Discovery of Bis-Imidazoline Derivatives as New CXCR4 Ligands.

The chemokine receptor CXCR4 and its ligand CXCL12 regulate leukocyte trafficking, homeostasis and functions and are potential therapeutic targets in many diseases such as HIV-1 infection and cancers. Here, we identified new CXCR4 ligands in the CERMN chemical library using a FRET-based high-throughput screening assay. These are bis-imidazoline compounds comprising two imidazole rings linked by an alkyl chain. The molecules displace CXCL12 binding with submicromolar potencies, similarly to AMD3100, the only marketed CXCR4 ligand. They also inhibit anti-CXCR4 mAb 12G5 binding, CXCL12-mediated chemotaxis and HIV-1 infection. Further studies with newly synthesized derivatives pointed out to a role of alkyl chain length on the bis-imidazoline properties, with molecules with an even number of carbons equal to 8, 10 or 12 being the most potent. Interestingly, these differ in the functions of CXCR4 that they influence. Site-directed mutagenesis and molecular docking predict that the alkyl chain folds in such a way that the two imidazole groups become lodged in the transmembrane binding cavity of CXCR4. Results also suggest that the alkyl chain length influences how the imidazole rings positions in the cavity. These results may provide a basis for the design of new CXCR4 antagonists targeting specific functions of the receptor.

Genetic Ablation of Nrf2 Exacerbates Neuroinflammation in Ocular Autoimmunity.

Experimental autoimmune uveoretinitis (EAU) is an animal model of non-infectious uveitis and is developed by immunization with retinal antigen, interphotoreceptor retinoid-binding protein (IRBP). Nuclear factor erythroid 2- (NF-E2-) related factor 2 (Nrf2) is responsible for regulating antioxidant and inflammatory responses. In this study, we investigated the role of Nrf2 on the development of EAU. Clinical and pathological examination demonstrated that retinal inflammation was exacerbated in Nrf2 knockout (Nrf2 KO) mice compared to wild type (WT) mice, and the expression of inflammatory cytokines (IFN-γ, IL-6, and IL-17) in the retina was significantly elevated in Nrf2 KO mice. GFAP positive cells (astrocytes) and Iba-1 positive cells (microglia cells) in the retina were more numerous in Nrf2 KO mice compared to WT mice. Furthermore, we examined the suppressive effect of the Nrf2 activator CDDO-Im (2-cyano-3,12 dioxooleana-1,9 dien-28-oyl imidazoline) on the development of EAU. The treatment with CDDO-Im significantly reduced the clinical and pathological score of EAU compared to those of vehicle-treated mice. These findings suggest that Nrf2 plays a regulatory role in the pathogenesis of autoimmune uveoretinitis and the activation of the Nrf2 system may have therapeutic potential for protecting vision from autoimmune neuroinflammation.

Diaza-1,3-butadienes as Useful Intermediate in Heterocycles Synthesis.

Many heterocyclic compounds can be synthetized using diaza-1,3-butadienes (DADs) as key structural precursors. Isolated and in situ diaza-1,3-butadienes, produced from their respective precursors (typically imines and hydrazones) under a variety of conditions, can both react with a wide range of substrates in many kinds of reactions. Most of these reactions discussed here include nucleophilic additions, Michael-type reactions, cycloadditions, Diels-Alder, inverse electron demand Diels-Alder, and aza-Diels-Alder reactions. This review focuses on the reports during the last 10 years employing 1,2-diaza-, 1,3-diaza-, 2,3-diaza-, and 1,4-diaza-1,3-butadienes as intermediates to synthesize heterocycles such as indole, pyrazole, 1,2,3-triazole, imidazoline, pyrimidinone, pyrazoline, -lactam, and imidazolidine, among others. Fused heterocycles, such as quinazoline, isoquinoline, and dihydroquinoxaline derivatives, are also included in the review.

Probing Alterations in MDM2 Catalytic Core Structure Effect of Garcinia Mangostana Derivatives: Insight from Molecular Dynamics Simulations.

