT2, and GLUT5 in breast cancer-effects of hypoxia.

Molecular Imaging of GLUT1 and GLUT5 in Breast Cancer: A Multitracer Positron Emission Tomography Imaging Study in Mice.

Use of [18F]FDG-positron emission tomography (PET) in clinical breast cancer (BC) imaging is limited mainly by insufficient expression levels of facilitative glucose transporter (GLUT)1 in up to 50% of all patients. Fructose-specific facilitative hexose transporter GLUT5 represents an alternative biomarker for PET imaging of hexose metabolism in BC. The goal of the present study was to compare the uptake characteristics of selected hexose-based PET radiotracers in murine BC model EMT6. Uptake of 1-deoxy-1-[18F]fluoro-d-fructose (1-[18F]FDF), 6-deoxy-6-[18F]fluoro-d-fructose (6-[18F]FDF), 1-deoxy-1-[18F]fluoro-2,5-anhydro-mannitol (1-[18F]FDAM), 2-deoxy-2-[18F]fluoro-d-glucose (2-[18F]FDG), and 6-deoxy-6-[18F]fluoro-d-glucose (6-[18F]FDG) was studied in EMT6 cells, tumors, and muscle and correlated to GLUT1 and GLUT5 expression levels. Fructose-derivative 6-[18F]FDF revealed greater tumor uptake than did structural analog 1-[18F]FDF, whereas 1-[18F]FDAM with locked anomeric configuration showed similar low tumor uptake to that of 1-[18F]FDF. Glucose-derivative 6-[18F]FDG reached maximum tumor uptake at 20 minutes, with no further accumulation over time. Uptake of 2-[18F]FDG was greatest and continuously increasing owing to metabolic trapping through phosphorylation by hexokinase II. In EMT6 tumors, GLUT5 mRNA expression was 20,000-fold lower compared with GLUT1. Whereas the latter was much greater in tumor than in muscle tissue (GLUT1 50:1), the opposite was found for GLUT5 mRNA expression (GLUT5 1:6). GLUT5 protein levels were higher in tumor versus muscle tissue as determined by Western blot and immunohistochemistry. Our data suggest that tumor uptake of fructose metabolism-targeting radiotracers 1-[18F]FDF, 6-[18F]FDF, and 1-[18F]FDAM does not correlate with GLUT5 mRNA levels but is linked to GLUT5 protein levels. In conclusion, our results highlight the importance of detailed biochemical studies on GLUT protein expression levels in combination with PET imaging studies for functional characterization of GLUTs in BC.

Targeting Prostate-Specific Membrane Antigen (PSMA) with F-18-Labeled Compounds: the Influence of Prosthetic Groups on Tumor Uptake and Clearance Profile.

PURPOSE: Prostate-specific membrane antigen (PSMA) is an important biomarker expressed in the majority of prostate cancers. The favorable positron emission tomography (PET) imaging profile of the PSMA imaging agent 2-(3-(1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl)-ureido)-pentane-dioic acid [18F]DCFPyL in preclinical prostate cancer models and in prostate cancer patients stimulated the development and validation of other fluorine-containing PSMA inhibitors to further enhance pharmacokinetics and simplify production methods. Here, we describe the synthesis and radiopharmacological evaluation of various F-18-labeled PSMA inhibitors which were prepared through different prosthetic group chemistry strategies. PROCEDURES: Prosthetic groups N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), 4-[18F]fluorobenzaldehyde, and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) were used for bioconjugation reactions to PSMA-binding lysine-urea-glutamate scaffold via acylation and oxime formation. All fluorine-containing PSMA inhibitors were tested for their PSMA inhibitory potency in an in vitro competitive binding assay in comparison to an established reference compound [125I]TAAG-PSMA. Tumor uptake and clearance profiles of three F-18-labeled PSMA inhibitors ([18F]4, [18F]7, and [18F]8) were studied with dynamic PET imaging using LNCaP tumor-bearing mice. RESULTS: F-18-labeled PSMA inhibitors were synthesized in 32-69 % radiochemical yields using (1) acylation reaction at the primary amino group of the lysine residue with [18F]SFB and (2) oxime formation with 4-[18F]fluorobenzaldehyde and [18F]FDG using the respective aminooxy-functionalized lysine residue. Compound 7 displayed an IC50 value of 6 nM reflecting very high affinity for PSMA. Compounds 4 and 8 showed IC50 values of 13 and 62 nM, respectively. The IC50 value of reference compound DCFPyL was 13 nM. Dynamic PET imaging revealed the following SUV60min for radiotracer uptake in PSMA(+) LNCaP tumors: 0.98 ([18F]DCFPyL), 2.11 ([18F]7), 0.40 ([18F]4), and 0.19 ([18F]8). CONCLUSION: The observed tumor uptake and clearance profiles demonstrate the importance of the selected prosthetic group on the pharmacokinetic profile of analyzed PSMA-targeting radiotracers. Radiotracer [18F]7 displayed the highest uptake and retention in LNCaP tumors, which exceeded uptake values of reference compound [18F]DCFPyL by more than 100 %. Despite the higher kidney and liver uptake and retention of compound [18F]7, the simple radiosynthesis and the exceptionally high tumor uptake (SUV60min 2.11) and retention make radiotracer [18F]7 an interesting alternative to radiotracer [18F]DCFPyL for PET imaging of PSMA in prostate cancer.

