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>

<ol>
	<li>Abbandonato, G., 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=Cis-trans+photoisomerization+properties+of+GFP+chromophore+analogs.">Cis-trans photoisomerization properties of GFP chromophore analogs.</a> European Biophysics Journal. 40 (11), 1205-1214 (2011).</li><li>Funakoshi, H., 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=Spectroscopic+studies+on+merocyanine+photoisomers.+IV.+Catalytic+isomerization+of+photoisomers+of+merocyanine+derivatives+in+protic+solvents.">Spectroscopic studies on merocyanine photoisomers. IV. Catalytic isomerization of photoisomers of merocyanine derivatives in protic solvents.</a> Nippon Kagaku Kaishi. (9), 1516-1522 (1989).</li><li>Puzicha, G., Shrout, D. 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=Synthesis+and+properties+of+homomologated+and+contracted+dipyrrinone+analogs+of+xanthobilirubic+acid.">Synthesis and properties of homomologated and contracted dipyrrinone analogs of xanthobilirubic acid.</a> Journal of Heterocyclic Chemistry. 27 (7), 2117-2123 (1990).</li><li>Bonnett, R., Hamzetash, D., Asuncion Valles, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propentdyopents+[5-(2-oxo-2H-pyrrol-5-ylmethylene)pyrrol-2(5H)-ones]+and+related+compounds.+Part+2.+The+Z+E+photoisomerization+of+pyrromethenone+systems.">Propentdyopents [5-(2-oxo-2H-pyrrol-5-ylmethylene)pyrrol-2(5H)-ones] and related compounds. Part 2. The Z E photoisomerization of pyrromethenone systems.</a> Journal of the Chemical Society, Perkins Transactions. 1 (6), 1383-1388 (1987).</li><li>Tikhomirova, K., Anisimov, A., Khoroshutin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biscyclohexane-Annulated+Diethyl+Dipyrrindicarboxylates:+Observation+of+a+Dipyrrin+Form+with+Absent+Visible+Absorption.">Biscyclohexane-Annulated Diethyl Dipyrrindicarboxylates: Observation of a Dipyrrin Form with Absent Visible Absorption.</a> European Journal of Organic Chemistry. 2012 (11), 2201-2207 (2012).</li><li>Brower, J. O., 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=Synthesis+and+spectroscopic+properties+of+a+new+class+of+strongly+fluorescent+dipyrrinones.">Synthesis and spectroscopic properties of a new class of strongly fluorescent dipyrrinones.</a> Journal of Organic Chemistry. 67 (8), 2713-2716 (2002).</li><li>Woydziak, Z. R., Boiadjiev, S. E., Norona, W. S., McDonagh, A. F., 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=Synthesis+and+Hepatic+Transport+of+Strongly+Fluorescent+Cholephilic+Dipyrrinones.">Synthesis and Hepatic Transport of Strongly Fluorescent Cholephilic Dipyrrinones.</a> Journal of Organic Chemistry. 70 (21), 8417-8423 (2005).</li><li>Jarvis, T., 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=Pyrrole+&#946;-amides:+Synthesis+and+characterization+of+a+dipyrrinone+carboxylic+acid+and+an+N-Confused+fluorescent+dipyrrinone.">Pyrrole &#946;-amides: Synthesis and characterization of a dipyrrinone carboxylic acid and an N-Confused fluorescent dipyrrinone.</a> Tetrahedron. 74 (14), 1698-1704 (2018).</li><li>Bodio, E., Denat, F., Goze, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BODIPYS+and+aza-BODIPY+derivatives+as+promising+fluorophores+for+in+vivo+molecular+imaging+and+theranostic+applications.">BODIPYS and aza-BODIPY derivatives as promising fluorophores for in vivo molecular imaging and theranostic applications.</a> Journal of Porphyrins and Phthalocyanines. 23, 1159-1183 (2019).</li><li>Acharya, 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=Photoinduced+Chemistry+in+Fluorescent+Proteins:+Curse+or+Blessing.">Photoinduced Chemistry in Fluorescent Proteins: Curse or Blessing.</a> Chemical Reviews. 117 (2), 758-795 (2017).</li><li>Romei, M. G., Lin, C. -. Y., Mathews, I. I., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrostatic+control+of+photoisomerization+pathways+in+proteins.">Electrostatic control of photoisomerization pathways in proteins.</a> Science. 367 (6473), 76-79 (2020).</li><li>Benson, N., Suleiman, O., Odoh, S. O., Woydziak, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ryrazole,+Imidazole,+and+Isoindolone+Dipyrrinone+Analogues:+pH-Dependent+Fluorophores+That+Red-Shift+Emission+Frequencies+in+a+Basic+Solution.">Ryrazole, Imidazole, and Isoindolone Dipyrrinone Analogues: pH-Dependent Fluorophores That Red-Shift Emission Frequencies in a Basic Solution.