The MDM2-p53 protein-protein interaction is a promising model for researchers to design, study, and discover new anticancer drugs. The design of therapeutically active compounds that can maintain or restore the binding of MDM2 to p53 has been found to limit the oncogenic activities of both. This led to the current development of a group of xanthone-core and cis-imidazoline analogs compounds, among which γ-Mangostin (GM), α-Mangostin (AM), and Nutlin exhibited their MDM2-p53 interaction inhibitory effects. Therefore, in this study, we seek to determine the mechanisms by which these compounds elicit MDM2-p53 interaction targeting. Unique to the binding of GM, AM, and Nutlin, from our findings, they share the same three active site residues Val76, Tyr50, and Gly41, which represent the top active side residues that contribute to high electrostatic energy. Consequently, the free binding energy contributed enormously to the binding of these compounds, which culminated in the high binding affinities of GM, AM, and Nutlin with high values. Furthermore, GM, AM, and Nutlin commonly interrupted the stable and compact conformation of MDM2 coupled with its active site, where Cα deviations were relatively high. We believe that our findings would assist in the design of more potent active anticancer drugs.

Combined Drug Targeting of p53-dependent and -independent Pathways Depletes Myelofibrosis Hematopoietic Stem/Progenitor Cells.

Current therapy for myelofibrosis (MF) results in a limited prolongation of patient survival. In order to improve treatment outcomes, we developed a strategy to effectively deplete MF hematopoietic stem/progenitor cells (HSPCs). In the present study, an imipridone, ONC201, was combined with RG7112, an antagonist of MDM2, a p53 negative regulator, to activate downstream events of the p53 and TNF-related apoptosis-inducing ligand (TRAIL)/death receptor (DR) pathways. As compared to treatment with the individual drugs, the combination of ONC201 and RG7112 promoted greater degrees of apoptosis of MF CD34+ cells through activation of both p53-dependent and -independent pathways. Importantly, treatment with ONC201-RG7112 not only decreased the number of JAK2V617F+ and calreticulin mutated colonies assayed from MF CD34+ cells, but allowed for the persistence or appearance of JAK2 wild type colonies. Treatment with ONC201 combined with RG7112 could be a potentially effective strategy for treating MF patients.

An aromatic imidazoline derived from chloroquinoline triggers cell cycle arrest and inhibits with high selectivity the Trypanosoma cruzi mammalian host-cells infection.

Trypanosoma cruzi is a hemoflagellated parasite causing Chagas disease, which affects 6-8 million people in the Americas. More than one hundred years after the description of this disease, the available drugs for treating the T. cruzi infection remain largely unsatisfactory. Chloroquinoline and arylamidine moieties are separately found in various compounds reported for their anti-trypanosoma activities. In this work we evaluate the anti-T. cruzi activity of a collection of 26 "chimeric" molecules combining choroquinoline and amidine structures. In a first screening using epimastigote forms of the parasite as a proxy for the clinically relevant stages, we selected the compound 7-chloro-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]quinoline (named here as A6) that performed better as an anti-T. cruzi compound (IC50 of 2.2 ± 0.3 µM) and showed a low toxicity for the mammalian cell CHO-K1 (CC50 of 137.9 ± 17.3 µM). We initially investigated the mechanism of death associated to the selected compound. The A6 did not trigger phosphatidylserine exposure or plasma membrane permeabilization. Further investigation led us to observe that under short-term incubations (until 6 hours), no alterations of mitochondrial function were observed. However, at longer incubation times (4 days), A6 was able to decrease the intracellular Ca2+, to diminish the intracellular ATP levels, and to collapse mitochondrial inner membrane potential. After analysing the cell cycle, we found as well that A6 produced an arrest in the S phase that impairs the parasite proliferation. Finally, A6 was effective against the infective forms of the parasite during the infection of the mammalian host cells at a nanomolar concentration (IC50(tryps) = 26.7 ± 3.7 nM), exhibiting a selectivity index (SI) of 5,170. Our data suggest that A6 is a promising hit against T. cruzi.

Acute effects of the imidacloprid metabolite desnitro-imidacloprid on human nACh receptors relevant for neuronal signaling.