Synthesis and pre-clinical evaluation of an (18)F-labeled single-chain antibody fragment for PET imaging of epithelial ovarian cancer.

Anti-CA125 antibodies have been used in immunoassays to quantify levels of shed antigen in the serum of patients who are under surveillance for epithelial ovarian cancer (EOC). However, there is currently no molecular imaging probe in the clinic for the assessment of CA125 expression in vivo. The present study describes the development of an (18)F-labeled single-chain variable fragment (scFv) for PET imaging of CA125 in preclinical EOC models. Anti-CA125 scFv was derived from MAb-B43.13 by recombinant expression of the fragment in E.coli. Fragment scFv-B43.13 was purified via immobilized metal affinity chromatography and characterized for antigen binding via immuno-staining and flow cytometry. Prosthetic group N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB) was used for radiolabeling of scFv-B43.13. Preclinical ovarian cancer models were developed based on ovarian cancer cell lines OVCAR3 (CA125-positive) and SKOV3 (CA125-negative) in NIH-III mice. The radiopharmacological profile of (18)F-labeled scFv-B43.13 ([(18)F]FBz-scFv-B43.13) was studied with PET. [(18)F]FBz-scFv-B43.13 was prepared in radiochemical yields of 3.7 ± 1.8% (n = 5) at an effective specific activity of 3.88 ± 0.76 GBq/µmol (n = 5). The radiotracer demonstrated selective uptake in CA125-positive OVCAR3 cells and virtually no uptake in CA125-negative SKOV3 cells. Standardized uptake values (SUV) of radioactivity uptake in OVCAR3 tumors was 0.5 (n = 3) and 0.3 (n = 2) in SKOV3 tumors after 60 min post injection (p.i.).