</a> Journal of Organic Chemistry. 84 (18), 11856-11862 (2019).</li><li>Xie, P., Gao, G., Liu, J., Jin, Q., Yang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Turn+on+Fluorescent+Probe+for+Selective+Detection+of+Cysteine/Homocysteine.">A New Turn on Fluorescent Probe for Selective Detection of Cysteine/Homocysteine.</a> Journal of Fluorescence. 25 (5), 1315-1321 (2015).</li><li>Alty, I. G., 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=Intramolecular+Hydrogen-Bonding+Effects+on+the+Fluorescence+of+PRODAN+Derivatives.">Intramolecular Hydrogen-Bonding Effects on the Fluorescence of PRODAN Derivatives.</a> Journal of Physical Chemistry A. 120 (20), 3518-3523 (2016).</li><li>Yang, Y., Li, D., Li, C., Liu, Y. F., Jiang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+bond+strengthening+induces+fluorescence+quenching+of+PRODAN+derivative+by+turning+on+twisted+intramolecular+charge+transfer.">Hydrogen bond strengthening induces fluorescence quenching of PRODAN derivative by turning on twisted intramolecular charge transfer.</a> Spectrochimica Acta, Part A. 187, 68-74 (2017).</li><li>Zhang, L., Liu, J., Gao, J., Zhang, F., Ding, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+solid+fluorescence+of+a+pyrazoline+derivative+through+hydrogen+bonding.">High solid fluorescence of a pyrazoline derivative through hydrogen bonding.</a> Molecules. 22 (8), 1 (2017).</li><li>Williams, A. T. R., Winfield, S. A., Miller, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+fluorescence+quantum+yields+using+a+computer-controlled+luminescence+spectrometer.">Relative fluorescence quantum yields using a computer-controlled luminescence spectrometer.</a> Analyst. 108 (1290), 1067-1071 (1983).</li><li>Eaton, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+materials+for+fluorescence+measurement.">Reference materials for fluorescence measurement.</a> Pure and Applied Chemistry. 60 (7), 1107-1114 (1988).</li><li>Dawson, W. R., Windsor, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+yields+of+aromatic+compounds.">Fluorescence yields of aromatic compounds.</a> Journal of Physical Chemistry. 72 (9), 3251-3260 (1968).</li><li>Zhang, X. -. F., Zhang, J., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fluorescence+Properties+of+Three+Rhodamine+Dye+Analogues:+Acridine+Red,+Pyronin+Y+and+Pyronin+B.">The Fluorescence Properties of Three Rhodamine Dye Analogues: Acridine Red, Pyronin Y and Pyronin B.</a> Journal of Fluorescence. 25 (4), 1151-1158 (2015).</li><li>Zanker, V., Rammensee, H., Haibach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+the+relative+quantum+yields+of+the+fluorescence+of+acridine+and+fluorescein+dyes.">Measurements of the relative quantum yields of the fluorescence of acridine and fluorescein dyes.</a> Zeitschrift f&#252;r Angewandte Physik. 10, 357-361 (1958).</li><li>Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J., Waggoner, A. 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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-ethyl-4-methyl-3-pyrrolin-2-one</td>
			<td>Combi-Blocks</td>
			<td>&#160;[766-36-9]</td>
			<td>Yellow solid reagent</td>
		</tr>
		<tr>
			<td>isoindolin-1-one</td>
			<td>ArkPharm</td>
			<td>&#160;[480-91-1]</td>
			<td>Off-white solid reagent</td>
		</tr>
		<tr>
			<td>5-bromoisoindolin-1-one</td>
			<td>Combi-Blocks</td>
			<td>&#160;[552330-86-6]</td>
			<td>Pink solid reagent</td>
		</tr>
		<tr>
			<td>2-formylimidazole</td>
			<td>Combi-Blocks</td>
			<td>&#160;[10111-08-7 ]</td>
			<td>Off-white solid reagent</td>
		</tr>
		<tr>
			<td>Imidazole-4-carbaldehyde</td>
			<td>ArkPharm</td>
			<td>&#160;[3034-50-2]</td>
			<td>Solid reagent</td>
		</tr>
		<tr>
			<td>1-H-pyrazole-4-carbaldehyde</td>
			<td>Oakwood Chemicals</td>
			<td>&#160;[35344-95-7]</td>
			<td>Solid reagent</td>
		</tr>
		<tr>
			<td>1-H-pyrazole-5-carbaldehyde</td>
			<td>Matrix Scientific</td>
			<td>&#160;[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 &#181;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-carboxalde...

