Z.R.W. and N.B. thank the NIH (2P20 GM103440-14A1) for their generous funding as well as Jungjae Koh and the University of Nevada, Las Vegas for their assistance in acquiring 1H and 13C NMR. Additionally, we would like to thank NSC visual media students, Arnold Placencia-Flores, Aubry Jacobs,&#160;and Alistair Cooper for their help in the filming and animation processes within the cinematography portions of this manuscript.

1. General Procedure for Synthesis of Dipyrrinone Analogs 16-25
<ol>
	<li>Dissolve pyrrolinone/isoindolone (1.00 mmol) and the corresponding pyrazole/imidazole aldehyde (1.00 mmol) in 5.0 mL of ethanol in a round-bottom flask.</li>
	<li>Add aqueous KOH (24.0 mmol, 10 M, 2.40 mL) to the flask in one portion.</li>
	<li>Stir and reflux the mixture until reaction completion is confirmed by TLC (See Table 1&#160;for a list of reaction times). A TLC eluent of 10% methanol in dichloromethane was used and the analogs have been observed to possess Rf values in or around the range of 0.62 to 0.86. Apply a sufficient amount of vacuum grease or use a PTFE sleeve at the interphase of glass joints to prevent the glass of the condenser and the round-bottom flask from seizing under high temperatures and basic conditions.</li>
	<li>Allow the reaction mixture to cool to room temperature and evaporate volatiles under reduced pressure using a rotary evaporator.</li>
	<li>Cool the reaction mixture to 0 &#176;C using an ice bath.</li>
	<li>Neutralize the remaining oily mixture by adding acetic acid (30.0 mmol, 1.70 mL) in one portion.</li>
	<li>Purify the resulting product material using vacuum filtration (see Step 2a, for compounds: 17, 18, 20-22, 24 and 25) or use liquid-liquid extraction/column chromatography (see Step 2b, for compounds: 16, 19, and 23).</li>
</ol>
2. Procedure Purification
<ol>
	<li>via Vacuum Filtration
	<ol>
		<li>Fit a Hirsch funnel to a side-arm flask using a fitted rubber adapter.</li>
		<li>Apply a piece of round filter paper to the Hirsch funnel and lightly wet using deionized water to allow adherence to the funnel.</li>
		<li>Connect a vacuum source to the side-arm of the flask and ensure that there is a sufficient vacuum seal by making sure none of the glassware can be pulled apart while under vacuum.</li>
		<li>Pour the flask containing the crystallized product over the vacuum filter and allow to filter. Rinse the crystals with 10 mL of ice-cold deionized water.</li>
		<li>Allow the filtered crystals to continue to dry on top of the filter paper, while still connected to the vacuum source, following the filtration of all liquids.</li>
		<li>Collect the filtered crystals and place in a 25 mL round-bottom flask.</li>
		<li>Attach the round-bottom flask to a fritted ground glass joint/vacuum line adapter. Secure the glass joint union with a keck clip.</li>
		<li>To the ribbed end of the glass high vacuum line adapter, attach a vacuum line that is routed to a high vacuum pump and adequately cool a vacuum trap (using coolant such as liquid nitrogen or dry ice/acetone) to condense any volatile materials that may evaporate from the crystalline material. Turn on the high vacuum pump to ensure complete evaporation of any trace amounts of remaining solvent from the crystals.</li>
		<li>Allow the crystals to dry under high vacuum for a minimum of 1 h. Remove the round-bottom flask from the vacuum line/adapter, turn off the high vacuum pump, and clean out the vacuum trap.</li>
	</ol>
	</li>
	<li>Procedure Purification via Column Chromatography
	<ol>
		<li>Dilute the acetic acid treated reaction mixture contents (from step 1.6) with 10 mL of deionized water and transfer into a separatory funnel. Add 10 mL of dichloromethane to the separatory funnel. Gently shake and vent the separatory funnel in order to separate the two layers.</li>
		<li>Extract the aqueous layer using subsequent portions of dichloromethane (3 x 5 mL). Combine the organic fractions and dry using anhydrous Na2SO4. Decant and remove all volatiles under reduced pressure using a rotary evaporator.</li>
		<li>Dilute the residue obtained from step 2.2.2. with 5 mL of CH2Cl2. Perform flash column chromatography using approximately 75 g of silica gel. Elute the sample with a solution of 10% methanol in dichloromethane.</li>
		<li>Remove the collected fractions of solvent under reduced pressure using a rotary evaporator. Transfer the solid residue to a 25 mL round-bottom flask using approximately 10 mL of CH2Cl2. Remove the solvent under reduced pressure using a rotary evaporator.</li>
		<li>Dry the remaining solid residue under high vacuum as previously described in steps 2.1.7 - 2.1.9.</li>
	</ol>
	</li>
</ol>
3. Molar Absorptivity Acquisition and UV/Vis pKa Studies for Analogs 16-25
<ol>
	<li>Create compound stock solutions for UV/Vis Spectrophotometry for analogs 16 - 25.
	<ol>
		<li>Weigh out 10 &#956;mol of the selected dipyrrinone analog (16 - 25) and add it to a 10 mL volumetric flask.</li>
		<li>Add DMSO to the 10.0 mL mark on the volumetric flask. 
		&#8203;NOTE: If the compound does not dissolve entirely, heat the flask using a heat gun and agitate the flask as needed to completely dissolve the compound.</li>
	</ol>
	</li>
	<li>Make phosphate buffered saline (PBS) solutions at various pH levels. The analogs were characterized in PBS buffers ranging in pH from approximately 4 to 15.
	<ol>
		<li>Using a 1 L volumetric cylinder, create 1 L of PBS stock solution by diluting 100 mL of PBS (x100) in 900 mL of deionized water.</li>
		<li>Transfer 50 mL of the prepared PBS stock solution (step 3.2.1) to a 100 mL beaker and add a magnetic stir bar. Then using a calibrated pH meter to monitor changes in pH, titrate the PBS buffer with either aqueous 1.0 M NaOH (to obtain buffers with pH &#62; 7.0) or 1.0 M HCl (to obtain buffers with pH &#60; 7.0). 
		NOTE: To obtain data that results in a well-defined titration curve, we recommend generating pH buffers in increments of 0.1 pH units within &#177; 0.5 of the anticipated point of inflection and increments of 0.5 outside of the anticipated point of inflection.</li>
	</ol>
	</li>
	<li>Acquire molar absorptivity spectra for analogs 16 - 25 in PBS (pH 7.0) and 1.0 M NaOH (pH 14.0) solutions.
	<ol>
		<li>Prepare a &#34;blank&#34; using a clean and dry quartz cuvette then add 2.0 mL of either PBS stock solution (pH 7.0) or aqueous 1.0 M NaOH to the cuvette using a 100 - 1000 &#181;L micropipette. 
		NOTE: It is important to the integrity of the blank solution data acquisition process to ensure that there are no air bubbles in the cuvette solution and to thoroughly wipe the sides of the cuvette with a Kim wipe to prevent light scattering resulting from dust or debris on the outside of the cuvette. If bubbles persist, gently and repeatedly tap the cuvette on a paper towel laid on a hard surface.</li>