Automated synthesis of [(18)F]DCFPyL via direct radiofluorination and validation in preclinical prostate cancer models.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is frequently overexpressed and upregulated in prostate cancer. To date, various (18)F- and (68)Ga-labeled urea-based radiotracers for PET imaging of PSMA have been developed and entered clinical trials. Here, we describe an automated synthesis of [(18)F]DCFPyL via direct radiofluorination and validation in preclinical models of prostate cancer. METHODS: [(18)F]DCFPyL was synthesized via direct nucleophilic heteroaromatic substitution reaction in a single reactor TRACERlab FXFN automated synthesis unit. Radiopharmacological evaluation of [(18)F]DCFPyL involved internalization experiments, dynamic PET imaging in LNCaP (PSMA+) and PC3 (PSMA-) tumor-bearing BALB/c nude mice, biodistribution studies, and metabolic profiling. In addition, reversible two-tissue compartmental model analysis was used to quantify pharmacokinetics of [(18)F]DCFPyL in LNCaP and PC3 tumor models. RESULTS: Automated radiosynthesis afforded radiotracer [(18)F]DCFPyL in decay-corrected radiochemical yields of 23 ± 5 % (n = 10) within 55 min, including HPLC purification. Dynamic PET analysis revealed rapid and high uptake of radioactivity (SUV5min 0.95) in LNCaP tumors which increased over time (SUV60min 1.1). Radioactivity uptake in LNCaP tumors was blocked in the presence of nonradioactive DCFPyL (SUV60min 0.22). The muscle as reference tissue showed rapid and continuous clearance over time (SUV60min 0.06). Fast blood clearance of radioactivity resulted in tumor-blood ratios of 1.0 after 10 min and 8.3 after 60 min. PC3 tumors also showed continuous clearance of radioactivity over time (SUV60min 0.11). Kinetic analysis of PET data revealed the two-tissue compartmental model as best fit with K 1 = 0.12, k 2 = 0.18, k 3 = 0.08, and k 4 = 0.004 min(-1), confirming molecular trapping of [(18)F]DCFPyL in PSMA+ LNCaP cells. CONCLUSIONS: [(18)F]DCFPyL can be prepared for clinical applications simply and in good radiochemical yields via a direct radiofluorination synthesis route in a single reactor automated synthesis unit. Radiopharmacological evaluation of [(18)F]DCFPyL confirmed high PSMA-mediated tumor uptake combined with superior clearance parameters. Compartmental model analysis points to a two-step molecular trapping mechanism based on PSMA binding and subsequent internalization leading to retention of radioactivity in PSMA+ LNCaP tumors.

Erratum: Automated synthesis and dosimetry of 6-deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF): a radiotracer for imaging of GLUT5 in breast cancer.

6-Deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF) is a promising PET radiotracer for imaging GLUT5 in breast cancer. The present work describes GMP synthesis of 6-[(18)F]FDF in an automated synthesis unit (ASU) and dosimetry calculations to determine radiation doses in humans. GMP synthesis and dosimetry calculations are important prerequisites for first-in-human clinical studies of 6-[(18)F]FDF. The radiochemical synthesis of 6-[(18)F]FDF was optimized and adapted to an automated synthesis process using a Tracerlab FXFN ASU (GE Healthcare). Starting from 30 GBq of cyclotron-produced n.c.a. [(18)F]fluoride, 2.9 ± 0.1 GBq of 6-[(18)F]FDF could be prepared within 50 min including HPLC purification resulting in an overall decay-corrected radiochemical yield of 14 ± 3% (n = 11). Radiochemical purity exceeded 95%, and the specific activity was greater than 5.1 GBq/µmol. Sprague-Dawley rats were used for biodistribution experiments, and dynamic and static small animal PET experiments. Biodistribution studies served as basis for allometric extrapolation to the standard man anatomic model and normal organ-absorbed dose calculations using OLINDA/EXM software. The calculated human effective dose for 6-[(18)F]FDF was 0.0089 mSv/MBq. Highest organ doses with a dose equivalent of 0.0315 mSv/MBq in a humans were found in bone. Injection of 370 MBq (10 mCi) of 6-[(18)F]FDF results in an effective whole body radiation dose of 3.3 mSv in humans, a value comparable to that of other (18)F-labeled PET radiopharmaceuticals. The optimized automated synthesis under GMP conditions, the good radiochemical yield and the favorable human radiation dosimetry estimates support application of 6-[(18)F]FDF in clinical trials for molecular imaging of GLUT5 in breast cancer patients.[This corrects the article on p. 248 in vol. 4.].

Automated synthesis and dosimetry of 6-deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF): a radiotracer for imaging of GLUT5 in breast cancer.