Several neonicotinoids have recently been shown to activate the nicotinic acetylcholine receptor (nAChR) on human neurons. Moreover, imidacloprid (IMI) and other members of this pesticide family form a set of diverse metabolites within crops. Among these, desnitro-imidacloprid (DN-IMI) is of special toxicological interest, as there is evidence (i) for human dietary exposure to this metabolite, (ii) and that DN-IMI is a strong trigger of mammalian nicotinic responses. We set out here to quantify responses of human nAChRs to DN-IMI and an alternative metabolite, IMI-olefin. To evaluate toxicological hazards, these data were then compared to those of IMI and nicotine. Ca2+-imaging experiments on human neurons showed that DN-IMI exhibits an agonistic effect on nAChRs at sub-micromolar concentrations (equipotent with nicotine) while IMI-olefin activated the receptors less potently (in a similar range as IMI). Direct experimental data on the interaction with defined receptor subtypes were obtained by heterologous expression of various human nAChR subtypes in Xenopus laevis oocytes and measurement of the transmembrane currents evoked by exposure to putative ligands. DN-IMI acted on the physiologically important human nAChR subtypes α7, α3ß4, and α4ß2 (high-sensitivity variant) with similar potency as nicotine. IMI and IMI-olefin were confirmed as nAChR agonists, although with 2-3 orders of magnitude lower potency. Molecular docking studies, using receptor models for the α7 and α4ß2 nAChR subtypes supported an activity of DN-IMI similar to that of nicotine. In summary, these data suggest that DN-IMI functionally affects human neurons similar to the well-established neurotoxicant nicotine by triggering α7 and several non-α7 nAChRs.

Synthesis, antiproliferative and antitrypanosomal activities, and DNA binding of novel 6-amidino-2-arylbenzothiazoles.

A series of 6-amidinobenzothiazoles, linked via phenoxymethylene or directly to the 1,2,3-triazole ring with a p-substituted phenyl or benzyl moiety, were synthesised and evaluated in vitro against four human tumour cell lines and the protozoan parasite Trypanosoma brucei. The influence of the type of amidino substituent and phenoxymethylene linker on antiproliferative and antitrypanosomal activities was observed, showing that the imidazoline moiety had a major impact on both activities. Benzothiazole imidazoline 14a, which was directly connected to N-1-phenyl-1,2,3-triazole, had the most potent growth-inhibitory effect (IC50 = 0.25 µM) on colorectal adenocarcinoma (SW620), while benzothiazole imidazoline 11b, containing a phenoxymethylene linker, exhibited the best antitrypanosomal potency (IC90 = 0.12 µM). DNA binding assays showed a non-covalent interaction of 6-amidinobenzothiazole ligands, indicating both minor groove binding and intercalation modes of DNA interaction. Our findings encourage further development of novel structurally related 6-amidino-2-arylbenzothiazoles to obtain more selective anticancer and anti-HAT agents.

An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression.

A majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

Solid-Phase Synthesis of an Insect Pyrokinin Analog Incorporating an Imidazoline Ring as Isosteric Replacement of a trans Peptide Bond.

A facile solid-phase synthetic method for incorporating the imidazoline ring motif, a surrogate for a trans peptide bond, into bioactive peptides is reported. The example described is the synthesis of an imidazoline peptidomimetic analog of an insect pyrokinin neuropeptide via a cyclization reaction of an iminium salt generated from the preceding amino acid and 2,4-diaminopropanoic acid (Dap).

A novel imidazolinone metformin-methylglyoxal metabolite promotes endothelial cell angiogenesis via the eNOS/HIF-1α pathway.

Peripheral arterial disease (PAD) is one of the major complications of diabetes due to an impairment in angiogenesis. Since there is currently no drug with satisfactory efficacy to enhance blood vessel formation, discovering therapies to improve angiogenesis is critical. An imidazolinone metabolite of the metformin-methylglyoxal scavenging reaction, (E)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl) guanidine (IMZ), was recently characterized and identified in the urine of type-2 diabetic patients. Here, we report the pro-angiogenesis effect of IMZ (increased aortic sprouting, cell migration, network formation, and upregulated multiple pro-angiogenic factors) in human umbilical vein endothelial cells. Using genetic and pharmacological approaches, we showed that IMZ augmented angiogenesis by activating the endothelial nitric oxide synthase (eNOS)/hypoxia-inducible factor-1 alpha (HIF-1α) pathway. Furthermore, IMZ significantly promoted capillary density in the in vivo Matrigel plug angiogenesis model. Finally, the role of IMZ in post-ischemic angiogenesis was examined in a chronic hyperglycemia mouse model subjected to hind limb ischemia. We observed improved blood perfusion, increased capillary density, and reduced tissue necrosis in mice receiving IMZ compared to control mice. Our data demonstrate the pro-angiogenic effects of IMZ, its underlying mechanism, and provides a structural basis for the development of potential pro-angiogenic agents for the treatment of PAD.