		<li>Using a UV/Vis spectrophotometer, blank the selected solution for a 200 - 800 nm range.</li>
		<li>Into a second clean and dry quartz cuvette add 2.00 mL of either PBS (pH 7.0) or 1.0 M NaOH (pH 14) followed by 10 &#181;L of the dipyrrinone analog (16 - 25) stock solution (see step 3.1) with a 5-50 &#181;L micropipette. Place a cap on the cuvette and shake well in addition to inverting the cuvette. 
		NOTE: It is important to the integrity of the sample solution data acquisition process to ensure that there are no air bubbles in the cuvette solution and to thoroughly wipe the sides of the cuvette with a Kim wipe to prevent light scattering resulting from dust or debris on the outside of the cuvette. If bubbles persist, gently and repeatedly tap the cuvette on a paper towel laid on a hard surface.</li>
		<li>Using the UV/Vis spectrophotometer, acquire an absorption spectrum for the dipyrrinone analog solution for a 200 - 800 nm range.</li>
		<li>To the same cuvette, add an additional 10 &#181;L of the dipyrrinone analog stock solution and repeat steps 3.3.3 and 3.3.4.</li>
		<li>Repeat step 3.3.5 until a total of 50 &#181;L of dipyrrinone analog stock solution has been added to the cuvette in order to acquire at least five excitation wavelength data points. Repeat steps 3.3.1 - 3.3.6 until all stock solutions of 16 - 25 have been obtained in both PBS (pH 7.0) and 1.0 M NaOH.</li>
	</ol>
	</li>
	<li>Obtain molar absorptivity values for 16 - 25 in PBS (pH 7.0) and 1.0 M NaOH using best fit linear regression analysis.
	<ol>
		<li>Using a graphing program such as GraphPad Prism 7, plot the measured absorbance (y-axis) against the dipyrrinone analog concentration (x-axis). Create a best-fit linear regression analysis for the five plotted points. A linear relationship should be observed and statistical analysis should show an R2 value &#8805; 0.98.</li>
		<li>Repeat step 3.4.1 for analogs 16 - 25 in PBS (pH 7.0) and 1.0 M NaOH.</li>
		<li>Calculate the molar absorptivity for 16 - 25 in PBS (pH 7.0) and 1.0 M NaOH using the extrapolated slope value from the best fit linear curve.</li>
	</ol>
	</li>
	<li>Determine the pKa values of 16 - 25 Studies Using UV/Vis Spectrophotometry
	<ol>
		<li>Into a clean and dry quartz cuvette, transfer 1,900 &#181;L of the PBS buffer at the selected pH level (prepared in Step 3.2) using a 100 - 1000 &#181;L micropipette. 
		&#8203;NOTE: We have noticed upon storage that a white precipitate can form in some of the buffers. To ensure that the buffer is completely homogenous and if any precipitate is visible, use gravity filtration to remove any precipitate immediately prior to use. See the note after step 3.3.1.</li>
		<li>Using the UV/Vis spectrophotometer, blank the selected PBS buffer solution for a 200 - 800 nm range.</li>
		<li>Into a second clean and dry quartz cuvette, transfer 1,900 &#181;L of the selected PBS buffer then add 100 &#181;L of the selected analog stock solution using a 5-50 &#181;L micropipette. Place a cap on the cuvette and shake well in addition to inverting the cuvette. 
		&#8203;NOTE: See the previous note after step 3.3.3.</li>
		<li>Using the UV/Vis spectrophotometer, acquire the absorption spectrum for the dipyrrinone analog for a range of 200 - 800 nm.</li>
		<li>Repeat steps 3.5.1 - 3.5.4 for 16 - 25 in each of the PBS buffers generated in step 3.2.</li>
	</ol>
	</li>
	<li>Determine the pKa values for 16 - 25 using a best fit sigmoidal curve fitting function.
	<ol>
		<li>Using a graphing program, graph the measured absorbance vs. wavelength (nm) for 16 - 25 at the various pH levels.</li>
		<li>Choose a wavelength between 380-415 nm where at lower pH levels (&#60; 7.0) absorbance is small (0-0.1 units) and at greater pH (&#62; 12.0) absorbance is considerably larger (0.8-1.0 units). Plot the absorbance at the chosen wavelength vs. the pH.</li>
		<li>Using a sigmoidal curve function, generate a best fit curve for each of the analogs 16 - 25. Report the extrapolated pH at half-height of the curve. This is the reported pKa value.</li>
	</ol>
	</li>
</ol>
4. Quantum Yield Acquisition and Fluorescence Studies
<ol>
	<li>Create fluorescence study stock solutions for dipyrrinone analogs 16 - 18 and 20 - 25.
	<ol>
		<li>Using a dipyrrinone analog stock solution created in step 3.1, perform a dilution of the stock solution by adding 10 &#181;L of the stock solution to a 1 mL volumetric flask using a 2 - 20 &#181;L micropipette, then add PBS (pH 7.0) buffer to the 1 mL mark. Place a cap on the volumetric flask and mix well by inverting and shaking the flask. This diluted stock solution will be used to generate the fluorescence spectra and will be referred to as the fluorescence stock solution.</li>
		<li>Repeat step 4.1.1 for analogs 16 - 18 and 20 - 25.</li>
	</ol>
	</li>
	<li>Acquire fluorescence emission spectra at varying concentrations, for analogs 16 - 18 and 20 - 25. For all analogs other than 18, acquire five spectra for each analog in solutions of pH 7 and 14 at concentrations of: 19.96, 39.84, 59.64, 79.37, and 99.01 nM. For analog 18, acquire five spectra in a solution of pH 7 at concentrations of: 49.75, 99.01, 147.8, 196.1, and 243.9 nM. In a solution of pH 14, acquire five spectra for analog 18 at concentrations of: 99.01, 196.1, 291.3, 384.6, 476.2 nM.
	<ol>
		<li>Into a transparent four-sided quartz cuvette, add 3.00 mL of PBS (pH 7.0) or 1.0 NaOH using a 100 -1,000 &#181;L micropipette in three 1,000 &#181;L increments. 
		&#8203;NOTE: See note after step 3.3.1.</li>
		<li>Using the fluorometer and the fluorometer software program FluorEssence, acquire an emission spectrum for the selected solution and label this as the solution &#34;blank&#34;.</li>
		<li>To the same cuvette, add 6 &#181;L of fluorescence stock solution for the selected dipyrrinone analog (Part 4.1) using a 0.5-10 &#181;L micropipette. Place the cap on the cuvette and mix well by inverting and gently shaking the cuvette. 
		NOTE: See note after step 3.3.3.</li>
		<li>Using the fluorometer, acquire an emission spectrum for the selected compound solution using &#955;&#8203;max abs as the excitation wavelength. Excitation intensity was measured over a 200 nm range starting at 15 nm beyond the excitation wavelength (typically a 200 nm range is required for the fluorescence intensity to return to baseline).</li>
		<li>Repeat steps 4.2.3 - 4.2.4 until a total of 30 &#181;L of fluorescence stock solution has been added to the cuvette.</li>
		<li>Repeat steps 4.2.1 - 4.2.5 for analogs 16 - 25 in PBS (pH 7.0) and 1.0 M NaOH. 
		NOTE: The cuvette concentrations were changed for analog 18 and data was acquired using: 2.0 mL of PBS with five consecutive 10 &#181;L increments of fluorescence stock solution for a neutral (protonated 18) solution and 2.0 mL of 1.0 M NaOH with five 20 &#181;L increments of added compound stock solution for a basic (deprotonated 18) solution.</li>
