Kamalika Mukherjee

4-Aminophenylalanine as a Biocompatible Nucleophilic Catalyst for Hydrazone-Ligations at Low Temperature and Neutral pH

Hydrazone formation and similar reactions are highly versatile and specific, but their application to biological systems has been limited by their characteristically slow reaction kinetics at neutral pH. Catalysis of these reactions through imine formation with aromatic amines such as aniline has broadened the applicability of these reactions to biomolecular labeling. High concentrations of the catalyst are necessary, which may be incompatible with the native structure of certain proteins. In this study, we investigated the utility of 4-aminophenylalanine (4a-Phe) as a catalyst for these reactions. We find that 4a-Phe is nearly as effective as aniline in catalyzing hydrazone formation between the reactive amino acid 3-formyltyrosine (3f-Tyr) and hydrazine-containing fluorophores, both free in solution and incorporated into the protein tubulin. The catalyst 4a-Phe maintains ∼70% of the catalytic efficacy of aniline and is less detrimental to the native structure of tubulin. Examination of the temperature dependence of imine formation between 3f-Tyr and 4a-Phe shows an increase in imine concentration accompanying a decrease in temperature, confirming the exothermic nature of the equilibrium reaction. Interestingly, decreasing the temperature of the 4a-Phe-catalyzed hydrazone reaction between 3f-Tyr and the fluorophore 7-hydrazinyl-4-methylcoumarin increases the overall rate of the reaction. This result indicates that the temperature dependence of the catalyst-aldehyde equilibrium is greater than the temperature dependence of the rate constant for hydrazone formation from this intermediate, and that the rate of hydrazone formation a direct function of the concentration of the intermediate imine. These results provide a platform for conducting nucleophilic catalysis under conditions that are more compatible with biomolecular targets than previously demonstrated, thereby expanding the utility of hydrazone ligations in biological systems.

Site-Specific Orthogonal Labeling of the Carboxy Terminus of α-Tubulin

A fluorescent probe has been attached to the carboxy terminus of the alpha-subunit of alpha,beta-tubulin by an enzymatic reaction followed by a chemical reaction. The unnatural amino acid 3-formyltyrosine is attached to the carboxy terminus of alpha-tubulin through the use of the enzyme tubulin tyrosine ligase. The aromatic aldehyde of the unnatural amino acid serves as an orthogonal electrophile that specifically reacts with a fluorophore containing an aromatic hydrazine functional group, which in this case is 7-hydrazino-4-methyl coumarin. Conditions for covalent bond formation between the unnatural amino acid and the fluorophore are mild, allowing fluorescently labeled tubulin to retain its ability to assemble into microtubules. A key feature of the labeling reaction is that it produces a red shift in the fluorophores absorption and emission maxima, accompanied by an increase in its quantum yield; thus, fluorescently labeled protein can be observed in the presence of unreacted fluorophore. Both the enzymatic and coupling reaction can occur in living cells. The approach presented here should be applicable to a wide variety of in vitro systems.

Aurones: Small Molecule Visible Range Fluorescent Probes Suitable for Biomacromolecules

Aurones, derivatives of 2-benylidenebenzofuran-3(2H)-one, are natural products that serve as plant pigments. There have been reports that some of these substances fluoresce, but little information about their optical properties is in the literature. In this report, series of aurone derivatives were synthesized as possible fluorescent probes that can be excited by visible light. We found that an amine substituent shifted the lowest energy absorption band from the near-UV to the visible region of the electromagnetic spectrum. Four amine-substituted aurone derivatives were synthesized to explore the effect of this substituent on the absorption and emission properties of the aurone chromophore. The emission maxima and intensities of the molecules are strongly dependent on the nature of the substituent and the solvent polarity. Overall, the emission intensity increases and the maximum wavelength decreases in less polar solvents; thus, the aurones may be useful probes for hydrophobic sites on biological molecules. A limited investigation with model protein, nucleic acid and fixed cells supports this idea. It is known that the sulfur analog of aurone can undergo photo-induced E/Z isomerization. This possibility was investigated for one of the aminoaurones, which was observed to reversible photoisomerize. The two isomers have similar absorption spectra, but the emission properties are distinct. We conclude that appropriately substituted aurones are potentially useful as biological probes and photoswitches.

Synthesis, Cytotoxic Properties and Tubulin Polymerization Inhibitory Activity of Novel 2-Pyrazoline Derivatives

A series of novel 1‐(3′,4′,5′‐trimethoxybenzoyl)‐3,5‐diarylpyrazoline derivatives were synthesized and evaluated for their cytotoxic properties on different cancer cell lines and tubulin polymerization inhibitory activity. Compounds 6d and 6e exhibited remarkable cytotoxic activity against different cancer cell lines with good tubulin polymerization inhibitory activity. Compound 6d exhibited moderate selectivity toward renal cancer and breast cancer subpanels with selectivity ratios of 3.06 and 5.11, respectively, at the cytostatic activity (TGI) level. Compounds 6e and 6d achieved good tubulin polymerization inhibitory activity with IC50 values of 17 and 40 µM, respectively. The photomicrographs made for compounds 6d and 6e on cellular microtubules indicated that the cytotoxicity of these compounds can be attributed to their ability to interfere with microtubule assembly. Molecular modeling studies involving compound 6e with the colchicine binding site of α,β‐tubulin revealed hydrogen‐bonding and hydrophobic interactions with several amino acids in the colchicine binding site of β‐tubulin.