New insight into the negative impact of imidazolium-based ionic liquid [C10mim]Cl on Hela cells: From membrane damage to biochemical alterations.

As an alternative to volatile organic solvents, ionic liquids (ILs) are known as "green solvents", and widely used in industrial applications. However, due to their high solubility and stability, ILs have tendency to persist in the water environment, thus having potential negative impacts on the aquatic ecosystem. For assessing the environmental risks of ILs, a fundamental understanding of the toxic effects and mechanisms of ILs is needed. Here we evaluated the cytotoxicity of 1-methyl-3-decylimidazolium chloride ([C10mim]Cl) and elucidated the main toxic mechanism of [C10mim]Cl in human cervical carcinoma (Hela) cells. Microstructural analysis revealed that [C10mim]Cl exposure caused the cell membrane breakage, swollen and vacuolated mitochondria, and spherical cytoskeletal structure. Cytotoxicity assays found that [C10mim]Cl exposure increased ROS production, decreased mitochondrial membrane potential, induced cell apoptosis and cell cycle arrest. These results indicated that [C10mim]Cl could induce damage to cellular membrane structure, affect the integrity of cell ultrastructure, cause the oxidative damage and ultimately lead to the inhibition of cell proliferation. Moreover, alterations of biochemical information including the increased ratios of unsaturated fatty acid and carbonyl groups to lipid, and lipid to protein, and the decreased ratios of Amide I to Amide II, and α-helix to ß-sheet were observed in [C10mim]Cl treated cells, suggesting that [C10mim]Cl could affect the structure of membrane lipid alkyl chain and cell membrane fluidity, promote the lipid peroxidation and alter the protein secondary structure. The findings from this work demonstrated that membrane structure is the key target, and membrane damage is involved in [C10mim]Cl induced cytotoxicity.

Chronic treatment with the (iso-)glutaminyl cyclase inhibitor PQ529 is a novel and effective approach for glomerulonephritis in chronic kidney disease.

Glomeruli and renal tubule injury in chronic kidney disease (CKD) is reported to involve induction of macrophage activation through the CCL2/CCR2 axis. The effects of inhibitors of the CCL2/CCR2 axis, such as anti-CCL2 antibody and CCR2 antagonist, on kidney function in animal models or humans with kidney dysfunction have been demonstrated. The N-terminal glutamine on immature CCL2 is replaced with pyroglutamate (pE) by glutaminyl cyclase (QC) and isoQC. pE-CCL2 is stable and resistant to peptidases. We hypothesized that inhibiting QC/isoQC activity would lead to the degradation of CCL2, thereby ameliorating CKD and reducing kidney inflammation. To test this hypothesis, we investigated the renoprotective properties of the QC/isoQC inhibitor PQ529 in anti-glomerular basement membrane (GBM) antibody-induced glomerulonephritis Wistar Kyoto (WKY) rats. Three-week repeated administration of PQ529 (30 and 100 mg/kg, twice daily) significantly reduced the serum and urine CCL2 and urinary protein excretion in a dose-dependent manner. Correlations between the urinary protein level and serum or urinary CCL2 levels were confirmed in tested animals. Repeated administration of PQ529 significantly reduced the expression of CD68, a macrophage marker, in the kidney cortex and mononuclear infiltration into the tubulointerstitium. In addition, decreased levels of urinary KIM-1, ß2 microglobulin, and clusterin were detected, suggesting the inhibition of inflammation in both the proximal and distal tubules. These results suggest that PQ529 suppresses the progression of inflammation-induced renal dysfunction by inhibiting the CCL2/CCR2 axis. Inhibition of QC/isoQC may thus be a viable alternative therapeutic approach for treating glomerulonephritis and CKD patients.

Nickel-Catalyzed Asymmetric Synthesis of α-Arylbenzamides.

