Turn-on Coumarin Precursor: From Hydrazine Sensor to Covalent Inhibition and Fluorescence Detection of Rabbit Muscle Aldolase.

Hydrazine, a highly toxic compound, demands sensitive and selective detection methods. Building upon our previous studies with pre-coumarin OFF-ON sensors for fluoride anions, we extended our strategy to hydrazine sensing by adapting phenol protecting groups (propionate, levulinate, and γ-bromobutanoate) to our pre-coumarin scaffold. These probes reacted with hydrazine, yielding a fluorescent signal with low micromolar limits of detection. Mechanistic studies revealed that hydrazine deprotection may be outperformed by a retro-Knoevenagel reaction, where hydrazine acts as a nucleophile and a base yielding a fluorescent diimide compound (6,6'-((1E,1'E)-hydrazine-1,2diylidenebis(methaneylylidene))bis(3(diethylamino)phenol, 7). Additionally, our pre-coumarins unexpectedly reacted with primary amines, generating a fluorescent signal corresponding to phenol deprotection followed by cyclization and coumarin formation. The potential of compound 3 as a theranostic Turn-On coumarin precursor was also explored. We propose that its reaction with ALDOA produced a γ-lactam, blocking the catalytic nucleophilic amine in the enzyme's binding site. The cleavage of the ester group in compound 3 induced the formation of fluorescent coumarin 4. This fluorescent signal was proportional to ALDOA concentration, demonstrating the potential of compound 3 for future theranostic studies in vivo.

Thioxobimanes.

Dioxobimanes, colloquially known as bimanes, are a well-established family of N-heterobicyclic compounds that share a characteristic core structure, 1,5-diazabicyclo[3.3.0]octadienedione, bearing two endocyclic carbonyl groups. By sequentially thionating these carbonyls in the syn and anti isomers of the known (Me,Me)dioxobimane, we were able to synthesize a series of thioxobimanes, representing the first heavy-chalcogenide bimane variants. These new compounds were extensively characterized spectroscopically and crystallographically, and their aromaticity was probed computationally. Their potential role as ligands for transition metals was demonstrated by synthesizing a representative gold(I)-thioxobimane complex.

Shining light on fluoride detection: a comprehensive study exploring the potential of coumarin precursors as selective turn-on fluorescent chemosensors.

In this study, we report a fluoride chemosensor based on the use of a non-fluorescent pre-coumarin, compound 1. This compound undergoes selective fluoride-triggered formation of coumarin 2, with a concomitant turn-on fluorescence signal. Although compound 1 exists as a mixture of alkene isomers (2 : 1 in favor of the E isomer), only the minor Z-isomer undergoes cyclization. Nonetheless, comprehensive computational and experimental studies provide evidence that in situ isomerization of E-1 to Z-1, followed by fluoride-triggered phenolate evolution and intramolecular cyclization, facilitates the generation of coumarin 2 in high yield. Moreover, this system is an effective turn-on fluorescence sensor for fluoride anions, which displays outstanding selectivity (limited response to other commonly occurring analytes), sensitivity (lowest reported limits of detection for this sensor class), and practicality (works in solution and on paper to generate both fluorometric and colorimetric responses). Ongoing efforts are focused on expanding this paradigm to other pre-coumarin scaffolds, which also undergo analyte-specific coumarin formation accompanied by turn-on fluorescence.

A two-step strategy to visually identify molecularly imprinted polymers for tagged proteins.

A practical and relatively simple method to identify molecularly imprinted polymers capable of binding proteins via the molecular tagging (epitope-like) approach has been developed. In our two-step method, we first challenge a previously obtained anti-tag molecularly imprinted polymer with a small molecule including the said tag of choice (a biotin derivative as shown here or other) connected to a linker bound to a second biotin moiety. An avidin molecule partially decorated with fluorescent labels is then allowed to bind the available biotin derivative associated with the polymer matrix. At the end of this simple process, and after washing off all the low-affinity binding molecules from the polymer matrix, only suitable molecularly imprinted polymers binding avidin through its previously acquired small molecule tag (or epitope-like probe, in a general case) will remain fluorescent. For confirmation, we tested the selective performance of the anti-biotin molecularly imprinted polymer binding it to biotinylated alkaline phosphatase. Residual chemical activity of the enzyme on the molecularly imprinted polymer solid support was observed. In all cases, the corresponding nonimprinted polymer controls were inactive.

Biolabile peptidyl delivery systems toward sequential drug release.

Compact carriers for peptidyl delivery systems (PDSs) loaded with various drugs were synthesized using a simple and convenient solid phase organic synthesis strategy, including semi-orthogonal functional group protection schemes. Each attachment point of the compact carrier can thus be bound to an anticancer agent through a biodegradable covalent link. Chemo- and biostability experiments of a model peptidyl platform loaded with three different drugs revealed pH and liver homogenate (metabolic) dependent sequential release behavior. The versatility of this approach will serve to expedite the preparation of PDS libraries. This approach may prove useful for applications suitable for personalized medicine where multiple drug delivery is required in a sequential and controlled fashion.

Automated docking with protein flexibility in the design of femtomolar "click chemistry" inhibitors of acetylcholinesterase.

The use of computer-aided structure-based drug design prior to synthesis has proven to be generally valuable in suggesting improved binding analogues of existing ligands. Here we describe the application of the program AutoDock to the design of a focused library that was used in the "click chemistry in-situ" generation of the most potent noncovalent inhibitor of the native enzyme acetylcholinesterase (AChE) yet developed (K(d) = ~100 fM). AutoDock version 3.0.5 has been widely distributed and successfully used to predict bound conformations of flexible ligands. Here, we also used a version of AutoDock which permits additional conformational flexibility in selected amino acid side chains of the target protein.