6-Deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF) is a promising PET radiotracer for imaging GLUT5 in breast cancer. The present work describes GMP synthesis of 6-[(18)F]FDF in an automated synthesis unit (ASU) and dosimetry calculations to determine radiation doses in humans. GMP synthesis and dosimetry calculations are important prerequisites for first-in-human clinical studies of 6-[(18)F]FDF. The radiochemical synthesis of 6-[(18)F]FDF was optimized and adapted to an automated synthesis process using a Tracerlab FXFN ASU (GE Healthcare). Starting from 30 GBq of cyclotron-produced n.c.a. [(18)F]fluoride, 2.9 ± 0.1 GBq of 6-[(18)F]FDF could be prepared within 50 min including HPLC purification resulting in an overall decay-corrected radiochemical yield of 14 ± 3% (n = 11). Radiochemical purity exceeded 95%, and the specific activity was greater than 5.1 GBq/µmol. Sprague-Dawley rats were used for biodistribution experiments, and dynamic and static small animal PET experiments. Biodistribution studies served as basis for allometric extrapolation to the standard man anatomic model and normal organ-absorbed dose calculations using OLINDA/EXM software. The calculated human effective dose for 6-[(18)F]FDF was 0.0089 mSv/MBq. Highest organ doses with a dose equivalent of 0.0315 mSv/MBq in a humans were found in bone. Injection of 370 MBq (10 mCi) of 6-[(18)F]FDF results in an effective whole body radiation dose of 3.3 mSv in humans, a value comparable to that of other (18)F-labeled PET radiopharmaceuticals. The optimized automated synthesis under GMP conditions, the good radiochemical yield and the favorable human radiation dosimetry estimates support application of 6-[(18)F]FDF in clinical trials for molecular imaging of GLUT5 in breast cancer patients.

Application of [18F]FDG in radiolabeling reactions using microfluidic technology.

Radiolabeling of peptides with the short-lived positron emitter fluorine-18 is usually a challenging endeavour. Conventional radiolabeling reactions mostly require fairly large amounts of peptides as labeling precursors, and extensive synthesis times. Intrinsic advantages of microfluidic technology permit to overcome these hurdles. Herein, we describe how microfluidic technology combined with [(18)F]FDG as readily available PET radiotracer allows for fast and high yielding radiolabeling reactions of peptides with fluorine-18.

Fluorine- and rhenium-containing geldanamycin derivatives as leads for the development of molecular probes for imaging Hsp90.

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone responsible for protein quality control in cells. Hsp90 has been shown to be overexpressed in many human cancers. This has prompted extensive research on Hsp90 inhibitors as novel anticancer agents and, more recently, the development of molecular probes for imaging Hsp90 expression in vivo. This work describes the development of various fluorine-containing and rhenium-containing geldanamycin derivatives as leads for the development of corresponding (18)F-labeled and (99m)Tc-labeled PET and SPECT probes for molecular imaging of Hsp90 expression. All compounds were evaluated in an in vitro ATPase activity assay using Hsp90 isoform Hsp82p. Fluorobenzoylated geldanamycin derivative 5 displayed comparable inhibitory potency like parent compound geldanamycin.

(18)F-Labeled phosphopeptide-cell-penetrating peptide dimers with enhanced cell uptake properties in human cancer cells.

INTRODUCTION: Phosphopeptides represent interesting compounds to study and elucidate cellular protein phosphorylation/dephosphorylation processes underlying various signal transduction pathways. However, studies of phosphopeptide action in cells are severely constrained by the negatively charged phosphate moiety of the phosphopeptide resulting in poor transport through the cell membrane. The following study describes the synthesis and radiopharmacological evaluation of two (18)F-labeled phosphopeptide-cell-penetrating peptide dimers. The polo-like kinase-1-binding hexaphosphopeptide H-Met-Gln-Ser-pThr-Pro-Leu-OH was coupled to cell-penetrating peptides (CPPs), either sC18, a cathelicidin-derived peptide, or the human calcitonin derivative hCT(18-32)-k7. METHODS: Radiolabeling was accomplished with the prosthetic group N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB) using both, conventional and microfluidic-based bioconjugation of [(18)F]SFB to N-terminal end of phosphopeptide part of the peptide dimers. Cellular uptake studies in human cancer cell lines HT-29 and FaDu cells at 4 °C and 37 °C and small animal PET in BALB/c mice were utilized for radiopharmacological characterization. RESULTS: Isolated radiochemical yields ranged from 2% to 4% for conventional bioconjugation with [(18)F]SFB. Significantly improved isolated radiochemical yields of up to 26% were achieved using microfluidic technology. Cellular uptake studies of radiolabeled phosphopeptide and phosphopeptide-CPP dimers indicate enhanced internalization of 50% ID/mg protein after 2 h for both phosphopeptide dimers compared to the phosphopeptide alone (<1% ID/mg protein). In vivo biodistribution of (18)F-labeled peptide dimers was determined with small animal PET revealing a superior biodistribution pattern of sC18-containing peptide dimer MQSpTPL-sC18 [(18)F]4. CONCLUSION: ([18)F]SFB labeling of the phosphopeptide-CPP dimers using a microfluidic system leads to an improved chemoselectivity towards the N-terminal NH(2) group compared to the conventional labeling approach. Cell-penetrating peptide sC18 can be considered as an ideal molecular shuttle for intracellular delivery of the Plk1-PBD-binding hexaphosphopeptide as demonstrated by its favourable radiopharmacological profile.