	</ol>
	</li>
	<li>Determine the quantum yield using the method of Williams, A. T. et al.17
	<ol>
		<li>Using a spreadsheet software program (i.e., Microsoft Excel), import the data (emission intensity data points) for the emission spectra for a single dipyrrinone analog (taken in either PBS [pH 7.0] or 1.0 M NaOH) at the various concentration levels.</li>
		<li>Import the data points from the emission spectra for the &#34;blank&#34; solution (steps 4.2.1 - 4.2.2) and subtract the &#34;blank&#34; emission intensity data points from the emission intensity data points, at the corresponding wavelengths, acquired at various concentration levels.</li>
		<li>Transfer the &#34;blank&#34; corrected emission intensity data points into a graphing program, such as GraphPad Prism 7, and plot emission vs. wavelength. Calculate the area under the curve for each of the curves obtained at the various concentration levels of dipyrrinone analog.</li>
		<li>Following the technique outlined by Williams, A. T. et al, calculate an extrapolated absorbance value for each of the varying concentration levels of dipyrrinone analog. This is accomplished by multiplying the calculated molar absorptivity value (from best-fit linear regression analysis, see step 3.4) by each concentration of dipyrrinone analog used in steps 4.2.3-4.2.5.</li>
		<li>Using a graphing program such as GraphPad Prism 7, create a plot of the analog&#39;s extrapolated absorbance (x-axis) against the calculated area under each concentration curve (step 4.3.4) for the emission wavelength corresponding to the greatest emission value. A linear relationship with a r2 &#8805; 0.96 should be observed.</li>
		<li>Perform steps analogous to 3.1-3.4 and 4.1-4.3.5 for quinine in 0.5 M H2SO4 (&#934;F = 0.55)18 and anthracene in ethanol (&#934;F = 0.27)18, 19 to obtain data for standards.</li>
		<li>Obtain the quantum yield values for 16 - 25 in PBS (pH 7.0) and 1.0 M NaOH by using the extrapolated slopes obtained from steps 4.3.5 and 4.3.6 in the following equation: 
		&#934;x =&#934;st(Gradx/Gradst)(&#951;&#8203;2x/&#951;2st) 
		where &#934;st represents the quantum yield of the standard, &#934;x&#160;represents the quantum yield of the unknown, Grad is the slope of the best linear fit, and &#951; is the refractive index of the solvent used (the refractive index ratio was calculated using &#951; = 1.36 for ethanol and &#951; = 1.35 for 0.5 M H2SO4).</li>
		<li>Report the quantum yields for 16 - 18 and 20 - 25 in PBS (pH 7.0) and 1.0 M NaOH as an average of the &#934;x obtained for quinine and anthracene.</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanine+dye+labeling+reagents:+Sulfoindocyanine+succinimidyl+esters.">Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters.</a> Bioconjugate Chemistry. 4 (2), 105-111 (1993).</li><li>Battersby, A. R., Dutton, C. J., Fookes, C. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+studies+relevant+to+biosynthetic+research+on+vitamin+B12.+Part+7.+Synthesis+of+(&#177;)-bonellin+dimethyl+ester.">Synthetic studies relevant to biosynthetic research on vitamin B12. Part 7. Synthesis of (&#177;)-bonellin dimethyl ester.</a> Journal of the Chemical Society, Perkin Transactions. 1 (6), 1569-1576 (1988).</li><li>Pfeiffer, W. P., Lightner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(m.n)-Homorubins:+syntheses+and+structures.">(m.n)-Homorubins: syntheses and structures.</a> Monatschfte f&#252;r Chemie. 145 (11), 1777-1801 (2014).</li><li>Huggins, M. T., Musto, C., Munro, L., Catalano, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+recognition+studies+with+a+simple+dipyrrinone.">Molecular recognition studies with a simple dipyrrinone.</a> Tetrahedron. 63 (52), 12994-12999 (2007).</li><li>Groselj, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+Spiro-&#948;2-Pyrrolin-4-One+Pseudo+Enantiomers+via+an+Organocatalyzed+Sulfa-Michael/Aldol+Domino+Sequence.">Synthesis of Spiro-&#948;2-Pyrrolin-4-One Pseudo Enantiomers via an Organocatalyzed Sulfa-Michael/Aldol Domino Sequence.</a> Advanced Synthesis &amp; Catalyst. 361 (22), 5118-5126 (2019).</li><li>El-Shwiniy, W. H., Shehab, W. S., Mohamed, S. F., Ibrahium, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+cytotoxic+evaluation+of+some+substituted+pyrazole+zirconium(IV)+complexes+and+their+biological+assay.">Synthesis and cytotoxic evaluation of some substituted pyrazole zirconium(IV) complexes and their biological assay.</a> Applied Organometallic Chemistry. 32 (10), (2018).</li><li>Murray, L., O'Farrell, A. -. M., Abrams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+indolinone+compounds+for+treatment+of+excessive+osteolysis.">Preparation of indolinone compounds for treatment of excessive osteolysis.</a> US Patent. , (2004).</li><li>Lozinskaya, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+biological+evaluation+of+3-substituted+2-oxindole+derivatives+as+new+glycogen+synthase+kinase+3&#946;+inhibitors.">Synthesis and biological evaluation of 3-substituted 2-oxindole derivatives as new glycogen synthase kinase 3&#946; inhibitors.</a> Bioorganic &amp; Medicinal Chemistry. 27 (9), 1804-1817 (2019).</li><li>Montforts, F. P., Schwartz, U. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+directed+synthesis+of+the+chlorin+system.">A directed synthesis of the chlorin system.</a> Liebigs Annalen der Chemie. (6), 1228-1253 (1985).</li><li>Uddin, M. I., Thirumalairajan, S., Crawford, S. M., Cameron, T. S., Thompson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+synthetic+route+to+C-ring+ester-functionalized+prodigiosenes.">Improved synthetic route to C-ring ester-functionalized prodigiosenes.</a> Synlett. (17), 2561-2564 (2010).</li><li>Brower, J. O., Lightner, D. A., McDonagh, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aromatic+congeners+of+bilirubin:+synthesis,+stereochemistry,+glucuronidation+and+hepatic+transport.">Aromatic congeners of bilirubin: synthesis, stereochemistry, glucuronidation and hepatic transport.</a> Tetrahedron. 57 (37), 7813-7827 (2001).</li><li>Clift, M. D., Thomson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Merged+Conjugate+Addition/Oxidative+Coupling+Sequence.+Application+to+the+Enantioselective+Total+Synthesis+of+Metacycloprodigiosin+and+Prodigiosin+R1.">Development of a Merged Conjugate Addition/Oxidative Coupling Sequence. Application to the Enantioselective Total Synthesis of Metacycloprodigiosin and Prodigiosin R1.</a> Journal of the American Chemical Society. 131 (40), 14579-14583 (2009).</li><li>Brower, J. O., Lightner, D. A., McDonagh, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+New+Lipophilic+Bilirubin.+Conformation,+Transhepatic+Transport+and+Glucuronidation.">Synthesis of a New Lipophilic Bilirubin. Conformation, Transhepatic Transport and Glucuronidation.</a> Tetrahedron. 56 (40), 7869-7883 (2000).</li></ol>

