Synthesis of pH Dependent Pyrazole, Imidazole, and Isoindolone Dipyrrinone Fluorophores using a Claisen-Schmidt Condensation Approach

The Claisen-Schmidt condensation reaction is an age-old reaction, first being reported by Claisen and Schmidt concurrently in 1881. It involves base-assisted enolate addition of a ketone or aldehyde, shown in blue, into an aromatic aldehyde, shown in red. Initially, the addition of the enolate results in the formation of an alcohol, as shown in the brackets.

However, a subsequent dehydration ultimately produces an enone. Since the aro-aldehyde does not contain an alpha hydrogen, it cannot form an enolate. As a result, often weak bases, such as hydroxide, can be used to generate the enolate.

The Claisen-Schmidt condensation has been used to generate a number of compounds over the years. However, it has been extensively used to join the aromatic ring systems of the chalcones and flavanones, which are shown in red and blue. The blue portion indicates that derived from the enolate and the red is of the aromatic portion.

The chalcones and flavanones are essential core to a range of biologically active molecules that possess a range of activities, such as anti-bacterial, anti-fungal, anti-inflammatory, and anti-tumor, depending on the substitution pattern. Another broad class of molecules generated from the Claisen-Schmidt condensation reaction is the methine-bridged compounds of which we give examples in this study. Our lab is interested in fluorescent constituents of the pigment bilirubin, which is naturally produces a degradation product of heme.

The synthesis of bilirubin and many of its constituents revolves around the Claisen-Schmidt type condensation, which can be visualized in the structures displayed by the blue enolate and the red aromatic aldehyde components. Typically, direct constituents of bilirubin, such as dipyrrinones, are non-fluorescent. However, if one bridges the two nitrogen groups with a methylene or carbonyl group, the resulting molecules become highly fluorescent, such as in the case of xanthoglows.

Normally, dipyrrinones absorb UV or blue light, which results in an Z to E isomerization process. Like unbridged dipyrrinones, the N in bridged dipyrrinones also absorb UV or blue light, but differ in that they relax in an excited state via fluorescence. We have recently discovered a series of dipyrrinone derivatives which actually fluoresce without a covalent bridge linking the two nitrogen groups.

Instead, a hydrogen bond appears to be deterring the Z to E isomerization process, leading to a mode of fluorescence. In addition, an unanticipated discovery was made that these molecules can be deprotonated in basic media, leading to redshifted absorption emission spectra in the deprotonated states. Consequently, these molecules may have value as ratiometric pH probes.

The fluorescent dipyrrinone derivatives are used, generating a slight adaptation in the traditional Claisen-Schmidt condensation reaction. This protocol deviates from the traditional Claisen-Schmidt condensation reaction in that a vinylogous enolate derived from a pyrrolinone or isoindolone is the nucleophilic source. The vinylogous enolate adds to either a pyrazole or imidazole aldehyde to generate a small library of dipyrrinone analogues.

The procedure which is used to create this library is illustrated in the video. However, analogous steps can be used to carry out the traditional Claisen-Schmidt reaction. While the Claisen-Schmidt reaction has been and is still a widely used synthetic reaction, this is the first video count of this method, to which we are aware.

To prepare for the synthesis of a fluorescent dipyrrinone analogue via aldol condensation, weigh out equal equivalents of the chosen nucleophile and electrophile. Then add them to a 25 milliliter round bottom flask containing a magnetic stir bar. Measure five milliliters of ethanol using a graduated cylinder.

Then add the ethanol to the round bottom flask. Measure 2.4 milliliters of previously prepared 10 molar potassium hydroxide using a graduated cylinder. Then add the potassium hydroxide to the flask.

To set the flask up to reflux, apply a sufficient amount of vacuum grease to the ground glass joint of a reaction condenser to prevent seizing of the ground glass joints. Connect the condenser to a cold water supply, then attach the greased joint of the condenser to the round bottom flask. Then place the flask in either an oil bath or on an aluminum heating block that can maintain a constant temperature via thermal couple with a hot plate stirrer.

Heat to reflux temperature while allowing the reaction to stir. The reaction mixture should be monitored by thin layer chromatography at one, three, six, 12 and 24 hours to gauge the reaction rate and to check for complete consumption of the starting materials. Allow the flask to cool to room temperature, then evaporate the ethanol solvent using a rotary evaporator.

Place the flask in an ice bath and allow the flask to equilibrate to the temperature of the ice bath over the course of five minutes. Neutralize any remaining potassium hydroxide in the flask by adding 1.7 milliliters of acetic acid in a single portion. If crystal formation occurred following neutralization, then follow the vacuum filtration purification procedure.

If no crystal formation was observed, then follow the flash column chromatography purification procedure. Place a round piece of filter paper on top of the funnel and lightly wet the paper using deionized water to adhere it to the funnel. To prepare for the vacuum filtration of crystals, fit a Hirsch or Buchner funnel to a side-arm flask using a fitted rubber adapter.

