Fingerprinting Biogenic Aldehydes through Pattern Recognition Analyses of Excitation-Emission Matrices.

Biogenic carbonyls, especially aldehydes, have previously demonstrated their potential to serve as early diagnostic biomarkers for disease and injury that have not been fully realized owing, in part, to the lack of a rapid and simple point-of-care method for aldehyde identification. The ability to determine which carbonyl compound is elevated and not just the total aldehydic load may provide more disease- or injury-specific diagnostic information. Toward this end, a novel fluorophore is presented that is able to form a complex with biogenic carbonyls under catalyst-free conditions so as to give a fluorescent fingerprint of the resulting hydrazone. The successful identification of bound carbonyls was accomplished with a newly described algorithm that applied principal curvature analysis of excitation-emission matrices to reduce surface features to ellipse representations, followed by a pattern-matching routine. With this algorithm, carbonyls were identified over a range of concentrations, and mixture components were successfully parsed. Overall, the results presented lay the groundwork for novel implementations of chemometrics to low-cost, rapid, and simple-to-implement point-of-care diagnostics.

Methyl 5-MeO-N-aminoanthranilate, a minimalist fluorogenic probe for sensing cellular aldehydic load.

Methyl 5-MeO-N-aminoanthranilate, a fluorogenic probe comprising a single substituted benzene ring has been applied towards the fluorescence detection of endogenous carbonyls through rapid, catalyst-free complexation of these bio-derived markers of cell stress under physiological conditions. The products formed during the reaction between the probe and aldehydic products of lipid peroxidation, including malondialdehyde and long-chain aliphatic aldehydes relevant to the oxidative decomposition of cell membranes, have been evaluated. Live cell imaging of diethyl maleate-induced oxidative stress with or without pretreatment with α-tocopherol was carried out, with the result suggesting that the presented molecule might serve as a minimalist molecular probe capable of cellular "Aldehydic Load" detection by fluorescence microscopy. This work also outlines functional constraints of the fluorogenic probe (i.e. intramolecular cyclization), providing a realistic evaluation of methyl 5-MeO-N-aminoanthranilate for fluorescence-based aldehyde detection.

Hydrazo-CEST: Hydrazone-Dependent Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Contrast Agents.

The rapid formation of hydrazones under physiological conditions was exploited for the detection of aldehydes through chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI). A metal-free, diamagnetic contrast agent derived from N-amino anthranilic acid was introduced, which selectively "turned-on" upon hydrazone formation through an effect termed Hydrazo-CEST. While the hydrazine form of the probe produced no CEST-MRI signal enhancement, the formation of the aryl hydrazone resulted in >20 % intensity decrease in the bulk water signal through the CEST effect, as measured by 300 MHz 1 H NMR, 3 T and 7 T MRI. Both the electronic contributions of the N-amino anthranilate and the aldehyde binding partner were shown to directly impact the exchange rate of the proton on the ring-proximal nitrogen, and thus the imaging signal. Additionally, the presence of the carboxylic acid moiety ortho to the hydrazine was necessary not only for contrast production, but also for rapid hydrazone formation and prolonged hydrazone product stability under physiological conditions. This work provided the first example of an MRI-based contrast agent capable of a "turn on" response upon reaction with bioactive aldehydes, and outlined both the structural and electronic requirements to expand on Hydrazo-CEST, a novel, hydrazone-dependent subtype of diamagnetic CEST-MRI.

Unusual C7- versus Normal 5'-O-Dimethoxytritylation of 6-Arylpyrrolocytidine Analogs.

Fluorescent deoxynucleosides possessing the modified bases 6-(2-benzo[b]furyl)- and 6-(2-furyl)pyrrolocytosine (BFpC and FpC) have been synthesized along with the quencher nucleosides possessing 6-{4-[(4-dimethylamino)azo]phenyl}pyrrolocytosine (DABCYLpC) and 6-(p-nitrophenyl)pyrrolocytosine (p-NO2-PhpC) nucleobase analogs. Standard treatment of BFpC, FpC, DABCYLpC, and p-NO2-PhpC with dimethoxytrityl chloride (DMT-Cl) led to the unusual substitution on the C7 of the pyrrolocytosine skeleton. The desired 5'-O-DMT-protected nucleoside analogs were synthesized from suitably protected 5'-O-DMT cytidines. Subsequent phosphitylation smoothly afforded BFpC-, FpC-, DABCYLpC-, and p-NO2-PhpC-derived monomers suitable for standard oligonucleotide synthesis.