The authors have nothing to disclose.

The Claisen-Schmidt condensation approach provides a fairly robust means of generating pyrazole, imidazole, and isoindolone dipyrrinone fluorophores through a relatively operationally simplistic protocol. While the synthesis of the fluorescent dipyrrinone analogs was the focus of this study, it should be noted that similar conditions can be applied to access other bicyclic methine-linked ring systems such as dipyrrinones23,24,25 and pyrrole-furan adducts26 as well as 3H-pyrazol-3-one-furan adducts27, isoindolone pyrrole adducts28, and 2H-Indol-2-one-pyrindine adducts29 which hold promise as potential pharmaceuticals. In general, the described procedure provides reaction products in moderate to high yields, however, it is important to note that continual monitoring of the reaction progress is essential for successful outcomes. In some of our preliminary trials, it was found that heating for excessive reaction times, well beyond (5 - 24 h) the completion of the reaction, led to decomposition products which can complicate the subsequent purification steps. For this reason, it is highly recommended that TLC analysis is performed at 1 h, 3 h, 6 h, 12 h, and 24 h time marks to monitor the reaction progress and to gain a sense of the reaction rate, as well as the rate of product decomposition.
Dipyrrinone analogs 16-25, in the protonated/neutral state, possess a range of solubility properties in commonly used organic solvents which can be problematic when studying photophysical, biological, and analytical properties. In general, 16-25 had varied solubility in water, alcohol solvents (methanol/ethanol), and CH2Cl2 but all had good solubility in highly polar aprotic solvents such as DMF, DMSO, and acetonitrile. Consequently, all stock solutions for UV/Vis (Step 3.1 of protocol) and fluorescence studies (Step 4.1 of protocol) and most NMR studies were carried out using DMSO or DMSO-d6. Though most compounds required gentle heating (using a heat gun) to completely solubilize in DMSO, once dissolved, 16-25 appear to remain soluble and can even be diluted in aqueous solutions without precipitating. Due to the highly polar nature of the ionic state, analogs 16-25 in basic solution are highly water soluble but have little solubility in organic solvents.
While the Claisen-Schmidt condensation reaction provides access to a range of methine-linked bicyclic aromatic compounds, beyond the dipyrrinone analogs describe within, the reaction conditions can limit the types of molecules produced through this method. As a fundamental requirement of the reaction, both an enolizable nucleophile (such as a pyrrolinone or isoindolone) and a non-enolizable aldehyde electrophile must react to enable the condensation. Failure to meet this basic requirement may result in the inability to link together the ring systems and/or the generation of competing side products. In addition, considerably basic conditions are used for generating the enolate nucleophile, which can create incompatibilities with functional groups (i.e., esters, nitriles, halides, etc.) that are susceptible to reactions with hydroxide. In such cases, it is possible to substitute hydroxide with nitrogenous bases or carbonate, as has been accomplished with 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU)30, triethylamine31, piperidine32, H&#252;nig&#39;s base33, and Na2CO334. In order to carry out an analogous reaction, we chose to use sodium hydroxide simply due to its availability and relative expense. While these constraints may require modifications to the procedure to access specific compounds or prevent access to others altogether, 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 analogs 16-25, the Claisen-Schmidt condensation has enabled one of the most accessible routes to pH dependent fluorophores described to date.
The Claisen-Schmidt condensation reaction has the potential to serve as a key reaction for the creation of a range of different bicyclic and tricyclic fluorophore systems. While this reaction has been critical to the development of 3H,5H-dipyrrolo[1,2-c:2&#39;,1&#39;-f]pyrimidin-3-ones (1), xanthoglows (2), pyrroloindolizinedione analogs (3, Figure 1), and most recently dipyrrinone analogs 16-25,it is possible to generate a range of completely novel fluorescent systems through pairing the Claisen-Schmidt condensation with molecular designs to restrict photoisomeric processes. More specific to the study at hand, future designs of dipyrrinone analogs will likely be developed using this outlined procedure in order to generate fluorescent compounds with stronger intramolecular hydrogen bonding capacity and lower pKa values. We 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.

A number of conjugated bicyclic systems, in which two aromatic rings are linked by a monomethine bridge, undergo isomerization via bond rotation, when excited with a photon (Figure 1A)1,2,3,4,5. The excited isomer will generally relax to the ground state through non-radiative decay processes6. If the energy barrier to bond rotation is increased to a large enough extent, it is possible to restrict or prevent the photoisomerization. Instead, photonic excitation results in an excited singlet state that often relaxes via fluorescence rather than non-radiative decay (Figure 1B). Restraining photoisomerization is most commonly accomplished by mechanically restricting bond rotation through tethering the two aromatic ring systems by covalent linkages, thereby locking the molecule into a particular isomeric state. This approach has been utilized to create several different fluorescent tricyclic dipyrrinone and dipyrrolemethane analogs such as: 3H,5H-dipyrrolo[1,2-c:2&#39;,1&#39;-f]pyrimidin-3-ones (1), xanthoglows (2)6,7, pyrroloindolizinedione analogs (3)8, and BODIPY analogs9 (4, Figure 2) whereby the pyrrolidine and/or pyrrole ring systems are tethered with methylene, carbonyl, or boron difluoro linkers. Typically, 1-4&#160;possess &#934;F&#160;&#62; 0.7 suggesting these systems are very efficient as fluorophore units.
It is also possible to restrict photoisomerization through means other than covalently linking the ring systems. For example, the phenolic and imidazolinone rings (Figure 2) of Green Fluorescent Protein (GFP) are restricted to rotation by the protein environment; the restrictive setting increases the quantum yield by three orders of magnitude in comparison to the same chromophore unit in free solution10. It is believed that the protein scaffold of GFP provides a rotational barrier through steric and electrostatic effects11. Recently, our group in collaboration with the Odoh group at the University of Nevada, Reno discovered another fluorophore system that bears structural similarity to the dipyrrinone-based xanthoglow systems (Figure 2)12. These dipyrrinone analogs, however, differ from the xanthoglow system in that intramolecular hydrogen bonds, rather than covalent bonds, deter photoisomerization and result in a fluorescent bicyclic system. Furthermore, the pyrazole, imidazole, and isoindolone dipyrrinone analogs can hydrogen bond in protonated and deprotonated states; deprotonation results in the red-shifting of both the excitation and emission wavelengths, likely due to a change in the electronic nature of the system. While hydrogen bonding has been reported to increase quantum yields though restricted rotation13,14,15,16, we are unaware of any other fluorophore system in which restricted isomerization serves as a mode of fluorescence in both protonated and deprotonated states of the molecule. Therefore, these pH dependent dipyrrinone fluorophores are unique in that respect.
In this video, we focus on the synthesis and chemical characterization of the fluorescent dipyrrinone analog series. In particular, there is an emphasis placed on the Claisen-Schmidt condensation methodology that was used to construct the complete series of fluorescent analogs. This reaction relies on the generation of a base-mediated vinylogous enolate ion which attacks an aldehyde group, to produce an alcohol that subsequently undergoes elimination. For the dipyrrinone analog series, a pyrrolinone/isoindolone is converted to an enolate to facilitate an attack upon an aldehyde group attached to a pyrazole or imidazole ring (Figure 3); after elimination a fully conjugated bicyclic system, linked by a methine-bridge, is formed. It is noteworthy that the entire series of dipyrrinone analogs can be constructed from readily available commercial materials and can be produced in a single one-pot reaction sequence typically in moderate to high yields (yields range from approximately 50-95%). Since most of the dipyrrinone analogs are highly crystalline in nature, very little purification outside of standard workup conditions is required to produce analytically pure samples. Consequently, this fluorophore system requires only a few steps to access from readily available commercial materials and can be synthesized, purified, and prepared for analytical or biological studies in a relatively short time frame.