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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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

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded synthesis strategy is used in which spherical seeds of CdSe are prepared first using a hot-injection technique. By controlling the crystal structure of the seed to be either wurtzite or zinc-blende, the subsequent hot-injection growth of CdS off of the seed results in either a rod-shaped or tetrapod-shaped nanocrystal, respectively. The phase and morphology of the synthesized nanocrystals are confirmed using X-ray diffraction and transmission electron microscopy, demonstrating that the nanocrystals are phase-pure and have a consistent morphology. The extinction coefficient and quantum yield of the synthesized nanocrystals are calculated using UV-Vis absorption spectroscopy and photoluminescence spectroscopy. The rods and tetrapods exhibit extinction coefficients and quantum yields that are higher than that of the bare seeds. This synthesis demonstrates the precise arrangement of materials that can be achieved at the nanoscale by using a seeded synthetic approach.

A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented.

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded ...

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms&#39; HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms&#39; frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljani&#263; groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte&#39;s structure and - because of cruciforms&#39; charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Cross-conjugated cruciform fluorophores based on 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole nuclei can be used to qualitatively identify diverse Lewis acidic and Lewis basic analytes. This method relies on the differences in emission colors of the cruciforms that are observed upon analyte addition. Structurally closely related species can be distinguished from each other.

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

The Synthesis of [Sn10(Si(SiMe3)3)4]2- Using a Metastable Sn(I) Halide Solution Synthesized via a Co-condensation Technique

The number of well-characterized metalloid tin clusters, synthesized by applying the disproportionation of a metastable Sn(I) halide in the presence of a sterically demanding ligand, has increased in recent years. The metastable Sn(I) halide is synthesized at&#160;&#34;outer space conditions&#34; via the preparative co-condensation technique. Thereby, the subhalide is synthesized in an oven at high temperatures, around 1,300 &#176;C, and at reduced pressure by the reaction of elemental tin with hydrogen halide gas (e.g., HCl). The subhalide (e.g., SnCl) is trapped within a matrix of an inert solvent, like toluene at -196 &#176;C. Heating the solid matrix to -78 &#176;C gives a metastable solution of the subhalide. The metastable subhalide solution is highly reactive but can be stored at -78 &#176;C for several weeks. On heating the solution to room temperature, a disproportionation reaction occurs, leading to elemental tin and the corresponding dihalide. By applying bulky ligands like Si(SiMe3)3, the intermediate metalloid cluster compounds can be trapped before complete disproportionation to elemental tin. Hence, the reaction of a metastable Sn(I)Cl solution with Li-Si(SiMe3)3 gives [Sn10(Si(SiMe3)3)4]2- 1 as black crystals in high yield. 1 is formed via a complex reaction sequence including salt metathesis, disproportionation, and degradation of larger clusters. Further, 1 can be analyzed by various methods like NMR or single crystal X-ray structure analysis.

The Synthesis of [Sn10SiSiMe334]2- Using a Metastable SnI Halide Solution Synthesized via a Co-condensation Technique

The disproportionation reaction of a metastable Sn(I) chloride solution, obtained via the preparative co-condensation technique, is used for the synthesis of a metalloid tin cluster compound.

The number of well-characterized metalloid tin clusters, synthesized by applying the disproportionation of a metastable Sn(I) halide in the presence of a ...

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 ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...