Pyrimidine-fused heterocyclic frameworks based on an N4-arylcytosine scaffold: synthesis, characterization, and PNA oligomerization of the fluorescent cytosine analogue 5,6-benzopC.

A synthesis of an intrinsically fluorescent cytosine analogue 5,6-benzopC has been developed utilizing the reductive Ni-mediated cyclization of an N4-aryl,N4-(Boc)cytosine intermediate as a key step. 5,6-BenzopC was found to possess interesting fluorescence properties (Φ = 0.79, EtOH; Stoke's shift 113 nm). Peptide nucleic acid (PNA) oligomerization of the 5,6-benzopC monomer was carried out, followed by hybridization studies with complementary deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which showed the modification to be well tolerated in the sequence contexts examined. Initial attempts to synthesize the heterocyclic skeleton present in 5,6-benzopC resulted in the discovery of routes to the pyrimido[1,6-a]benzimidazole, pyrimido[1,6-a]quinazoline, and pyrimido[1,6-a]benzo[b]6-bora-1,3-diazine heterocyclic frameworks.

Tracking the fate of bacteria-derived site-specific immunomodulators by positron emission tomography.

INTRODUCTION: Site-specific immunomodulators (SSIs) are a novel class of therapeutics made from inactivated bacterial species designed to regulate the innate immune system in targeted organs. QBECO is a gut-targeted SSI that is being advanced clinically to treat and/or prevent inflammatory bowel disease, cancer, and serious infections of the gastrointestinal (GI) tract and proximal organs, and QBKPN is a lung-targeted SSI that is in clinical development for the treatment and/or prevention of chronic inflammatory lung disease, lung cancers and respiratory tract infections. While these SSIs have demonstrated both safety and proof-of-concept in preclinical and clinical studies, detailed understanding of their trafficking and biodistribution is yet to be fully characterized. METHODS: QBECO and QBKPN were radiolabeled with [89Zr] and injected subcutaneously into healthy mice. The mice underwent Positron Emission Tomography (PET) imaging every day for eight days to track biodistribution of the SSIs. Tissue from the site of injection was collected and immunohistologically probed for immune cell infiltration. RESULTS: Differential biodistribution of the two SSIs was seen, adhering to their site-specific targeting. QBKPN appeared to migrate from the site of injection (abdomen) to the cervical lymph nodes which are nearer to the respiratory tract and lungs. QBECO remained in the abdominal region, with lymphatic trafficking to the inguinal lymph nodes, which are nearer to GI-proximal tissues/organs. Immune infiltration at the site of injection comprised of neutrophils for both SSIs, and macrophages for only QBKPN. CONCLUSION: Radiolabeling of SSIs allows for longitudinal in vivo imaging of biodistribution and trafficking. PET imaging revealed differential biodistribution of the SSIs based on the organotropism of the bacteria from which the SSI is derived. Trafficking from the site of injection to the targeted site is in part mediated via the lymphatics and involves macrophages and neutrophils.

It's a Trap! Aldolase-Prescribed C4 Deoxyradiofluorination Affords Intracellular Trapping and the Tracing of Fructose Metabolism by PET.

Fructose metabolism has been implicated in various diseases, including metabolic disorders, neurodegenerative disorders, cardiac disorders, and cancer. However, the limited availability of a quantitative imaging radiotracer has hindered its exploration in pathology and diagnostic imaging. Methods: We adopted a molecular design strategy based on the catalytic mechanism of aldolase, a key enzyme in fructolysis. We successfully synthesized a radiodeoxyfluorinated fructose analog, [18F]4-fluoro-4-deoxyfructose ([18F]4-FDF), in high molar activity. Results: Through heavy isotope tracing by mass spectrometry, we demonstrated that C4-deoxyfluorination of fructose led to effective trapping as fluorodeoxysorbitol and fluorodeoxyfructose-1-phosphate in vitro, unlike C1- and C6-fluorinated analogs that resulted in fluorolactate accumulation. This observation was consistent in vivo, where [18F]6-fluoro-6-deoxyfructose displayed substantial bone uptake due to metabolic processing whereas [18F]4-FDF did not. Importantly, [18F]4-FDF exhibited low uptake in healthy brain and heart tissues, known for their high glycolytic activity and background levels of [18F]FDG uptake. [18F]4-FDF PET/CT allowed for sensitive mapping of neuro- and cardioinflammatory responses to systemic lipopolysaccharide administration. Conclusion: Our study highlights the significance of aldolase-guided C4 radiodeoxyfluorination of fructose in enabling effective radiotracer trapping, overcoming limitations of C1 and C6 radioanalogs toward a clinically viable tool for imaging fructolysis in highly glycolytic tissues.