A nickel-catalyzed asymmetric reductive hydroarylation of vinyl amides to produce enantioenriched α-arylbenzamides is reported. The use of a chiral bisimidazoline (BIm) ligand, in combination with diethoxymethylsilane and aryl halides, enables the regioselective introduction of aryl groups to the internal position of the olefin, forging a new stereogenic center α to the N atom. The use of neutral reagents and mild reaction conditions provides simple access to pharmacologically relevant motifs present in anticancer, SARS-CoV PLpro inhibitors, and KCNQ channel openers.

Imidazoline ligand BU224 reverses cognitive deficits, reduces microgliosis and enhances synaptic connectivity in a mouse model of Alzheimer's disease.

BACKGROUND AND PURPOSE: Activation of type 2 imidazoline receptors has been shown to exhibit neuroprotective properties including anti-apoptotic and anti-inflammatory effects, suggesting a potential therapeutic value in Alzheimer's disease (AD). Here, we explored the effects of the imidazoline-2 ligand BU224 in a model of amyloidosis. EXPERIMENTAL APPROACH: Six-month-old female transgenic 5XFAD and wild-type (WT) mice were treated intraperitoneally with 5-mg·kg-1 BU224 or vehicle twice a day for 10 days. Behavioural tests were performed for cognitive functions and neuropathological changes were investigated by immunohistochemistry, Western blot, elisa and qPCR. Effects of BU224 on amyloid precursor protein (APP) processing, spine density and calcium imaging were analysed in brain organotypic cultures and N2a cells. KEY RESULTS: BU224 treatment attenuated spatial and perirhinal cortex-dependent recognition memory deficits in 5XFAD mice. Fear-conditioning testing revealed that BU224 also improved both associative learning and hippocampal- and amygdala-dependent memory in transgenic but not in WT mice. In the brain, BU224 reduced levels of the microglial marker Iba1 and pro-inflammatory cytokines IL-1ß and TNF-α and increased the expression of astrocytic marker GFAP in 5XFAD mice. These beneficial effects were not associated with changes in amyloid pathology, neuronal apoptosis, mitochondrial density, oxidative stress or autophagy markers. Interestingly, ex vivo and in vitro studies suggested that BU224 treatment increased the size of dendritic spines and induced a threefold reduction in amyloid-ß (Aß)-induced functional changes in NMDA receptors. CONCLUSION AND IMPLICATIONS: Sub-chronic treatment with BU224 restores memory and reduces inflammation in transgenic AD mice, at stages when animals display severe pathology.

Discovery, synthesis and characterization of a series of 7-aryl-imidazo[1,2-a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors.

The activin-like kinases are a family of kinases that play important roles in a variety of disease states. Of this class of kinases, ALK2, has been shown by a gain-of-function to be the primary driver of the childhood skeletal disease fibrodysplasia ossificans progressiva (FOP) and more recently the pediatric cancer diffuse intrinsic pontine glioma (DIPG). Herein, we report our efforts to identify a novel imidazo[1,2-a]pyridine scaffold as potent inhibitors of ALK2 with good in vivo pharmacokinetic properties suitable for future animal studies.

A protein-independent fluorescent RNA aptamer reporter system for plant genetic engineering.

Reporter systems are routinely used in plant genetic engineering and functional genomics research. Most such plant reporter systems cause accumulation of foreign proteins. Here, we demonstrate a protein-independent reporter system, 3WJ-4 × Bro, based on a fluorescent RNA aptamer. Via transient expression assays in both Escherichia coli and Nicotiana benthamiana, we show that 3WJ-4 × Bro is suitable for transgene identification and as an mRNA reporter for expression pattern analysis. Following stable transformation in Arabidopsis thaliana, 3WJ-4 × Bro co-segregates and co-expresses with target transcripts and is stably inherited through multiple generations. Further, 3WJ-4 × Bro can be used to visualize virus-mediated RNA delivery in plants. This study demonstrates a protein-independent reporter system that can be used for transgene identification and in vivo dynamic analysis of mRNA.

Core-Shell Imidazoline-Functionalized Mesoporous Silica Superparamagnetic Hybrid Nanoparticles as a Potential Theranostic Agent for Controlled Delivery of Platinum(II) Compound.