<table><tbody><tr><td>3-ethyl-4-methyl-3-pyrrolin-2-one</td><td>Combi-Blocks</td><td>&amp;nbsp;[766-36-9]</td><td>Yellow solid reagent</td></tr><tr><td>isoindolin-1-one</td><td>ArkPharm</td><td>&amp;nbsp;[480-91-1]</td><td>Off-white solid reagent</td></tr><tr><td>5-bromoisoindolin-1-one</td><td>Combi-Blocks</td><td>&amp;nbsp;[552330-86-6]</td><td>Pink solid reagent</td></tr><tr><td>2-formylimidazole</td><td>Combi-Blocks</td><td>&amp;nbsp;[10111-08-7 ]</td><td>Off-white solid reagent</td></tr><tr><td>Imidazole-4-carbaldehyde</td><td>ArkPharm</td><td>&amp;nbsp;[3034-50-2]</td><td>Solid reagent</td></tr><tr><td>1-H-pyrazole-4-carbaldehyde</td><td>Oakwood Chemicals</td><td>&amp;nbsp;[35344-95-7]</td><td>Solid reagent</td></tr><tr><td>1-H-pyrazole-5-carbaldehyde</td><td>Matrix Scientific</td><td>&amp;nbsp;[3920-50-1]</td><td>Solid reagent</td></tr><tr><td>Solid KOH Pellets</td><td>BeanTown Chemicals</td><td>[1310-58-3]</td><td>White solid pellets</td></tr><tr><td>Siliflash Silica Gel</td><td>Scilicycle</td><td>R12030B</td><td>Fine white powder</td></tr><tr><td>Phosphate Buffered Saline (PBS) (x10)</td><td>Growcells</td><td>MRGF-6235</td><td>Colorless translucent liquid</td></tr><tr><td>Beckman Coulter DU-800 UV/Vis Spectrophotometer and Software</td><td>Beckman Coulter</td><td>N/A</td><td>Spectroscopy Instrument and Software</td></tr><tr><td>Fluoromax-4 Spectrofluorometer</td><td>Horiba Scientific</td><td>N/A</td><td>Spectroscopy Instrument</td></tr><tr><td>FluorEssence Fluoremetry Software V3.5</td><td>Horiba Scientific</td><td>N/A</td><td>Spectroscopy Software</td></tr><tr><td>Finnpipette II Micropipette (sizes: 100-1,000, 20-200, and 0.5-10 &amp;micro;L)</td><td>Fischerbrand</td><td>N/A</td><td>Equipment</td></tr><tr><td>Wilmad-LabGlass Rotary Evaporator (Model: WG-EV311-V-PLUS)</td><td>SP Scienceware</td><td>N/A</td><td>Equipment</td></tr><tr><td>DuoSeal Vacuum Pump (Model Number: 1405)</td><td>Welch</td><td>N/A</td><td>Equipment</td></tr><tr><td>GraphPad Prism 4</td><td>GraphPad</td><td>N/A</td><td>Data Analysis Software</td></tr><tr><td>SympHony pH Meter (Model: Sb70P)</td><td>VWR</td><td>N/A</td><td>Equipment</td></tr></tbody></table>

synthesis of ph dependent pyrazole, imidazole, and isoindolone dipyrrinone fluorophores using a claisen-schmidt condensation approach

Methine-bridged conjugated bicyclic aromatic compounds are common constituents of a range of biologically relevant molecules such as porphyrins, dipyrrinones, and pharmaceuticals. Additionally, restricted rotation of these systems often results in highly to moderately fluorescent systems as observed in 3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidin-3-ones, xanthoglows, pyrroloindolizinedione analogs, BODIPY analogs, and the phenolic and imidazolinone ring systems of Green Fluorescent Protein (GFP). This manuscript describes an inexpensive and operationally simple method of performing a Claisen-Schmidt condensation to generate a series of fluorescent pH dependent pyrazole/imidazole/isoindolone dipyrrinone analogs. While the methodology illustrates the synthesis of dipyrrinone analogs, it can be translated to produce a wide range of conjugated bicyclic aromatic compounds. The Claisen-Schmidt condensation reaction utilized in this method is limited in scope to nucleophiles and electrophiles that are enolizable under basic conditions (nucleophile component) and non-enolizable aldehydes (electrophile component). Additionally, both the nucleophilic and electrophilic reactants must contain functional groups that will not inadvertently react with hydroxide. Despite these limitations, this methodology offers access to completely novel systems that can be employed as biological or molecular probes.