Simultaneous in vivo pH and temperature mapping using a PARACEST-MRI contrast agent.

Altered tissue temperature and/or pH is a common feature in pathological conditions, where metabolic demand exceeds oxygen supply such as in tumors and following stroke. Therefore, in vivo tissue temperature and pH may become valuable biomarkers for disease detection and the monitoring of disease progression or treatment response in conditions with altered metabolic demand. In this study, pH is measured using the amide protons of a thulium (Tm(3+)) complex with a DOTAM-Glycine-Lysine (ligand: Tm(3+)-DOTAM-Gly-Lys). The pH was uniquely determined from the linewidth of the asymmetry curve of the chemical exchange saturation transfer spectrum, independent of contrast agent concentration, or temperature for a given saturation pulse. pH maps with an inter-pixel standard deviation of less than 0.1 pH units were obtained in 10 mM Tm(3+)-DOTAM-Gly-Lys solutions with pH ranging from 6.0 to 8.0 pH units at 37°C. Temperature maps were simultaneously obtained using the chemical shift of the chemical exchange saturation transfer peak. Temperature and pH maps are demonstrated in the mouse leg (N = 3), where the mean and standard deviation for pH was 7.2 ± 0.2 pH unit and temperature was 37.4 ± 0.5°C.

Direct mapping of kidney function by DCE-MRI urography using a tetrazinanone organic radical contrast agent.

Chronic kidney disease (CKD) and acute kidney injury (AKI) are ongoing global health burdens. Glomerular filtration rate (GFR) is the gold standard measure of kidney function, with clinical estimates providing a global assessment of kidney health without spatial information of kidney- or region-specific dysfunction. The addition of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to the anatomical imaging already performed would yield a 'one-stop-shop' for renal assessment in cases of suspected AKI and CKD. Towards urography by DCE-MRI, we evaluated a class of nitrogen-centered organic radicals known as verdazyls, which are extremely stable even in highly reducing environments. A glucose-modified verdazyl, glucoverdazyl, provided contrast limited to kidney and bladder, affording functional kidney evaluation in mouse models of unilateral ureteral obstruction (UUO) and folic acid-induced nephropathy (FAN). Imaging outcomes correlated with histology and hematology assessing kidney dysfunction, and glucoverdazyl clearance rates were found to be a reliable surrogate measure of GFR.

In vivo detection of MRI-PARACEST agents in mouse brain tumors at 9.4 T.

Paramagnetic chemical exchange saturation transfer (PARACEST) contrast agents are under development for biological target identification by magnetic resonance imaging. Image contrast associated with PARACEST agents can be generated by radiofrequency irradiation of the chemically shifted protons bound to a PARACEST contrast agent molecule or by direct irradiation of the on-resonance bulk water protons. The observed signal change in a magnetic resonance image after the administration of a PARACEST contrast agent is due to both altered relaxation time constants and the CEST effect. Despite high sensitivity in vitro, PARACEST agents have had limited success in vivo where sensitivity is reduced by the magnetization transfer effect from endogenous macromolecules. The purpose of this study was to demonstrate the in vivo detection of a PARACEST contrast agent using the on-resonance paramagnetic chemical exchange effect (OPARACHEE) in a mouse glioblastoma multiforme tumor model and to isolate the OPARACHEE effect from the changes in relaxation induced by the PARACEST agent. Three mice with tumors were imaged on a 9.4 T MRI scanner following tail vein injection of 150 µL 50 mM Tm(3+)-DOTAM-glycine-lysine. A fast low angle shot pulse sequence with a low power radiofrequency pulse train (WALTZ-16) as the preparation pulse was used to generate OPARACHEE contrast. To study the dynamics of agent uptake, reference images (without the preparation pulse) and OPARACHEE images were acquired continuously in an alternating fashion before, during and after agent injection. Signal intensity decreased by more than 10% in tumor in the control images after agent administration. Despite these changes, a clear OPARACHEE contrast of 1-5% was also observed in brain tumors after contrast agent injection and maintained in the hour following injection. This result is the first in vivo observation of OPARACHEE contrast in brain tumors with correction of T(1) and T(2) relaxation effects.

Synthesis of natural/13C-enriched d-tagatose from natural/13C-enriched d-fructose.