Measurement of in vitro microtubule polymerization by turbidity and fluorescence.

Tubulin polymerization may be conveniently monitored by the increase in turbidity (optical density, or OD) or by the increase in fluorescence intensity of diamidino-phenylindole. The resulting data can be a quantitative measure of microtubule (MT) assembly, but some care is needed in interpretation, especially of OD data. Buffer formulations used for the assembly reaction significantly influence the polymerization, both by altering the critical concentration for polymerization and by altering the exact polymer produced-for example, by increasing the production of sheet polymers in addition to MT. Both the turbidity and the fluorescence methods are useful for demonstrating the effect of MT-stabilizing or -destabilizing additives.

Benzocoumarin Hydrazine: A Large Stokes Shift Fluorogenic Sensor for Detecting Carbonyls in Isolated Biomolecules and in Live Cells

Detection and quantification of biomolecule carbonylation, a critical manifestation of oxidative stress, allows better understanding of associated disease states. Existing approaches for such analyses require further processing of cells and tissues, which leads to loss of both spatial and temporal information about carbonylated biomolecules in cells. Live cell detection of these species requires sensors that are nontoxic, sufficiently reactive with the biocarbonyl in the intracellular milieu, and detectable with commonly available instrumentation. Presented here is a new fluorescent sensor for biomolecule carbonyl detection: a hydrazine derivative of a benzocoumarin, 7-hydrazinyl-4-methyl-2H-benzo[h]chromen-2-one (BzCH), which meets these requirements. This probe is especially well suited for live cell studies. It can be excited by a laser line common to many fluorescence microscopes. The emission maximum of BzCH undergoes a substantial red shift upon hydrazone formation (from ∼430 to ∼550 nm), which is the result of fluorophore disaggregation. Additionally, the hydrazone exhibits an exceptionally large Stokes shift (∼195 nm). The latter properties eliminate self-quenching of the probe and the need to remove unreacted fluorophore for reliable carbonyl detection. Thus, biomolecule carbonylation can be detected and quantified in cells and in cell extracts in a one-step procedure using this probe.

Detection of oxidative stress-induced carbonylation in live mammalian cells

Oxidative stress is often associated with etiology and/or progression of disease conditions, such as cancer, neurodegenerative diseases, and diabetes. At the cellular level, oxidative stress induces carbonylation of biomolecules such as lipids, proteins, and DNA. The presence of carbonyl-containing biomolecules as a hallmark of these diseases provides a suitable target for diagnostic detection. Here, a simple, robust method for detecting cellular aldehydes and ketones in live cells using a fluorophore is presented. A hydrazine-functionalized synthetic fluorophore serves as an efficient nucleophile that rapidly reacts with reactive carbonyls in the cellular milieu. The product thus formed exhibits a wavelength shift in the emission maximum accompanied by an increase in emission intensity. The photochemical characteristics of the fluorophore enable the identification of the fluorophore-conjugated cellular biomolecules in the presence of unreacted dye, eliminating the need for removal of excess fluorophore. Moreover, this fluorophore is found to be nontoxic and is thus appropriate for live cell analysis. Utility of the probe is demonstrated in two cell lines, PC3 and A549. Carbonylation resulting from serum starvation and hydrogen peroxide-induced stress is detected in both cell lines using fluorescence microscopy and a fluorescence plate reader. The fluorescent signal originates from carbonylated proteins and lipids but not from oxidized DNA, and the majority of the fluorescence signal (>60%) is attributed to fluorophore-conjugated lipid oxidation products. This method should be useful for detecting cellular carbonylation in a high-content assay or high-throughput assay format.

Site-specific fluorescent labeling of tubulin.

Fluorescent tubulin can be prepared in which a fluorophore is covalently bound to the protein at only the carboxy terminus of the α-subunit of the αβ-tubulin dimer. This two-step procedure consists of an enzymatic reaction followed by a bioorthogonal chemical reaction. In the first step of the process, the enzyme tubulin tyrosine ligase is used to attach a reactive tyrosine derivative, 3-formyltyrosine, to the protein. In the second step of the procedure, a fluorophore possessing a complementary reactive functional group, such as a hydrazine, hydrazide, or hydroxylamine, is allowed to react with the protein under conditions that are compatible with native tubulin. Polymerization-competent, fluorescently labeled tubulin can be prepared in just a few hours using this protocol. The method described here should be useful for attaching virtually any probe or material to tubulin at this site.