A dipodal bimane-ditriazole-diCu(II) complex serves as an ultrasensitive water sensor.

An ultrasensitive fluorescent water sensor based on a dipodal bimane-Cu(II) complex is reported here. This complex, which is non-fluorescent in the absence of water, demonstrates a remarkable turn-on fluorescence in the presence of extremely low (0.000786% v/v) concentrations of water, via highly selective water-induced displacement of copper and restoration of the innate bimane fluorescence.

Highly Sensitive Water Detection Through Reversible Fluorescence Changes in a syn-Bimane Based Boronic Acid Derivative.

Reported herein is a fluorometric and colorimetric sensor for the presence of trace amounts of water in organic solvents, using syn-bimane based boronate ester 1. This sensor responds to the presence of water with a highly sensitive turn-off fluorescence response, with detection limits as low as 0.018% water (v/v). Moreover, analogously high performance was observed when compound 1 was adsorbed on filter paper, with the paper-based sensor responding both to the presence of liquid water and to humid atmospheres. Reusability of the paper-based sensor up to 11 cycles was demonstrated, albeit with progressive decreases in the performance, and 1H NMR and mass spectrometry analyses were used to explain the observed, hydrolysis-based sensor response.

Expedient synthesis and anticancer evaluation of dual-action 9-anilinoacridine methyl triazene chimeras.

The efficient synthesis of molecular hybrids including a DNA-intercalating 9-anilinoacridine (9-AnA) core and a methyl triazene DNA-methylating moiety is described. Nucleophilic aromatic substitution (SN Ar) and electrophilic aromatic substitution (EAS) reactions using readily accessible starting materials provide a quick entry to novel bifunctional anticancer molecules. The chimeras were evaluated for their anticancer activity. Chimera 7b presented the highest antitumor activity at low micromolar IC50 values in antiproliferative assays performed with various cancer cell lines. In comparison, compound 7b outperformed DNA-intercalating drugs like amsacrine and AHMA. Mechanistic studies of chimera 7b suggest a dual mechanism of action: methylation of the DNA-repairing protein MGMT associated with the triazene structural portion and Topo II inhibition by intercalation of the acridine core.

Highly sensitive detection of cobalt through fluorescence changes in ß-cyclodextrin-bimane complexes.

A supramolecular complex of syn-(methyl,methyl)bimane (1) and ß-cyclodextrin demonstrates a sensitive (limit of detection = 0.60 nM) and selective fluorescence turn-off response in the presence of cobalt in aqueous media, with calibration curves enabling quantitation in solution and using filter papers on which bimane and cyclodextrin were adsorbed. 1H NMR spectroscopy provides insight into interactions underlying the sensor performance.

syn-Bimane as a chelating O-donor ligand for palladium(ii).

A cationic Pd(ii) complex containing syn-(Me,Me)bimane as a ligand was prepared and fully characterized. This complex represents the first well-defined case of a bimane scaffold coordinated to a metal center. The strongly-fluorescent syn-bimane chelates the Pd(ii) center via its carbonyl oxygen atoms, affording a non-fluorescent complex. The crystal structure of this complex shows that the coordinated bimane departs from planarity, with its bicyclic framework bent about the N-N bond. Spectroscopic evidence demonstrates that bimane coordination is reversible in solution.

α-Aminoisobutyric Acid Leads a Fluorescent syn-bimane LASER Probe Across the Blood-brain Barrier.

Penetration of the blood brain barrier (BBB) by appropriate fluorescent probes remains a challenge in optical imaging and diagnostics. We designed, synthesized and observed the in vivo BBB penetration of a LASER syn-bimane probe. Results demonstrate that the Aib transporter unit in our probe may lead a fluorescent bimanyl moiety across the BBB.

Model of the alphaLbeta2 integrin I-domain/ICAM-1 DI interface suggests that subtle changes in loop orientation determine ligand specificity.

The interaction of the alphaLbeta2 integrin with its cellular ligand the intercellular adhesion molecule-1 (ICAM-1) is critical for the tight binding interaction between most leukocytes and the vascular endothelium before transendothelial migration to the sites of inflammation. In this article we have modeled the alphaL subunit I-domain in its active form, which was computationally docked with the D1 domain of the ICAM-1 to probe potential protein-protein interactions. The experimentally observed key interaction between the carboxylate of Glu 34 in the ICAM-1 D1 domain and the metal ion-dependent adhesion site (MIDAS) in the open alphaL I-domain was consistently reproduced by our calculations. The calculations reveal the nature of the alphaLbeta2/ICAM-1 interaction and suggest an explanation for the increased ligand-binding affinity in the "open" versus the "closed" conformation of the alphaL I-domain. A mechanism for substrate selectivity among alphaL, alphaM, and alpha2 I-domains is suggested whereby the orientation of the loops within the I-domain is critical in mediating the interaction of the Glu 34 carboxylate of ICAM-1 D1 with the MIDAS.

Mutants of 4-oxalocrotonate tautomerase catalyze the decarboxylation of oxaloacetate through an imine mechanism.

A designed single amino acid substitution can alter the catalytic activity and mechanism of 4-oxalocrotonate tautomerase (4-OT). While the wild-type enzyme catalyzes only the tautomerization of oxalocrotonate, the Pro1Ala mutant (P1A) catalyzes two reactions--the original tautomerization reaction and the decarboxylation of oxaloacetate. Although the N-terminal amine group of P1A is involved in both reactions, our results support a nucleophilic mechanism for the decarboxylase activity, in contrast to the general acid/base mechanism that has been previously established for the tautomerase activity. These findings demonstrate that a single catalytic group in a 4-OT mutant can catalyze two reactions by two different mechanisms.