A concise, easily scalable synthesis of a rare ketohexose, d-tagatose, was developed, that is compatible with the preparation of d-[UL-13C6]tagatose. Epimerization of the widely available and inexpensive ketohexose d-fructose at the C-4 position via an oxidation/reduction (Dess-Martin periodinane/NaBH4) was a key step in the synthesis. Overall, fully protected natural d-tagatose (3.21 g) was prepared from d-fructose (9 g) on a 50 mmol scale in 23% overall yield, after five steps and two chromatographic purifications. d-[UL-13C6]Tagatose (92 mg) was prepared from d-[UL-13C6]fructose (465 mg, 2.5 mmol) in 16% overall yield after six steps and four chromatographic purifications.

Mapping vitamin B6 metabolism by hydrazoCEST magnetic resonance imaging.

A new CEST-MRI contrast agent, 2-HYNIC, capable of sensing aromatic aldehydes is reported. Pyridoxal 5'-phosphate, a key Vitamin B6 metabolite necessary for >140 biotransformations was mapped by CEST-MRI in vitro and in vivo in lung cancer. 2-HYNIC provided access to this key biomarker associated with a variety of human diseases.

In vivo detection of PARACEST agents with relaxation correction.

Several pulse sequences have been used to detect paramagnetic chemical exchange saturation transfer (PARACEST) contrast agents in animals to quantify the uptake over time following a bolus injection. The observed signal change is a combination of relaxation effects and PARACEST contrast. The purpose of the current study was to isolate the PARACEST effect from the changes in bulk water relaxation induced by the PARACEST agent in vivo for the fast low-angle shot pulse sequence. A fast low-angle shot-based pulse sequence was used to acquire continuous images on a 9.4-T MRI of phantoms and the kidneys of mice following PARACEST agent (Tm(3+)-DOTAM-Gly-Lys) injection. A WALTZ-16 pulse was applied before every second image to generate on-resonance paramagnetic chemical exchange effects. Signal intensity changes of up to 50% were observed in the mouse kidney in the control images (without a WALTZ-16 preparation pulse) due to altered bulk water relaxation induced by the PARACEST agent. Despite these changes, a clear on-resonance paramagnetic chemical exchange effect of 4-7% was also observed. A four-pool exchange model was used to describe image signal intensity. This study demonstrates that in vivo on-resonance paramagnetic chemical exchange effect contrast can be isolated from tissue relaxation time constant changes induced by a PARACEST agent that dominate the signal change.

Synthesis of MRI contrast agents derived from DOTAM-Gly-L-Phe-OH incorporating a disulfide bridge: conjugation to a cell penetrating peptide and preparation of a dimeric agent.

A cell penetrating peptide conjugate and dimeric PARACEST MRI contrast agents, based on the DOTAM-Gly-L-Phe-OH scaffold have been prepared in moderate yields using diethyl azodicarboxylate (DEAD) or iodine-mediated disulfide bridge formation as a key step. Magnetic (PARACEST) properties of these agents have been evaluated.

A paramagnetic chemical exchange-based MRI probe metabolized by cathepsin D: design, synthesis and cellular uptake studies.

Overexpression of the aspartyl protease cathepsin D is associated with certain cancers and Alzheimer's disease; thus, it is a potentially useful imaging biomarker for disease. A dual fluorescence/MRI probe for the potential detection of localized cathepsin D activity has been synthesized. The probe design includes both MRI and optical reporter groups connected to a cell penetrating peptide by a cathepsin D cleavable sequence. This design results in the selective intracellular deposition (determined fluorimetrically) of the MRI and optical reporter groups in the presence of overexpressed cathepsin D. The probe also provided clearly detectable in vitro MRI contrast by the mechanism of paramagnetic chemical exchange effects (OPARACHEE).

Discovery of inhibitors of brassinin oxidase based on the scaffolds of the phytoalexins brassilexin and wasalexin.

Inhibitors of brassinin oxidase (BOLm), a unique phytoalexin detoxifying enzyme produced by the plant pathogenic fungus Leptosphaeria maculans (asexual stage Phoma lingam), were designed based on scaffolds of the phytoalexins brassilexin and wasalexin. Evaluation of these compounds using purified BOLm established that the inhibitory effect of brassilexin and derivatives decreased as follows: 6-chlorobrassilexin approximately 6-bromobrassilexin>5-bromobrassilexin approximately 5-chlorobrassilexin approximately 6-fluorobrassilexin>8-methylbrassilexin>brassilexin approximately 5-fluorobrassilexin. 6-Chlorobrassilexin was determined to be the best competitive inhibitor of BOLm discovered to date, with a K(i)=31microM. Importantly, brassilexin and derivatives did not appear to induce BOLm in fungal cultures. Overall, these results suggest that the brassilexin scaffold is a good lead for further development of paldoxins against L. maculans, as it inhibits competitively BOLm without apparent induction.