The Claisen-Schmidt condensation reaction provided access to dipyrrinone analogs (16-25, Figure 4) using the one-pot procedure described in the protocol section (see step 1). Analogs 16-25 were all generated by condensing pyrrolinone 9, bromoisoindolone 10, or isoindolone 11 with 1H-imidazole-2-carboxaldehyde (12), 1H-imidazole-5-carboxaldehyde (13), 1H-pyrazole-3-carboxaldehyde (14), or 1H-pyrazole-4-carboxaldehyde (15); the combinations produced ten different analogs including a control compound, 19, which is incapable of forming intramolecular hydrogen bonds (Table 1). Reaction times typically necessitated 24 h of reflux for completion, however, in the case of 20 only 6 h were required, whereas, for 23 and 24 slightly longer times of 30 h and 27 h respectively were needed. Product yields ranged from 41% to 96%, as illustrated in Table 1, which follow traditional trends of analogous condensation reactions for dipyrrinones. Compounds 17, 18, 20-22,24 and 25, due to their highly crystalline nature, were all purified by simple vacuum filtration methods; only compounds 16, 19, and 23 required chromatography for purification.
The photophysical properties of compounds 16-25, obtained from performing steps 3 - 4 in the protocol section, are summarized in Table 2. The pKa values measured for each compound ranged from 12 to &#62; 13.5, suggesting sufficiently basic conditions are needed to completely deprotonate each dipyrrinone analog. Due to differing photophysical properties in the protonated and deprotonated states of each compound, spectra were acquired using neutral (pH 7.0 PBS) and basic (1.0 M NaOH) solutions of 16-25. In neutral pH (protonated state), compounds 16-25 have &#955;max abs ranging from 324 nm to 365 nm, which are all blue-shifted by 10 to 37 nm in comparison to the deprotonated states. The molar absorptivities range from 15,000 to 30,000 but do not appear to substantially deviate amongst the protonated and deprotonated states of a given analog. Analog 19 did not display any detectable fluorescence, however, 16-18 and 20-25 emitted light with &#955;max em ranging from 409 - 457 nm at neutral pH and 443 - 482 nm at basic pH; a similar red-shifting trend to that of the maximum protonated/deprotonated absorbance wavelengths is observed for the emission wavelengths as well. The &#934;F ranged from 0.01 to 0.30 in both neutral and basic aqueous solutions, which are considerably lower than comparable xanthoglows, but compounds 16, 20, and 25 fall in the similar region of heavily used fluorophores such as rhodamine B (&#934;F = 0.23),20 acridine orange (&#934;F = 0.36),21 pyronin Y (&#934;F = 0.22),20 and most of the cyanine dye series (typically &#934;F = 0.12-0.28).22
Dipyrrinone analogs 16-25 were all chemically characterized using melting point analysis, IR spectroscopy, 1H NMR and 13C NMR spectroscopy, and high-resolution mass spectrometry in addition to the UV/Vis and fluorescence spectroscopy experiments summarized in Table 2. Chemical characterization and original 1H NMR and 13C NMR spectra can be found from the original literature source,12 however, for convenience, characterization for compounds 16, 20, and 23, which possess the largest quantum yields&#160;are reported below:
(Z)-5-((1H-imidazol-2-yl)methylene)-3-ethyl-4-methyl-1,5-dihydro-2H-pyrrol-2-one (16). Decomposes at 160 &#176;C; 1H NMR (400 MHz, DMSO-d6) &#948; 12.3 (brs, 1H), 9.87 (s, 1H), 7.13 (apps, 2H), 5.93 (s, 1H), 2.23 (q, J = 7.5 Hz, 2H), 2.00 (s, 3H), 0.98 (t, J = 7.5 Hz, 3H); 13C NMR (101 MHz, DMSO- d6) &#948; 170.7, 144.8, 140.0, 139.6, 133.9, 130.2, 117.6, 94.1, 16.7, 13.6, 9.33; IR (thin film) 3742, 3148, 3063, 2924, 2353, 1651, 1543, 1450, 771, 717 cm-1; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C11H13N3ONa 226.0956, Found 226.0956.
(Z)-5-((1H-pyrazol-4-yl)methylene)-3-ethyl-4-methyl-1,5-dihydro-2H-pyrrol-2-one (19). Decomposes at 202 &#176;C; 1H NMR (400 MHz, 20% CD3OD in CDCl3) &#948; 1H NMR (400 MHz, Chloroform-d) &#948; 7.74 (s, 2H), 6.01 (s, 1H), 2.27 (q, J = 7.4 Hz, 2H), 2.02 (s, 3H), 1.02 (t, J = 7.4 Hz, 3H); 13C{1H} NMR (101 MHz, CDCl3) &#948; 173.7, 141.9, 136.0, 133.0, 116.1, 105.0, 100.8, 16.9, 13.4, 9.61; IR (thin film) 3163, 3117, 3048, 2963, 2362, 1674, 1558, 1512, 1396, 1257, 1157, 948, 871, 794, 702 cm-1; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C11H13N3ONa 226.0956, Found 226.0955.
(Z)-3-((1H-imidazol-2-yl)methylene)-5-bromoisoindolin1-one (20). Decomposes at 213 &#176;C; 1H NMR (400 MHz, DMSO-d6) &#948; 7.97 (s, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 8.0, 1H), 7.06 (s, 2H), 6.57 (s, 1H); 13C NMR (101 MHz, DMSO-d6) &#948; 167.7, 146.6, 140.2, 134.5, 131.7, 129.1, 125.8, 125.03, 124.99, 123.6, 96.5; IR (thin film) 3742, 3240, 2314, 1682, 1543, 1520, 1435, 1312, 1080, 826, 694 cm-1; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C12H8BrN3ONa 311.9749, Found 311.9752.
(Z)-3-((1H-imidazol-2-yl)methylene)isoindolin-1-one (23). Decomposes at 228 &#176;C; 1H NMR (400 MHz, DMSO-d6) &#948; 12.34 (s, 1H), 10.74 (s, 1H), 7.90 (d, J = 7.5 Hz, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.63 (dd, J = 7.6, 7.2 Hz, 1H), 7.50 (dd, J = 7.6, 7.3 Hz, 1H), 7.17 (s, 2H), 6.46 (s, 1H); 13C NMR (101 MHz, DMSO-d6) &#948; 167.3, 144.9, 137.3, 135.1, 132.8, 129.9, 129.3, 123.6, 121.0, 117.6, 92.9; IR (thin film) 3741, 3201, 3086, 2361, 2322, 1682, 1543, 1520, 1119, 748, 687 cm-1; HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for C12H9N3ONa 234.0643, Found 234.0641.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Entry</td>
			<td>Pyrrolinone/Isoindolone</td>
			<td>Aldehyde</td>
			<td>Yield (%)b</td>
			<td>Time (h)</td>
			<td>Product</td>
		</tr>
		<tr>
			<td>1</td>
			<td>9</td>
			<td>12</td>
			<td>80</td>
			<td>24</td>
			<td>16</td>
		</tr>
		<tr>
			<td>2</td>
			<td>9</td>
			<td>13</td>
			<td>41</td>
			<td>24</td>
			<td>17</td>
		</tr>
		<tr>
			<td>3</td>
			<td>9</td>
			<td>14</td>
			<td>79</td>
			<td>24</td>
			<td>18</td>
		</tr>
		<tr>
			<td>4</td>
			<td>9</td>
			<td>15</td>
			<td>61</td>
			<td>24</td>
			<td>19</td>
		</tr>
		<tr>
			<td>5</td>
			<td>10</td>
			<td>12</td>
			<td>96</td>
			<td>6</td>
			<td>20</td>
		</tr>
		<tr>
			<td>6</td>
			<td>10</td>
			<td>13</td>
			<td>70</td>
			<td>24</td>
			<td>21</td>
		</tr>
		<tr>
			<td>7</td>
			<td>10</td>
			<td>14</td>
			<td>66</td>
			<td>24</td>
			<td>22</td>
		</tr>
		<tr>
			<td>8</td>
			<td>11</td>
			<td>12</td>
			<td>49</td>
			<td>30</td>
			<td>23</td>
		</tr>
		<tr>
			<td>9</td>
			<td>11</td>
			<td>13</td>
			<td>49</td>
			<td>27</td>
			<td>24</td>
		</tr>
		<tr>
			<td>10</td>
			<td>11</td>
			<td>14</td>
			<td>94</td>
			<td>24</td>
			<td>25</td>
		</tr>
	</tbody>
</table>
Table 1. Conditions and reaction yields for the synthesis of 16-25a
a Reactions performed on a 1 mmol scale in 5 mL of EtOH. b Isolated Yield.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Abs. &#955;max (nm)</td>
			<td>&#949; (M-1 cm-1)</td>
			<td>Fluor. &#955;max (nm)</td>
			<td>&#934;b</td>
			<td>pKa</td>
		</tr>
		<tr>
			<td>16</td>
			<td>351 (384)</td>
			<td>24500 (22800)</td>
			<td>451 (482)</td>
			<td>0.30 (0.30)</td>
			<td>12.7</td>
		</tr>
		<tr>
			<td>17</td>
			<td>338 (380)</td>
			<td>18600 (18600)</td>
			<td>442 (462)</td>
			<td>0.01 (0.03)</td>
			<td>12.8</td>
		</tr>
		<tr>
			<td>18</td>
			<td>324 (349)</td>
			<td>29800 (25700)</td>
			<td>455 (465)</td>
			<td>0.01 (0.02)</td>
			<td>13</td>
		</tr>
		<tr>
			<td>19</td>
			<td>326 (358)</td>
			<td>29900 (21300)</td>
			<td>&#8211;a</td>
			<td>&#8211;a</td>
			<td>12.9</td>
		</tr>
		<tr>
			<td>20</td>
			<td>365 (378)</td>
			<td>15000 (15500)</td>
			<td>457 (475)</td>
			<td>0.22 (0.20)</td>
			<td>12.5</td>
		</tr>
		<tr>
			<td>21</td>
			<td>355 (380)</td>
			<td>15100 (16800)</td>
			<td>409 (443)</td>
			<td>0.03 (0.01)</td>
			<td>12.9</td>
		</tr>
		<tr>
			<td>22</td>
			<td>341 (363)</td>
			<td>19800 (23100)</td>
			<td>427 (452)</td>
			<td>0.02 (0.01)</td>
			<td>&#62;13.5</td>
		</tr>
		<tr>
			<td>23</td>
			<td>360 (373)</td>
			<td>29000 (21300)</td>
			<td>449 (474)</td>
			<td>0.25 (0.26)</td>
			<td>12</td>
		</tr>
		<tr>
			<td>24</td>
			<td>351 (373)</td>
			<td>17200 (19400)</td>
			<td>432 (454)</td>
			<td>0.07 (0.05)</td>
			<td>12.8</td>
		</tr>
		<tr>
			<td>25</td>
			<td>340 (357)</td>
			<td>20200 (23500)</td>
			<td>410 (449)</td>
			<td>0.02 (0.02)</td>
			<td>&#62;13.5</td>
		</tr>
	</tbody>
</table>
Table 2. Photophysical properties and pKa values of 6-14 and 22 in pH 7.0 PBS buffer and 1 M NaOH (given in parenthesis).
a Fluorescence was not detectable for 19. b Quinine (Q = 0.55)15 and Anthracene (Q = 0.27)15, 16 were used as standards.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61944/61944fig01.jpg" /> 
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<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61944/61944fig02.jpg" /> 
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<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61944/61944fig03.jpg" /> 
Figure 3: <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61944/61944fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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Watch this Scientific Journal Video about Synthesis of pH Dependent Pyrazole, Imidazole, and Isoindolone Dipyrrinone Fluorophores using a Claisen-Schmidt Condensation Approach at JoVE.com