INTRODUCTION: As widely used chemotherapeutic agents, platinum compounds have several therapeutic challenges, such as drug resistance and adverse effects. Theranostic systems, macromolecular or colloidal therapeutics with companion diagnostics, not only address controlled drug delivery but also enable real-time monitoring of tumor sites. METHODS: Synthesis of magnetic mesoporous silica nanoparticles (MMSNs) was performed for dual magnetic resonance imaging and drug delivery. MMSN surfaces were modified by imidazoline groups (MMSN-Imi) for cisplatin (Cis-Pt) conjugation via free N-termini to achieve well-controlled drug-release kinetics. Cis-Pt adsorption isotherms and drug-release profile at pH 5 and 7.4 were investigated using inductively coupled plasma atomic emission spectroscopy. RESULTS: MMSN-Imi showed a specific surface area of 517.6 m2 g-1, mean pore diameter of 3.26 nm, and saturated magnetization of 53.63 emu/g. A relatively high r2/r1 relaxivity value was obtained for MMSN-Imi. The nanoparticles provided high Cis-Pt loading with acceptable loading capacity (~30% w:w). Sustained release of Cis-Pt under acidic conditions led to specific inhibitory effects on the growth of human epithelial ovarian carcinoma cells, determined using MTT assays. Dual acridine orange-propidium iodide staining was investigated, confirming induction of apoptosis and necrotic cell death. CONCLUSION: MMSN-Imi exhibited potential for applications in cancer chemotherapy and combined imaging.

Deciphering Imidazoline Off-targets by Fishing in the Class A of GPCR field.

Based on the finding that a central antihypertensive agent with high affinity for I1-type imidazoline receptors - rilmenidine, shows cytotoxic effects on cultured cancer cell lines, it has been suggested that imidazoline receptors agonists might have a therapeutic potential in the cancer therapy. Nevertheless, potential rilmenidine side effects caused by activation of α-adrenoceptors, or other associated receptors and enzymes, might hinder its therapeutic benefits. Considering that human α-adrenoceptors belong to the rhodopsin-like class A of G-protein-coupled receptors (GPCRs) it is reasonable to assume that imidazolines might have the affinity for other receptors from the same class. Therefore, to investigate possible off-target effects of imidazoline ligands we have prepared a reverse docking protocol on class A GPCRs, using imidazoline ligands and their decoys. To verify our in silico results, three ligands with high scores and three ligands with low scores were tested for antagonistic activity on α2 - adrenoceptors.

Synthesis of 2'-Deoxyuridine Modified with a 3,5-Difluoro-4-Methoxybenzylidene Imidazolinone Derivative for Incorporation into Oligonucleotide Probes for Detection of HER2 Breast Cancer Marker.

Nucleoside intercalator conjugates (NICs) describe an innovative methodology developed in our research group for preparation of fluorescence turn-on DNA hybridization probes targeting specific mRNA sequences (e.g., breast cancer markers). In this methodology, we conjugate a non-fluorescent intercalator to the base of a nucleic acid (e.g., uracil) via a flexible spacer. This modified monomer can be incorporated into oligonucleotides by solid-phase synthesis and a large fluorescence enhancement is observed when the modified oligonucleotide is hybridized with its complementary strand due to intercalation of the fluorophore between the two strands. 5-(6-p-Methoxybenzylidene imidazolinone-1-hexene)-2'-deoxyuridine (dUMBI ) is a synthetic monomer to which 4-methoxybenzylidene imidazolinone (MBI), the fluorescent chromophore of green fluorescent protein (GFP), has been conjugated via a flexible spacer. The detection of human epidermal growth factor receptor 2 (HER2) mRNA by this probe has already been established by our group. The fluorescent intensity of the single-strand DNA can be considered as negligible due to the free rotation of the fluorophore. Upon hybridization, however, the flexible spacer allows for the intercalation of the fluorophore between the hybridized strands, giving rise to enhanced fluorescence and indicating the presence of target mRNA. 3,5-Difluoro-4-methoxybenzylidene (DFMBI) has enhanced photophysical properties compared to MBI fluorophore. This protocol describes a simple, reliable, efficient, and general method for the synthesis of improved derivative dUDFMBI as a monomer of fluorescent turn-on DNA hybridization probe with application for detection of HER2 mRNA. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Synthesis of 5-[(6)-3,5-difluoro-4-methoxybenzylidene imidazolinone-1-hexene]-2'-deoxyuridine.