Optimized MRI contrast for on-resonance proton exchange processes of PARACEST agents in biological systems.

Image contrast associated with paramagnetic chemical exchange saturation transfer agents can be generated by off-resonance irradiation of agent-bound water or amide protons or on-resonance irradiation of bulk water. Previously, a four-pool model was developed to describe an in vivo system. The model incorporated the magnetization transfer effect from macromolecules when using off-resonance irradiation. In the current study, this four-pool model is modified to describe the in vivo system when using on-resonance irradiation. The influences of pulse power, pulse duration, the chemical shift of bound water, the proton exchange rate between bulk water and bound water, and agent concentration on the on-resonance paramagnetic agent chemical exchange effects were simulated using a WALTZ-16 pulse train in the absence and presence of the macromolecule pool. The results demonstrated that while contrast increases with pulse duration in aqueous solution, there is an optimal pulse duration that maximizes on-resonance paramagnetic agent chemical exchange effects contrast in vivo. This predication was verified by experimental spectroscopic and imaging results from aqueous solution, bovine serum albumin phantoms, and a tissue phantom containing thulium-DOTAM (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide)-glycine-lysine. This model can be used to optimize sequence parameters to maximize in vivo on-resonance paramagnetic agent chemical exchange effects contrast.

Mapping aldehydic load in vivo by positron emission tomography with [18F]NA3BF3.

A new radiotracer, [18F]NA3BF3, capable of rapid, stable, and catalyst-free complexation of aldehydes in vivo is reported. [18F]NA3BF3 was shown to bind aldehydes in live subjects using locally administered aldehyde-presenting microparticles, and was then applied to mapping aldehydic load in a mouse model of sepsis. [18F]NA3BF3 may enable the direct investigation of the chemical biology of aldehydes in living subjects, and may open avenues for the adoption of endogenous aldehydic load as an imaging biomarker of inflammatory pathology.

Detection of Active Caspase-3 in Mouse Models of Stroke and Alzheimer's Disease with a Novel Dual Positron Emission Tomography/Fluorescent Tracer [68Ga]Ga-TC3-OGDOTA.

Apoptosis is a feature of stroke and Alzheimer's disease (AD), yet there is no accepted method to detect or follow apoptosis in the brain in vivo. We developed a bifunctional tracer [68Ga]Ga-TC3-OGDOTA containing a cell-penetrating peptide separated from fluorescent Oregon Green and 68Ga-bound labels by the caspase-3 recognition peptide DEVD. We hypothesized that this design would allow [68Ga]Ga-TC3-OGDOTA to accumulate in apoptotic cells. In vitro, Ga-TC3-OGDOTA labeled apoptotic neurons following exposure to camptothecin, oxygen-glucose deprivation, and ß-amyloid oligomers. In vivo, PET showed accumulation of [68Ga]Ga-TC3-OGDOTA in the brain of mouse models of stroke or AD. Optical clearing revealed colocalization of [68Ga]Ga-TC3-OGDOTA and cleaved caspase-3 in brain cells. In stroke, [68Ga]Ga-TC3-OGDOTA accumulated in neurons in the penumbra area, whereas in AD mice [68Ga]Ga-TC3-OGDOTA was found in single cells in the forebrain and diffusely around amyloid plaques. In summary, this bifunctional tracer is selectively associated with apoptotic cells in vitro and in vivo in brain disease models and represents a novel tool for apoptosis detection that can be used in neurodegenerative diseases.

A new synthesis of cystamine modified Eu3+ DOTAM-Gly-Phe-OH: a conjugation ready temperature sensitive MRI contrast agent.

Several approaches towards asymmetrically derivatized peptide-decorated cyclens that yield lanthanide metal chelators, in which three of the nitrogen atoms of cyclen share a common substituent and the fourth nitrogen atom is differentially substituted, have been evaluated. The most effective route consisted of selective monoalkylation followed by peralkylation with a second different electrophile. The unique substituent also possessed a masked sulfanyl group that was suitable for subsequent chemoselective conjugation chemistry.