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

The Claisen-Schmidt condensation reaction is an important methodology for the generation of methine-bridged conjugated bicyclic aromatic compounds. Through utilizing a base-mediated variant of the aldol reaction, a range of fluorescent and/or biologically relevant molecules can be accessed through a generally inexpensive and operationally simple synthetic approach.

Methine-bridged conjugated bicyclic aromatic compounds are common constituents of a range of biologically relevant molecules such as porphyrins, ...

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&alpha;-Carbon Chemistry: Enols, Enolates, and Enamines

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5.9K Views.  Nevada State College. The Claisen-Schmidt condensation reaction is an important methodology for the generation of methine-bridged conjugated bicyclic aromatic compounds. Through utilizing a base-mediated variant of the aldol reaction, a range of fluorescent and/or biologically relevant molecules can be accessed through a generally inexpensive and operationally simple synthetic approach.

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

Metal-free Synthesis of Ynones from Acyl Chlorides and Potassium Alkynyltrifluoroborate Salts

Ynones are a valuable functional group and building block in organic synthesis. Ynones serve as a precursor to many important organic functional groups and scaffolds. Traditional methods for the preparation of ynones are associated with drawbacks including harsh conditions, multiple purification steps, and the presence of unwanted byproducts. An alternative method for the straightforward preparation of ynones from acyl chlorides and potassium alkynyltrifluoroborate salts is described herein. The adoption of organotrifluoroborate salts as an alternative to organometallic reagents for the formation of new carbon-carbon bonds has a number of advantages. Potassium organotrifluoroborate salts are shelf stable, have good functional group tolerance, low toxicity, and a wide variety are straightforward to prepare. The title reaction proceeds rapidly at ambient temperature in the presence of a Lewis acid without the exclusion of air and moisture. Fair to excellent yields may be obtained via reaction of various aryl and alkyl acid chlorides with alkynyltrifluoroborate salts in the presence of boron trichloride.

The goal of this manuscript is to demonstrate a straightforward method for the preparation of ynones from acyl chloride and potassium alkynyltrifluoroborate salt starting materials. The one-pot reaction proceeds rapidly in the presence of boron trichloride without exclusion of air and moisture.

Ynones are a valuable functional group and building block in organic synthesis. Ynones serve as a precursor to many important organic functional groups ...

Facile Preparation of 4-Substituted Quinazoline Derivatives

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted 2-aminobenzophenones and thiourea in the presence of dimethyl sulfoxide (DMSO). This is a unique complementary reaction system in which thiourea undergoes thermal decomposition to form carbodiimide and hydrogen sulfide, where the former reacts with 2-aminobenzophenone to form 4-phenylquinazolin-2(1H)-imine intermediate, whilst hydrogen sulfide reacts with DMSO to give methanethiol or other sulfur-containing molecule which then functions as a complementary reducing agent to reduce 4-phenylquinazolin-2(1H)-imine intermediate into 4-phenyl-1,2-dihydroquinazolin-2-amine. Subsequently, the elimination of ammonia from 4-phenyl-1,2-dihydroquinazolin-2-amine affords substituted quinazoline derivative. This reaction usually gives quinazoline derivative as a single product arising from 2-aminobenzophenone as monitored by GC/MS analysis, along with small amount of sulfur-containing molecules such as dimethyl disulfide, dimethyl trisulfide, etc. The reaction usually completes in 4-6 hr at 160 &#186;C in small scale but may last over 24 hr when carried out in large scale. The reaction product can be easily purified by means of washing off DMSO with water followed by column chromatography or thin layer chromatography.

A protocol for facile preparation of 4-substituted quinazoline derivatives from 2-aminobenzophenones, thiourea and dimethyl sulfoxide is presented.

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted ...

Scale-up Chemical Synthesis of Thermally-activated Delayed Fluorescence Emitters Based on the Dibenzothiophene-S,S-Dioxide Core

We report a procedure to linearly scale-up the synthesis of 2,8-bis(3,6-di-tert-butyl-9H-carbazol-9-yl)dibenzothiophene-S,S-dioxide (compound 4) and 2,8-bis(10H-phenothiazin-10-yl)dibenzothiophene-S,S-dioxide (compound 5) using Buchwald-Hartwig cross-coupling reaction conditions. In addition, we demonstrate a scaled-up synthesis of all non-commercially available starting materials that are required for the amination cross-coupling reaction. In the present article, we provide the detailed synthetic procedures for all of the described compounds, alongside their spectral characterization. This work shows the possibility to produce organic molecules for optoelectronic applications on a large scale, which facilitates their implementation into real world devices.

Scale-up synthesis of highly efficient thermally activated delayed fluorescence emitters is described in the presented article.

We report a procedure to linearly scale-up the synthesis of 2,8-bis(3,6-di-tert-butyl-9H-carbazol-9-yl)dibenzothiophene-S,S-dioxide (compound 4) and ...

Preparation of N-(2-alkoxyvinyl)sulfonamides from N-tosyl-1,2,3-triazoles and Subsequent Conversion to Substituted Phthalans and Phenethylamines

Decomposition of N-tosyl-1,2,3-triazoles with rhodium(II) acetate dimer in the presence of alcohols forms synthetically versatile N-(2-alkoxyvinyl)sulfonamides, which react under a variety of conditions to afford useful N- and O-containing compounds. Acid-catalyzed addition of alcohols or thiols to N-(2-alkoxyvinyl)sulfonamide-containing phthalans provides access to ketals and thioketals, respectively. Selective reduction of the vinyl group in N-(2-alkoxyvinyl)sulfonamide-containing phthalans via hydrogenation yields the corresponding phthalan in good yield, whereas reduction with sodium bis(2-methoxyethoxy)aluminumhydride generates a ring-opened phenethylamine analogue. Because the N-(2-alkoxyvinyl)sulfonamide functional group is synthetically versatile, but often hydrolytically unstable, this protocol emphasizes key techniques in preparing, handling, and reacting these pivotal substrates in several useful transformations.