To avoid clogging of the filter paper which can impede filtration, we used a larger Hirsch or Buchner funnel than is typical for similar scale filtration processes. Pour the contents from the round bottom flask over the filter paper and allow the mixture to filter. Rinse the crystals during the filtration process using 10 milliliters of ice-cold deionized water.

Following filtration, transfer the crystals to a 25 milliliter round bottom flask. Apply a light coat of vacuum grease to a high power vacuum line glass adapter, then connect the adapter to the round bottom flask. Secure the glass joint with a Keck clip.

To prepare the high power vacuum for drying the crystals of any residual solvent, adequately cool the glass vacuum trap with a mixture of dry ice and acetone. Connect a high power vacuum line to the glass adapter attached to the round bottom flask. Turn on the high power vacuum pump and allow the crystals to dry for at least one hour.

Once the crystals have been sufficiently dried under vacuum, turn the vacuum pump off and release the vacuum seal in order to remove the round bottom flask. Weigh the dried crystals to report the reaction percent yield. To prepare for purification via flash column chromatography, add the acid-treated mixture that did not form crystals from the synthesis procedure to a separatory funnel.

Measure 10 milliliters of dichloromethane using a graduated cylinder and use this to dilute the acid-treated mixture in the separatory funnel. Close and gently shake the separatory funnel while making sure to vent frequently. Following this, two separate layers should be visible in the separatory funnel.

Extract the aqueous layer using an additional five milliliters of dichloromethane. Complete this step two more times. Combine all the organic fractions and add a sufficient amount of anhydrous sodium sulfate in order to dry the organic fractions.

Transfer the dried organic fractions to a round bottom flask and remove the dichloromethane using a rotary evaporator. Dilute the remaining residue with an additional five milliliters of dichloromethane. Prepare a column using approximately 75 grams of silica gel and use this to perform flash column chromatography on the sample, using 10%methanol in dichloromethane as the eluent.

Evaporate the eluent from the collected fractions using a rotary evaporator. Prepare the high power vacuum pump and glass solvent trap, as previously described in the vacuum filtration purification procedure, and allow the collected solid to dry under high vacuum for at least one hour. Once the crystals have been sufficiently dried under vacuum, weigh the dried crystals to report the reaction percent yield.

To confirm the structure of each of the dipyrrinone analogues in the library, several spectroscopic methods were utilized in combination, including nuclear magnetic resonance spectroscopy, infrared spectroscopy, and high resolution mass spectrometry. UV-Vis and fluorescent spectroscopy were used in the photophysical characterization of the fluorescent dipyrrinone analogues. Using the Claisen-Schmidt condensation reaction, we were able to synthesize a small library of 10 compounds, including that of a control compound that cannot engage in intermolecular hydrogen bonds.

Yields for dipyrrinone analogues varied from roughly 40%to nearly quantitative and are listed below each molecule. The compounds with the highest quantum yields, both in the protonated and deprotonated forms, were derived from 2-formylimidazole and are displayed in pink boxes. The control compound, which does not fluoresce, is in a cyan box.

The dipyrrinone derivatives under a standard 365 nanometer long wavelength lamp gives the observed fluorescence. One can visually observe the redshifted fluorescence that results from deprotonation. Through the color of the vials, they transition from blue to cyan in color.

For more quantitative data on the photophysical and other physical properties of the dipyrrinone derivatives, we direct viewers to table two in the written portion of the manuscript. Overall, the Claisen-Schmidt condensation reaction provides access to a range of methine-linked bicyclic aromatic compounds. However, there are some limitations.

The reaction is dependent upon the use of both an enolizable nucleophile and a non-enolizable aldehyde electrophile, such as an aro-aldehyde, in order to undergo successful condensation. In failing to meet this basic requirement, attempts to perform this reaction will likely result in the inability to link together the ring systems and/or the generation of competing side products. Another consideration is that basic conditions are used for generating the enolate nucleophile which can create incompatibilities with functional groups that are susceptible to reactions with hydroxide.

In such cases, it’s possible to substitute hydroxide with nitrogenous bases or carbonate that has been accomplished with DBU, triethylamine, piperidine, Hunig’s base and sodium carbonate. We simply chose to use potassium hydroxide due to its availability and relative expense. Despite these limitations, the method outlined in the protocol can provide a means of coupling aromatic rings for numerous systems through a procedurally simple and cost-effective single-step reaction.

In the case of dipyrrinone analogues we have synthesized, the Claisen-Schmidt condensation has enabled one of the most accessible routes to pH-dependent fluorophores described to date. Nevertheless, future designs of dipyrrinone analogues will be developed using the outlined procedure in order to generate fluorescent compounds with stronger intermolecular hydrogen bonding capacity and lower pKa values. Anticipate these enhanced pH-dependent probes will possess higher quantum yields while enabling the visualization of pH fluctuations for a wider range of intracellular events.