Microfluidic technology: an economical and versatile approach for the synthesis of O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET).

A new synthesis of O-(2-[(18)F]fluoroethyl)-L-tyrosine [(18)F]FET was developed using a NanoTek® microfluidic synthesis system (Advion BioSciences, Inc.). Optimal reaction conditions were studied through screening different reaction parameters like temperature, flow rate, reaction time, concentration of the labeling precursor, and the applied volume ratio between the labeling precursor and [(18)F]fluoride. [(18)F]FET was obtained after HPLC purification with 50% decay-corrected radiochemical yield starting from as little as 40 µg of labeling precursor. Small animal PET studies in EMT-6 tumor bearing mice showed radioactivity accumulation in the tumor (SUV(60min) 1.21±0.2) resulting in an slightly increasing tumor-to-muscle ratio over time.

Copper-free click chemistry with the short-lived positron emitter fluorine-18.

The copper-free strain-promoted click chemistry between (18)F-labeled aza-dibenzocyclooctyne [(18)F]FB-DBCO and various azides is described. [(18)F]FB-DBCO was prepared in 85% isolated radiochemical yield (decay-corrected) through acylation of amino aza-dibenzocyclooctyne 1 with N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB). [(18)F]FB-DBCO showed promising radiopharmacological profil with fast blood clearance as assessed with dynamic small animal PET studies. Metabolic stability of [(18)F]FB-DBCO was 60% of intact compound after 60 min post injection in normal Balb/C mice and blood clearance half-life was determined to be 53 s based on the time-activity-curve (TAC). Copper-free click chemistry was performed with various azides at low concentrations (1-2 µM) which differed in their structural complexity in different solvents (methanol, water, phosphate buffer and in bovine serum albumin (BSA) solution). Reaction proceeded best in methanol (>95% yield after 15 min at room temperature), whereas reaction in BSA required longer reaction times of 60 min and 40 °C upon completion.

Synthesis of hypoxia imaging agent 1-(5-deoxy-5-fluoro-α-D-arabinofuranosyl)-2-nitroimidazole using microfluidic technology.