Preparation of N-2-alkoxyvinylsulfonamides from N-tosyl-1,2,3-triazoles and Subsequent Conversion to Substituted Phthalans and Phenethylamines

Representative experimental procedures for the synthesis of N-(2-alkoxyvinyl)sulfonamides and subsequent conversion to phthalan and phenethylamine derivatives are presented in detail.

Decomposition of N-tosyl-1,2,3-triazoles with rhodium(II) acetate dimer in the presence of alcohols forms synthetically versatile ...

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of &#945;-Imino &#947;-Lactones and Alkylidene Pyrazolones

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. Recently, we have developed an efficient organocatalytic strategy, which provides facile access to a variety of enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons. In this paper, we demonstrate a detailed protocol for the asymmetric synthesis of drug-like bispirocyclic compounds with two spirocyclic carbon centers via an organocatyltic 1,3-dipolar cycloaddition reaction. Spirocyclization synthons &#945;-imino &#947;-lactones and alkylidene pyrazolones are prepared first, which are then subjected to a cycloaddition reaction in the presence of a bifunctional squaramide organocatalyst to afford the desired bispirocycles in high yields and excellent stereoselectivities. Chiral high-performance liquid chromatography (HPLC) is carried out to determine the enantiomeric purity of the products, and the d.r. value is examined by proton nuclear magnetic resonance (1H NMR). The absolute configuration of the product is assigned according to an X-ray crystallographic analysis. This synthetic strategy allows scientists to prepare a diversity of bispirocyclic scaffolds in high yields and excellent diastereo- and enantioselectivities.

Enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons are asymmetrically synthesized through a simple organocatalytic 1,3-dipolar cycloaddition reaction.

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. ...

[(DPEPhos)(bcp)Cu]PF6: A General and Broadly Applicable Copper-Based Photoredox Catalyst

Our group recently reported the use of [(DPEPhos)(bcp)Cu]PF6 as a general copper-based photoredox catalyst which proved efficient to promote the activation of a broad variety of organic halides, including unactivated ones. These can then participate in various radical transformations such as reduction and cyclization reactions, as well as in the direct arylation of several (hetero)arenes. These transformations provide a straightforward access to a range of small molecules of interest in synthetic chemistry, as well as to biologically active natural products. Altogether, [(DPEPhos)(bcp)Cu]PF6 acts as a convenient photoredox catalyst which appears to be an attractive, cheap and complementary alternative to the state-of-the-art iridium- and ruthenium-based photoredox catalysts. Here, we report a detailed protocol for the synthesis of [(DPEPhos)(bcp)Cu]PF6, as well as NMR and spectroscopic characterizations, and we illustrate its use in synthetic chemistry for the direct arylation of (hetero)arenes and radical cyclization of organic halides. In particular, the direct arylation of N-methylpyrrole with 4-iodobenzonitrile to afford&#160;4-(1-methyl-1H-pyrrol-2-yl)benzonitrile and the radical cyclization of N-benzoyl-N-[(2-iodoquinolin-3-yl)methyl]cyanamide to afford natural product luotonin A are detailed. The scope and limitations of this copper-based photoredox catalyst are also briefly discussed.

[DPEPhosbcpCu]PF6: A General and Broadly Applicable Copper-Based Photoredox Catalyst

Detailed and general protocols are presented for the synthesis of [(DPEPhos)(bcp)Cu]PF6, a general copper-based photoredox catalyst, and for its use in synthetic chemistry for the direct arylation of C-H bonds in (hetero)arenes and radical cyclization of organic halides.

Our group recently reported the use of [(DPEPhos)(bcp)Cu]PF6 as a general copper-based photoredox catalyst which proved efficient to promote the ...

Microwave-Assisted Preparation of 1-Aryl-1H-pyrazole-5-amines

A synthetic process for the preparation of a variety of 1-aryl-1H-pyrazole-5-amines was developed. The microwave-mediated nature of this method makes it efficient in both time and resources and utilizes water as the solvent. 3-Aminocrotononitrile or an appropriate &#945;-cyanoketone is combined with an aryl hydrazine and dissolved in 1 M HCl. The mixture is then heated in a microwave reactor at 150 &#176;C, typically for 10-15 min. The product can be readily obtained by basifying the solution with 10% NaOH and isolating the desired compound with a simple vacuum filtration. The use of water as a solvent in this reaction lends to its ease and utility in production, and this method is easily reproducible with a variety of functional groups. Typical isolated yields range from 70-90%, and reactions can be performed on the milligram to gram scale with little to no change in observed yields. Some of the applications of these molecules and their derivatives include pesticides, anti-malarials, and chemotherapeutics, among many others.

Microwave-Assisted Preparation of 1-Aryl-1H-pyrazole-5-amines

1-Aryl-1H-pyrazole-5-amines are prepared from aryl hydrazines combined with either 3-aminocrotononitrile or an &#945;-cyanoketone in a 1 M HCl solution using a microwave reactor. Most reactions are done in 10-15 minutes and pure product can be obtained via vacuum filtration with typical isolated yields of 70-90%.

A synthetic process for the preparation of a variety of 1-aryl-1H-pyrazole-5-amines was developed. The microwave-mediated nature of this method makes it ...

Cercosporin-Photocatalyzed [4+1]- and [4+2]-Annulations of Azoalkenes Under Mild Conditions

The interest on nitrogen-containing heterocycles has expanded rapidly in the synthetic community since they are important motifs for new drugs. Traditionally, they were synthesized through thermal cycloaddition reactions, whereas today, photocatalysis is preferred due to the mild and efficient conditions. With this focus, a new photocatalytic method for the synthesis of nitrogen-containing heterocycles is highly desired. Here, we report a protocol for the biosynthesis of cercosporin, which could function as a metal-free photocatalyst. We then illustrate cercosporin-photocatalyzed protocols for the synthesis of nitrogen-containing heterocycles 1,2,3-thiadiazoles through annulation of azoalkenes with KSCN, and synthesis of 1,4,5,6-tetrahydropyridazines [4+2] through cyclodimerization of azoalkenes under mild conditions, respectively. As a result, there is a new bridge between the microbial fermentation method and organic synthesis in a mild, cost-effective, environmentally friendly and sustainable manner.

New routes for the synthesis of nitrogen-containing heterocycles utilizing cercosporin as a metal-free photocatalyst were developed.

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

Summary

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Chapters in this video

Metal-free Synthesis of Ynones from Acyl Chlorides and Potassium Alkynyltrifluoroborate Salts

Facile Preparation of 4-Substituted Quinazoline Derivatives

Scale-up Chemical Synthesis of Thermally-activated Delayed Fluorescence Emitters Based on the Dibenzothiophene-S,S-Dioxide Core

Preparation of N-(2-alkoxyvinyl)sulfonamides from N-tosyl-1,2,3-triazoles and Subsequent Conversion to Substituted Phthalans and Phenethylamines

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of α-Imino γ-Lactones and Alkylidene Pyrazolones

[(DPEPhos)(bcp)Cu]PF₆: A General and Broadly Applicable Copper-Based Photoredox Catalyst

Microwave-Assisted Preparation of 1-Aryl-1H-pyrazole-5-amines

Cercosporin-Photocatalyzed [4+1]- and [4+2]-Annulations of Azoalkenes Under Mild Conditions