INTRODUCTION: Microfluidic technology allows fast reactions in a simple experimental setup, while using very low volumes and amounts of starting material. Consequently, microfluidic technology is an ideal tool for radiolabeling reactions involving short-lived positron emitters. Optimization of the complex array of different reaction conditions requires knowledge of the different reaction parameters linked to the microfluidic system as well as their influence on the radiochemical yields. 1-(5-Deoxy-5-fluoro-α-d-arabinofuranosyl)-2-nitroimidazole ([(18)F]FAZA) is a frequently used radiotracer for PET imaging of tumor hypoxia. The present study describes the radiosynthesis of [(18)F]FAZA by means of microfluidic technology and subsequent small animal PET imaging in EMT-6 tumor-bearing mice. METHODS: Radiosyntheses were performed using the NanoTek Microfluidic Synthesis System (Advion BioSciences, Inc.). Optimal reaction conditions were studied through screening different reaction parameters like temperature, flow rate, residency time, concentration of the labeling precursor (1-(2,3-di-O-acetyl-5-O-tosyl-α-d-arabinofuranosyl)-2-nitroimidazole) and the applied volume ratio between the labeling precursor and [(18)F]fluoride. RESULTS: Optimized reaction conditions at low radioactivity levels (1 to 50 MBq) afforded 63% (decay-corrected) of HPLC-purified [(18)F]FAZA within 25 min. Higher radioactivity levels (0.4 to 2.1 GBq) gave HPLC-purified [(18)F]FAZA in radiochemical yields of 40% (decay-corrected) within 60 min at a specific activity in the range of 70 to 150 GBq/µmol. Small animal PET studies in EMT-6 tumor-bearing mice showed radioactivity accumulation in the tumor (SUV(20min) 0.74 ± 0.08) resulting in an increasing tumor-to-muscle ratio over time. CONCLUSIONS: Microfluidic technology is an ideal method for the rapid and efficient radiosynthesis of [(18)F]FAZA for preclinical radiopharmacological studies. Careful analysis of various reaction parameters is an important requirement for the understanding of the influence of different reaction parameters on the radiochemical yield using microfluidic technology. Exploration of microfluidic technology for the radiosynthesis of other PET radiotracers in clinically relevant radioactivity levels is currently in progress.

A short and economical synthesis of orthogonally protected C-linked 2-deoxy-2-acetamido-alpha-D-galactopyranose derivatives.

A short and high-yielding synthesis has been devised to prepare C-linked 2-deoxy-2-acetamido-alpha-D-galactopyranose derivative 3. One of the main advantages of this approach is that it employs commercially available and inexpensive d-glucosamine as the starting material. The key steps include a highly stereoselective C-allylation followed by epimerization of the C-4 hydroxyl group. Building block 3 and orthogonally protected C-linked 2-deoxy-2-acetamido-alpha-D-galactopyranose derivative 2 were obtained in 44% overall yield (six steps) and 29% overall yield (eight steps), respectively. This represents a significant improvement over previously reported syntheses.

Aggregation of antifreeze glycoprotein fraction 8 and its effect on antifreeze activity.

Antifreeze glycoproteins (AFGPs) have many potential applications ranging from the cryopreservation and hypothermic storage of tissues and organs to the preservation of various frozen food products. Since supplying native AFGP for these applications is a labor-intensive and costly process, the rational design and synthesis of functional AFGP analogues is a very attractive alternative. While structure-function studies have implicated specific structural motifs as essential for antifreeze activity in AFGP, the relationship between solution conformation and antifreeze activity is poorly understood. Toward this end, we have analyzed AFGP8 in aqueous solutions using dynamic light scattering (DLS) and circular dichroism (CD). Our results indicate that AFGP8 forms discrete aggregates in solution. These aggregates are predominantly composed of dimers that form at solution concentrations greater than 20 mM. CD spectroscopy indicates that the preferred solution conformation of AFGP8 is consistent with that of random coil. However, significant beta-sheet and alpha-helix character is observed in more concentrated solutions, indicating that these glycopeptides are highly flexible in solution. Aggregation appears to have a minimal effect on the overall solution conformation. Thermal hysteresis (TH) activity of the aggregated solutions is much higher than that of less concentrated solutions that do not form aggregates. While cooperative functioning between lower and higher molecular weight AFGPs has been reported, this is the first instance where cooperative functioning in lower molecular weight AFGPs has been observed.

Antifreeze glycoproteins: structure, conformation, and biological applications.

Antifreeze glycoproteins (AFGPs) are a novel class of biologically significant compounds that possess the ability to inhibit the growth of ice both in vitro and in vivo. Any organic compound that possesses the ability to inhibit the growth of ice has many potential medical, industrial, and commercial applications. In an effort to elucidate the molecular mechanism of action, various spectroscopic and physical techniques have been used to investigate the solution conformations of these glycoproteins. This review examines the characterization of AFGPs and potential biological applications relating to stabilization of lipid membranes and vitrification adjuvants.